Abstract
Calcium-mediated agonists have long been known to stimulate transepithelial ion and fluid transport across a wide array of epithelial tissues. A key component of this response are the Ca2+-activated K+ channels, whose activation results in a hyperpolarization of the basolateral and apical membranes, thereby maintaining the electrochemical driving force for ion transport. In 1997, the intermediate conductance, Ca2+-activated K+ channel, KCa3.1, was cloned and subsequently shown to be localized to both the basolateral and apical membranes of secretory epithelia where it is activated by Ca2+-mediated agonists. Herein, we review the data confirming the critical role that KCa3.1 plays in transepithelial ion transport as well as the regulation, gating, trafficking of this channel, and this channel’s role in cell proliferation. Finally, we summarize the evidence for the role of KCa3.1 in epithelial diseases.
Keywords
1 Introduction
1.1 Early Evidence for Ca2+-Mediated Regulation of Transepithelial Transport
The evidence that parasympathetic stimulation is important in intestinal salt and water secretion was first obtained by several investigators in the 1880s when they demonstrated that injection of the muscarinic agonist pilocarpine, in large doses, could induce colonic secretion (Florey et al. 1941). While this early work suggested an important role for parasympathetic innervation of the intestine, very little additional work was done in this area until the 1940s. In a classic paper, Wright et al. (1940) demonstrated that direct vagal stimulation resulted in the flow of intestinal juices that continued only as long as the stimulation was maintained. It was further shown that this vagally induced secretion was blocked by the classic muscarinic antagonist, atropine (Wright et al. 1940). This potentially fruitful field of investigation again lays dormant until the 1960s when Tidball (1961) demonstrated, in the anesthetized dog, that administration of the cholinergic agonist, bethanechol, resulted in a change in the direction of intestinal water and Cl− movement from absorption to secretion. It was further demonstrated that Cl− was being secreted against both electrical and chemical gradients, demonstrating that secretion must be an active process (Tidball 1961). Similar increases in the transepithelial potential difference (PDte) and Cl− transport were reported for both in vivo (Hardcastle and Eggenton 1973; Hubel 1976, 1977) and in vitro (Hardcastle and Eggenton 1973) rat intestinal preparations. Isaacs et al. (1976) confirmed the above results in a stripped ileal mucosa preparation from human intestine and further demonstrated, using isotopic flux measurements, that the increase in PDte was due to an increase in the unidirectional flux of Cl− from serosa to mucosa. Although the above results clearly demonstrated that parasympathetic stimulation was involved in secretion, all of these results were from small intestinal preparations. It was not until 1977 that acetylcholine (Ach) was shown to cause fluid and electrolyte secretion from the colon, both in vivo and in vitro (Browning et al. 1977).
Initial evidence that sympathetic stimulation of ion and water secretion was due to direct innervation of epithelia came from staining for acetylcholinesterase, thereby localizing the nerve endings at the mucosa (Jacobowitz 1965; Browning et al. 1977; Isaacs et al. 1976). The functional release of neurotransmitter in the proximity of colonic epithelial cells was demonstrated by showing the uptake of radiolabeled choline and the subsequent synthesis and release of labeled Ach (Wu et al. 1982). This localization of functional nerve endings at the mucosa suggested that the physiological response associated with parasympathetic stimulation was due to the actions of Ach directly on the epithelial cells. This was confirmed by Rimele et al. (1981) when they demonstrated the existence of muscarinic receptors on intestinal epithelial cells. Zimmerman and Binder (1982) further demonstrated that the ability of muscarinic agonists to increase the short-circuit current (I sc) was directly related to agonist binding to specific muscarinic receptors on colonic epithelial cells.
While these early studies confirmed that parasympathetic stimulation of the intestine resulted in the direct stimulation of Cl− and fluid secretion from the epithelia, a great many additional studies were required to elucidate the mechanisms by which this fluid secretion occurred, as detailed in earlier chapters in this volume. Drawing upon a wealth of electrophysiological data, a general Cl− secretory model has been elucidated (Welsh et al. 1982; Reuss et al. 1983; Greger et al. 1984; Suzuki and Petersen 1985) as shown in Fig. 20.1. This model includes (a) the uptake of Cl− from the interstitial fluid by an electroneutral Na+-K+-2Cl− cotransporter; (b) the recycling of Na+ and K+ across the basolateral (serosal) membrane by the Na+/K+-ATPase and K+ channel, respectively; and (c) the movement of Cl− across the apical (mucosal) membrane through a Cl− channel by passive diffusion. Each of these transporters and channels is covered in detail in additional chapters in this volume. In the present chapter, we focus our attention on the basolateral, Ca2+-activated K+ channel activated by Ca2+-mediated agonists, including Ach.
Early electrophysiological evidence for the role of an increased basolateral K+ conductance during stimulation came from cAMP-mediated agonists, rather than Ca2+-mediated agonists, using canine trachea (Welsh et al. 1982) and later spiny dogfish rectal gland (Greger and Schlatter 1984a). In canine trachea, intracellular microelectrode measurements demonstrated that epinephrine caused an initial depolarization of membrane potential, due to Cl− exit across the apical membrane, followed approximately 20 s later by a repolarization that was associated with a decrease in basolateral membrane resistance due to the activation of a K+ conductance (Welsh et al. 1982). Later studies demonstrated that this agonist-induced Cl− secretory response could be blocked by either raising serosal K+ or adding the nonspecific K+ channel blocker, Ba2+ (Greger and Schlatter 1984b; Smith and Frizzell 1984), both of which will depolarize the basolateral membrane and, by electrical coupling, the apical membrane, thereby decreasing the electrochemical driving force for Cl− exit across the apical membrane.
1.2 Basolateral Membrane Ca2+-Activated K+ Channels
The first direct demonstration of a basolateral Ca2+-activated K+ conductance in epithelial cells was made by Maruyama et al. (1983a) in salivary acinar cells. The relatively easy access of the basolateral membrane of acinar cells to patch-clamp electrodes allowed others to quickly follow up on these observations by identifying additional Ca2+-activated K+ channels. Petersen and colleagues (Maruyama et al. 1983b; Maruyama and Petersen 1984) identified a 200 pS K+ channel activated by Ach and cholecystokinin in pig pancreatic acinar cells and later identified a 50 pS channel in the basolateral membrane. Early on, Ca2+-activated K+ channels were also identified in lacrimal (Trautmann and Marty 1984) and parotid (Foskett et al. 1989) acinar cells. In contrast to acinar cells, basolateral membrane Ca2+-activated K+ channels were only identified later in intestinal cells. Chang and Dawson (1988) identified a Ca2+-activated K+ conductance in permeabilized turtle colon, whereas Sepulveda and Mason (1985) characterized 36 pS and 90 pS Ca2+-activated K+ conductances in rabbit enterocytes, while Morris et al. (1986) identified a 250 pS K+ channel in rat enterocytes. However, these initial observations were all made on absorptive cells of the intestine rather than the secretory crypts. Subsequently, Loo and Kaunitz (1989) patch-clamped the basolateral membrane of crypt cells from rabbit distal colon and identified a 130 pS K+ channel that was activated by both Ca2+ and cAMP. Further, Burckhardt and Gogelein (1992) identified a 12 pS K+ channel in rat distal colonic crypts. In 1993, we identified a Ca2+-activated K+ channel in the T84 colonic cell line that was activated by Ach and taurodeoxycholate (Devor and Frizzell 1993; Devor et al. 1993). We had previously shown that bile acids increase intracellular Ca2+ via an IP3-mediated mechanism, such that this activation of KCa3.1 was identical to the Ach-mediated activation of the channel (Devor et al. 1993). Further, this channel was inwardly rectified, with chord conductances of 10 pS at +100 mV and 35 pS at −100 mV in symmetric K+. This rectification was independent of voltage and the channel was blocked by extracellular charybdotoxin (CTX) in a voltage-dependent manner (Devor and Frizzell 1993). This colonic Ca2+-activated K+ channel appeared to be identical to that described by Welsh and colleagues in tracheal epithelia (Welsh and McCann 1985; McCann et al. 1990) in which they characterized a channel with a slope conductance of 19 pS at 0 mV that was activated by intracellular Ca2+ and blocked by extracellular CTX. To the best of our knowledge, these studies represented the first identification of the Ca2+-activated K+ channel in epithelia that would eventually be realized to be the KCNN4 gene product , KCa3.1 (syn, IK1, SK4, KCa4).
2 Cloning of KCa3.1
As is apparent from the above, there are multiple forms of Ca2+-activated K+ channels. These have historically been grouped into (a) small conductance, SK channels, with single channel conductances of ~5–20 pS, many of which are sensitive to block by the bee venom, apamin; (b) the intermediate conductance, IK channels, having single channel conductances of ~20–80 pS and which are blocked by clotrimazole and charybdotoxin; and (c) the large conductance, maxi-K channels (also called BK ), having single channel conductances of ~100–250 pS that are blocked by charybdotoxin, iberiotoxin, and paxilline. The maxi-K channels (KCa1.1) have been discussed in Chap. 21 and will not be discussed herein. In 1996, Kohler et al. described the cloning of the SK family of channels , including SK1, SK2, and SK3, now referred to as KCa2.1, KCa2.2, and KCa2.3, respectively. These channels were predicted to have a classic 6-transmembrane domain architecture with both the N- and C-termini being cytosolic and the pore region being between the fifth and sixth transmembrane domains (S5 and S6), similar to the voltage-gated K+ channels (Kv), although these channels reside on distinct evolutionary branches. Kohler et al. (1996) demonstrated that these SK channels were Ca2+ dependent, being half-maximally activated at ~600–700 nM intracellular Ca2+ with a very steep dependence on Ca2+, having Hill coefficients of between 4 and 5. These authors further demonstrated the SK channels were voltage independent and that SK2 and SK3 were apamin sensitive, whereas SK1 was insensitive to apamin block. In total, these characteristics were completely consistent with these channels being responsible for the wide array of physiological events attributed to SK channels previously. As KCa2.x (SK) channels have rarely been reported to be expressed in epithelia as of the writing of this chapter, these channels will not be discussed in any detail, but rather we will contrast some of their attributes with KCa3.1 where appropriate. Interested readers are referred to Adelman et al. (2012) for further details about KCa2.x channels.
Based on the SK channel clones, Joiner et al. (1997) and Ishii et al. (1997) screened the expressed sequence tag database and identified a fourth family member. Joiner et al. (1997) referred to this clone as SK4, based on its homology to the rest of the SK family members, whereas Ishii et al. (1997) dubbed this channel IK1 based on this clone having both conductance and blocker characteristics historically associated with IK channels. Logsdon et al. (1997) also cloned this channel from T lymphocytes and referred to it as KCa4. Based on the standard nomenclature derived from the International Union of Pharmacology (IUPHAR ; Gutman et al. 2003), this channel is now referred to as KCa3.1. Full-length KCa3.1 is 427 amino acids in length with an architecture identical to that for the SK and Kv channels (Fig. 20.2). Indeed, KCa3.1 is ~40 % identical to the KCa2.x channels, with the highest degree of homology being in the transmembrane domains, pore region, and the proximal C-terminus (Joiner et al. 1997; Ishii et al. 1997). In contrast, the cytosolic N-terminus and distal C-terminus exhibit virtually no homology within the gene family, and numerous unique regulatory and trafficking motifs have been identified in this region of KCa3.1, as detailed below. KCa3.1 was shown to activate in response to increasing intracellular Ca2+, with a half-maximal concentration reported anywhere between 100 and 600 nM and a Hill coefficient of ~2 (Joiner et al. 1997; Ishii et al. 1997; Gerlach et al. 2000; Bailey et al. 2010). Note that these rather disparate K0.5 values for Ca2+ are likely associated with differences in posttranslational modifications (see below). At the single channel level, KCa3.1 was shown to exhibit slight inward rectification in symmetric K+, having chord conductances of ~10 pS at +100 mV and ~35 pS at −100 mV (Fig. 20.3), similar to what had previously been reported for IK channels in epithelia (McCann et al. 1990; Devor and Frizzell 1993). Further, in these initial reports, it was demonstrated that KCa3.1 was blocked by clotrimazole and charybdotoxin (Fig. 20.4), known blockers of IK channels in epithelia (McCann et al. 1990; Devor and Frizzell 1993; Rufo et al. 1996; Devor et al. 1997). Importantly, transcripts for KCa3.1 were found in numerous epithelial containing tissues, including the placenta, lung, trachea, salivary gland, kidney, pancreas, colon, bladder, stomach, and prostate (Joiner et al. 1997; Ishii et al. 1997; Logsdon et al. 1997; Jensen et al. 1998). Warth et al. (1999) and Gerlach et al. (2000) subsequently identified KCa3.1 by RT-PCR and Northern blot, respectively, in the T84 cell line, a human colonic crypt cell model. Gerlach et al. (2000) also identified KCa3.1 in Calu-3 cells , which are a model for serous cells from human airway. These results clearly implicated KCa3.1 as being the previously characterized IK1 channel in airway and colonic epithelia, although additional studies would be required to confirm this speculation.
3 Role of Basolateral KCa3.1 in Transepithelial Ion Transport
As is clear from the above, Ca2+-dependent K+ channels are required in the basolateral membrane to maintain the electrochemical driving force for trans epithelial Cl− secretion during Ca2+-mediated agonist secretion. Below, we will detail the experimental results that have led to our understanding of the role of KCa3.1 in the basolateral membrane of colonic, airway, salivary acinar, and pancreatic duct epithelia.
3.1 KCa3.1 in the Basolateral Membrane of Intestinal Epithelium
An important breakthrough in the study of Ca2+-mediated transepithelial Cl− secretion came from the work of Dharmsathaphorn and colleagues when they characterized the T84 cell line as a colonic crypt model system which formed high-resistance monolayers capable of vectorial electrolyte transport (Dharmsathaphorn et al. 1984, 1985, 1989; Weymer et al. 1985; Dharmsathaphorn and Pandol 1986; Mandell et al. 1986; Madara et al. 1987; Wasserman et al. 1988). Using 86Rb+ efflux measurements (note that Rb+ is often used as a surrogate for K+ in these studies), Dharmsathaphorn and Pandol (1986) demonstrated that both the cAMP-mediated agonists , vasoactive intestinal peptide (VIP ) and prostaglandin E1 , as well as the Ca2+-mediated agonist, carbachol, increased K+ efflux across the basolateral membrane, while having no effect on K+ efflux across the apical membrane, in T84 cells. These authors further demonstrated that Ba2+ blocked the VIP-stimulated basolateral K+ efflux pathway, whereas the carbachol-dependent pathway was insensitive to Ba2+ block. Finally, these two K+ efflux pathways were additive in nature, indicative of them being unique conductances. Duffey and colleagues (Devor et al. 1990, 1991; Devor and Duffey 1992) characterized this carbachol-induced K+ conductance using the whole-cell patch-clamp technique , demonstrating that it was activated by the increase in intracellular Ca2+ induced by carbachol and insensitive to block by Ba2+ as predicted based on the 86Rb+ efflux experiments of Dharmsathaphorn and Pandol (1986). Further studies by Devor and Frizzell (1993), using on-cell and excised patch-clamp techniques , defined the basolateral membrane, carbachol-activated K+ channel, in T84 cells as being Ca2+ dependent, inwardly rectifying and blocked by extracellular charybdotoxin.
Further evidence for the role of this channel in transepithelial Cl− secretion came when Devor et al. (1996a, b) characterized the first known pharmacological opener of this channel, 1-ethyl-2-benzimidazolinone (1-EBIO). 1-EBIO was subsequently shown to activate KCa3.1 by numerous labs (Devor et al. 1996a, b; Pedersen et al. 1999; Syme et al. 2000; Jensen et al. 2001), as detailed in Chap. 26; thus the pharmacology of these modulators will not be discussed herein. Critically, 1-EBIO induced a sustained transepithelial Cl− secretory response in T84 cells that was blocked by the basolateral addition of charybdotoxin, with an inhibitory constant of 3.6 nM, indicative of IK (KCa3.1) being the basolateral K+ channel activated. In contrast, the chromanol, 293B (Lohrmann et al. 1995), an inhibitor of the cAMP-dependent K+ channel (now known to be KCNQ1), had no effect on this 1-EBIO -induced current. These results, as illustrated in Fig. 20.5, clearly demonstrated, using selective K+ channel blockers , that the Ca2+- and cAMP-mediated basolateral K+ conductances were distinct, as initially proposed based upon their Ba2+ sensitivity. Similar results were observed in rat colonic epithelium, consistent with this IK channel playing a key role in native ex vivo tissue (Devor et al. 1996a, b). Another critical piece of evidence for these IK channels being the basolateral K+ channel activated by Ca2+-dependent agonists came when Rufo et al. (1996) demonstrated that clotrimazole inhibited transepithelial Cl− secretion across T84 cells via an inhibition of 86Rb+ efflux across the basolateral membrane with no effect on other transporters critical to this Cl− secretory process. These authors further demonstrated that clotrimazole inhibited the basolateral K+ conductance in T84 cells using nystatin-permeabilized monolayers (Rufo et al. 1997). Finally, Devor et al. (1997) demonstrated a direct effect of clotrimazole on IK channels in excised patch-clamp experiments from T84 cells and further demonstrated that clotrimazole inhibited the 1-EBIO -induced Cl− secretory response. Subsequently, Hamilton et al. (1999) demonstrated that 1-EBIO stimulated transepithelial Cl− secretion across mouse jejunum and further demonstrated that 1-EBIO directly activated IK channels in the basolateral membrane of isolated jejunal crypts using the on-cell patch-clamp technique. Similarly, Warth et al. (1999) demonstrated in rat and rabbit colonic mucosa that 1-EBIO stimulated clotrimazole-sensitive Cl− secretion as well as activated IK channels in excised patches from the basolateral membrane of rat colonic crypts. These authors also demonstrated that antisense probes to KCa3.1 attenuated the carbachol-induced Cl− secretory current across T84 cells, confirming the critical role of this channel in Ca2+-mediated Cl− secretion in these cells (Warth et al. 1999). In an additional series of studies, Cuthbert and colleagues (Cuthbert et al. 1999; Cuthbert 2001; MacVinish et al. 2001) demonstrated that 1-EBIO stimulated Cl− secretion across mouse colon and that this was dependent upon a charybdotoxin-sensitive K+ conductance in the basolateral membrane. As noted above, subsequent to the cloning of KCa3.1 and the realization that this was the IK channel previously reported, Warth et al. (1999) and Gerlach et al. (2000) confirmed by RT-PCR and Northern blot, respectively, that this was the channel expressed in T84 cells. Finally, Flores et al. (2007) carried out studies on KCa3.1 knockout mice and demonstrated that Ca2+-mediated Cl− secretion was completely eliminated in both distal colon and small intestinal epithelium which resulted in a marked reduction in water content in the stools. In total, these studies unequivocally demonstrate that KCa3.1 is the basolateral membrane K+ chan nel activated by Ca2+-mediated agonists as a means of maintaining transepithelial Cl− secretion across intestinal epithelia.
3.2 KCa3.1 in the Basolateral Membrane of Airway Epithelium
Similar to colonic epithelia, early microelectrode data demonstrated that cAMP-mediated agonists activate a basolateral K+ conductance necessary for the maintenance of transepithelial Cl− secretion (Welsh et al. 1982) and that Ba2+ applied to the basolateral side blocked this conductance, resulting in a diminution of Cl− secretion (Greger and Schlatter 1984b; Smith and Frizzell 1984). Welsh and McCann (1985) first characterized the Ca2+-activated K+ channel in primary cultures of canine tracheal epithelial cells, demonstrating that it was activated by epinephrine, which increases Ca2+ in tracheal epithelia. These authors further demonstrated that increasing Ca2+ induced 86Rb+ efflux from subconfluent canine tracheal epithelial cells that was blocked by charybdotoxin. Additional patch-clamp studies showed that the K+ channel was blocked by charybdotoxin (McCann et al. 1990). Finally, these authors (McCann and Welsh 1990) confirmed these studies on primary cultures of canine tracheal epithelial cells grown on semipermeable supports, thereby demonstrating that this IK channel (called KCLIC by these authors) was the basolateral membrane Ca2+-activated K+ channel in airway epithelia.
With the identification of 1-EBIO as a dir ect activator of IK channels, Devor et al. (1996a, b) also demonstrated that this compound stimulated transepithelial Cl− secretion across murine tracheal epithelium. Subsequent studies in both the serous cell model cell line, Calu-3 (Devor et al. 1999), and primary cultures of human bronchial epithelia (Devor et al. 2000) demonstrated that 1-EBIO and the higher affinity analogue, DCEBIO (5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazol-2-one) (Singh et al. 2001), stimulated transepithelial Cl− secretion that was blocked by basolateral addition of either charybdotoxin or clotrimazole. Additional patch-clamp (Devor et al. 1999) and Northern blot analysis (Gerlach et al. 2000) confirmed expression of KCa3.1 in Calu-3 cells. Subsequent studies by Cowley and Linsdell (2002) and Mall et al. (2003) confirmed expression of KCa3.1 in Calu-3 cells and native human nasal epithelia from normal and cystic fibrosis (CF) patient samples by RT-PCR. Similar studies in the 16HBE14o- (Bernard et al. 2003) and H441 (Wilson et al. 2006) human airway cell lines confirmed expression of KCa3.1 by RT-PCR and that this channel was activated by 1-EBIO resulting in sustained transepithelial Cl− secretion that was blocked by clotrimazole. In total, these studies clearly demonstrate expression of KCa3.1 in the basolateral membrane of airway epithelia and that activation of this channel is required for Ca2+-mediated Cl− secretion.
Interestingly, Smith and Welsh (1992) demonstrated that increasing cAMP stimulated HCO3 − secretion across normal airway epithelia but not in airway from CF airway epithelia, while Ashton et al. (1991) also suggested that pancreatic duct epithelial cells could be differentially stimulated to secrete either Cl− or HCO3 −. In this regard, it has been shown that the cystic fibrosis transmembrane conductance regulator (CFTR ) conducts both Cl− and HCO3 − (Gray et al. 1990; Linsdell et al. 1997; Ishiguro et al. 2009). Further, Devor et al. (1999) found that activation of KCa3.1 acted as an “ionic switch,” dictating whether the human airway serous cell line, Calu-3, secreted HCO3 − or Cl−. That is, stimulation of Calu-3 cells with forskolin , to increase cAMP, resulted in a HCO3 − secretory response as assessed by both the lack of bumetanide sensitivity and isotopic flux experiments. In contrast, stimulating Calu-3 cells with the KCa3.1 opener, 1-EBIO, resulted in Cl− secretion. Finally, stimulation with both forskolin and 1-EBIO resulted in a primarily Cl− secretory response with a decrease in HCO3 − secretion relative to forskolin alone. Thus, these results suggest that activation of basolateral KCa3.1 and the concomitant basolateral hyperpolarization dictate the anion transport properties of the epithelium.
Finally, Devor et al. (2000) s howed that activation of basolateral KCa3.1 also increased the amiloride-sensitive transepithelial Na+ absorption (Fig. 1) across primary cultures of human bronchial epithelia (HBE) expressing ΔF508 CFTR. These results indicate that the KCa3.1-induced hyperpolarization was sufficient to drive Na+ entry though the epithelial Na+ channel (ENaC) in the apical membrane of HBEs. Gao et al. (2001) confirmed that 1-EBIO increased transepithelial Na+ transport in monolayers of the ΔF508 CFTR-expressing human airway cell line, CFT1. These authors further demonstrated that 1-EBIO did not activate apical ENaC in basolateral membrane amphotericin B-permeabilized CFT1 monolayers or in Xenopus oocytes expressing ENaC, confirming that activation of basolateral KCa3.1 results in an increased transepithelial Na+ absorption. In contrast to these studies, Devor et al. (1996a) demonstrated that 1-EBIO did not influence Na+ transport in either rat colonic or murine tracheal epithelia. These apparently disparate results are likely the result of the unique physiologies of human airway versus mouse airway and rat colon such that the rate-limiting conductance for Na+ absorption is different in these cell types.
3.3 KCa3.1 in the Basolateral Membrane of Salivary Acinar and Pancreatic Duct Epithelium
Salivary acinar cells secrete electrolytes and fluid by mechanisms similar to those described above and recently reviewed by Catalan et al. (2014). Importantly, both maxi-K (KCa1.1) and KCa3.1 channels have been molecularly described in human and rodent salivary gland acinar cells (Nehrke et al. 2003; Begenisich et al. 2004). Surprisingly, genetic knockout of either KCa1.1 or KCa3.1 alone in mice had no effect on salivary gland fluid secretion, indicating that expression of either of these channels is sufficient to maintain fluid secretion (Begenisich et al. 2004; Romanenko et al. 2006). This result was explained by the observation that knockout of either of these channels did not affect the ability of the acinar cells to hyperpolarize toward the K+ reversal potential (EK). In contrast, mice lacking both KCa1.1 and KCa3.1 channels exhibited a severely reduced fluid secretion rate in response to Ca2+-mediated agonists, and this was paralleled by an inhibition of the membrane hyperpolarization toward EK (Romanenko et al. 2006). These results clearly demonstrate a role for KCa3.1 in salivary acinar cell physiology but also demonstrate that these cells can bypass deficits in KCa3.1 function by recruiting KCa1.1 channels.
Finally, Nguyen and Moody (1998) demonstrated that an 1-EBIO activated and charybdotoxin and clotrimazole inhibited IK channel in primary cultures of dog pancreatic duct epithelial cells that were localized to the basolateral membrane based on both 86Rb+ efflux studies and blocker sidedness. Similar to the above, this channel has now been positively identified as KCa3.1 in rodent and human pancreatic duct cell lines (Capan-1, PANC-1, CFPAC-1) as well as in ducts isolated from rodent pancreas (Hede et al. 2005; Hayashi et al. 2012; Wang et al. 2013). In these cells, DCEBIO stimulated, and clotrimazole inhibited, anion secretion, although it was not determined whether this was HCO3 − or Cl− secretion. As Ashton et al. (1991) have suggested that pancreatic duct epithelial cells can be differentially stimulated to secrete either Cl− or HCO3 −, it will be important to determine whether activation of KCa3.1 regulates this process as was described in human airway (Devor et al. 2000). The study of the regulation and function of KCa3.1 in the pancreatic ducts is still in its early stages and the next few years are likely to shed additional light on this important subject.
4 Role of KCa3.1 in the Apical Membrane
In addition to K+ channels playing a pivotal role in the basolateral membrane to modulate transepithelial Cl− secretion, it is also well known that epithelia of the airway, pancreas, kidney, parotid gland, and intestine secrete K+ across the apical membrane, as detailed in Chap. 3. While the K+ channels that have been historically shown to be involved in this K+ secretory process include the maxi-K (KCa1.1, KCNMA1, Chap. 21) and ROMK (Kir1.1, KCNJ1, Chap. 19) channels, evidence has accumulated to implicate KCa3.1 in this process as well. Indeed, Bernard et al. (1993) demonstrated that the KCa3.1 blocker , clotrimazole (Rufo et al. 1996; Devor et al. 1997; Wulff and Castle 2010), inhibited 86Rb+ efflux across both the basolateral and apical membranes of the human bronchial cell line , 16HBE14o-. Further, 1-EBIO stimulated 86Rb+ efflux across both basolateral and apical membranes in a clotrimazole-dependent manner, implicating a role for KCa3.1 in the apical membrane of airway epithelia (Bernard et al. 1993). Similarly, Joiner et al. (2003) demonstrated that the apical addition of clotrimazole completely inhibite d the carbachol-induced 86Rb+ secretion across rat proximal colon. These authors also isolated apical versus basolateral membrane and demonstrated that KCa3.1 was expressed in both membrane fractions by immunoblot. Further, it was demonstrated that rats deprived of K+ in their diet exhibited a loss of KCa3.1 mRNA that correlated with 86Rb+ secretion being abolished (Joiner et al. 2003). At the same time, Neylon and colleagues (Furness et al. 2003) demonstrated, by immunofluorescent localization, that KCa3.1 was expressed in both apical and basolateral membranes of rat intestine . Finally, Almassy et al. (2012) demonstrated expression of KCa3.1 in the apical membrane of parotid acinar cells . In these experiments, caged Ca2+ was released in close proximity to the apical membrane while simultaneously recording whole-cell K+ currents. These authors demonstrated that the release of Ca2+ exclusively adjacent to the apical membrane resulted in the activation of K+ currents that were partially blocked by both paxilline and TRAM-34, inhibitors of maxi-K and KCa3.1 channels, respectively. Further, in parotid acinar cells from maxi-K null mice, apical TRAM-34-sensitive KCa3.1 currents were still observed following release of Ca2+ at the apical membrane, providing compelling evidence for KCa3.1 in the apical membrane of parotic acinar cells. Taken together, these results implicate KCa3.1 in K+ secretion across the apical membrane of various epithelial tissues.
While there is clear data supporting a role fo r KCa3.1 in both the basolateral and apical membranes of secretory epithelia, these data beg the question of how this channel is targeted to both apical and basolateral membranes. The answer to this question began to come into focus in 2010 when Rajendran and colleagues (Barmeyer et al. 2010) identified a splice variant of KCa3.1, KCNN4c from rat colon that lacked exon 2 which encodes 29 amino acids, including 5 amino acids in the S1–S2 extracellular linker as well as all of S2 and 2 amino acids in the proposed intracellular S2–S3 linker. As expected, deletion of S2 resulted in a channel that failed to traffic to the plasma membrane when heterologously expressed. However, when KCNN4c was co-expressed with the maxi-K β1-subunit, the channel was expressed at the membrane in Xenopus oocytes (see Fig. 20.2). KCNN4c was confirmed to form a functional channel by measuring 86Rb+ efflux from oocytes that was activated by the KCa3.1 channel opener, DCEBIO (Singh et al. 2001), and inhibited by the KCa3.1 blocker, TRAM-34 (Wulff et al. 2000), although the sensitivity of TRAM-34 block was significantly reduced in the KCNN4c channel, being 0.6 μM for full-length KCa3.1 and 7.8 μM for KCNN4c plus β1-subunit (Barmeyer et al. 2010). Further studies demonstrated that the KCNN4c/β1 channels had single channel properties that are similar to full-lengt h KCa3.1, i.e., they display inward rectification and have a chord conductance of 31 pS in symmetric K+ (Basalingappa et al. 2011). Interestingly, these authors also demonstrated that KCNN4c did not co-assemble and, therefore, inhibit expression of the full-length KCa3.1 channel (Barmeyer et al. 2010). Importantly, immunoblots of apical versus basolateral membranes demonstrated an ~37 kDa band in the apical membrane lysates, while an ~40 kDa band was detected in the basolateral membrane lysates, suggesting that this lower molecular mass product corresponds to KCNN4c being expressed in the apical membrane of rat colonic epithelia (Barmeyer et al. 2010).
Further studies by Singh et al. (2012) demonstrated that a Na+-free diet , which increases circulating aldosterone levels, increases KCNN4c mRNA and protein expression in rat distal colon. These authors also demonstrated that in the presence of aldosterone, a TRAM-34-dependent K+ secretory pathway was revealed, consistent with an increase in apical membrane expression of KCNN4c under these conditions. More recent studies have identified mineralocorticoid response elements in KCNN4 introns that respond to aldosterone (O’Hara et al. 2014). In an exciting series of studies, Hoque and colleagues demonstrated that binding of cAMP to exchange protein directly activated by cAMP (Epac) activates a PKA-independent, Ca2+-dependent Cl− secretory process in T84 cells that involves stimulation of Rap2-phospholipase Cε (Hoque et al. 2010). More recently, these authors demonstrated that Epac1 regulates the apical surface expression of KCNN4c in T84 cells (Sheikh et al. 2013). That is, knockdown of Epac1 both significantly decreased the TRAM-34-dependent apical potassium conductance (GK) and also resulted in the redistribution of KCNN4c into subapical vesicles. Furthermore, inhibitors of both RhoA (GGT1298) and Rho-associated kinase (ROCK; H1152) reduced apical GK, and ROCK inhibition also caused KCNN4c to redistribute out of the apical membrane (Sheikh et al. 2013). To our knowledge, these studies represent the first demonstration of a dynamic redistribution of KCNN4c into and out of the apical memb rane of polarized epithelia.
5 Gating of KCa3.1
The gating of an ion channel can be simply thought of as the process by which the pore transitions between nonconducting (closed) and conducting (open) states . As outlined above, KCa3.1 gating is dictated by changes in the intracellular Ca2+ concentration. Calmodulin is constitutively bound to the calmodulin-binding domain (CaMBD) of KCa3.1 (see Fig. 20.2), located in the C-terminus just distal to S6 (Fanger et al. 1999; Schumacher et al. 2001, 2004). When Ca2+ binds to calmodulin, this results in a conformational change in calmodulin and, therefore, the CaMBD, allowing the channel to transition from the closed to the open conformation (Keen et al. 1999; Li et al. 2009). The mechanism by which Ca2+ binding is translated to KCa3.1 gating has received considerable attention, as detailed below.
5.1 A Gating Model for KCa3.1
The first attempt to define the open-closed gating kinetics of KCa3.1 was carried out by Grygorczyk and Schwarz (1985) using the single channel patch-clamp technique on human red blood cells. These authors identified a single open state, as assessed by fitting an open-time (τ o) distribution plot to a single exponential, as well as two closed states. However, the authors speculated an additional long closed time (τ c) that was >60 ms in duration that could not be resolved during their recordings to fully account for their entire data set. The τ o was shown to increase with increasing Ca2+, whereas the two τ c did not significantly depend on Ca2+, although the mean closed time was decreased by increasing Ca2+, indicative of the long, unresolved τ c being reduced in duration. In 1992, Leinders et al. (1992) again evaluated the gating kinetics of KCa3.1 in human erythrocytes. These authors found that both the τ o and τ c distributions were fit to two exponentials, indicating two open as well as two closed states. However, these authors similarly proposed a long-lived third τ c to account for all of their data. While Leinders et al. (1992) found no dependence of τ o on Ca2+, distinct from Grygorczyk and Schwarz (1985), they proposed that the long-lived third τ c was dependent on Ca2+, similar to Grygorczyk and Schwarz (1985). Dunn (1998) conducted a gating study on KCa3.1 in human erythrocytes and identified two τ o and three τ c, indicative of two open and three closed gating steps. Indeed, this third τ c averaged 155 ms in agreement with previous reports. However, Dunn did not evaluate the effect of Ca2+ on these gating kinetics. Syme et al. (2000) studied KCa3.1 heterologously expressed in HEK cells and also identified two τ o and three τ c. However, based on an analysis of the relative amplitudes of each closed component, the data they could resolve would have accounted for a P o of 0.3, when the single channel patches used to obtain the data had an actual P o of 0.18. Thus, similar to the authors above, Syme et al. (2000) concluded that a longer fourth τ c of ~200 ms, which could not be resolved, must be present. Again, these authors did not evaluate the effect of changing Ca2+ on these gating parameters.
A significant advancement in our understanding of the gating of the KCNN gene family members came in 1998 when Hirschberg et al. (1998) studied the single channel gating kinetics of KCa2.2 heterologously expressed in Xenopus oocytes. An analysis of the single channel behavior revealed two τ o and three τ c. Further, it was demonstrated that neither of the τ o nor the shorter τ c were Ca2+ dependent. Rather, it was the long τ c that was shown to decrease with increasing concentrations of Ca2+, similar to what was described by Grygorczyk and Schwarz (1985) for KCa3.1. When Hirschberg et al. (1998) attempted to mathematically model KCa2.2 channel gating kinetics, they found that a model with two τ o and three τ c would only yield a reasonable fit to their experimental data if they assumed that the derived rate constants had a nonlinear dependence on Ca2+ concentration. To obtain the desired linear dependence on Ca2+ concentration, these authors required a fourth τ c that was not experimentally resolved, similar to what had been described for KCa3.1, to accurately model their data. The six-state open-closed gating model for KCa2.2 is shown in Fig. 20.6a with the forward transitions between closed states being Ca2+ dependent.
Bailey et al. (2010) undertook a study to model the Ca2+ dependence of KCa3.1 gating behavior based upon the model of Hirschberg et al. (1998) described above. In contrast to previous studies on single channel patches, Bailey et al. (2010) utilized macro-patches containing hundreds or thousands of channels heterologously expressed in HEK cells and carried out rapid step changes in Ca2+ (~5 ms) from 0.4 to 10 μM, after which the macroscopic currents were fit to a series of exponentials and gating kinetics modeled. Consistent with other reports, the activation kinetics of KCa3.1 were found to be Ca2+ dependent, whereas the deactivation kinetics were Ca2+ independent. Using the six-state model of Hirschberg et al. (1998), Bailey et al. (2010) obtained an accurate description of their entire data set except for the activation rate at saturating Ca2+ (Fig. 20.6c). Based on this, these authors assumed that the Ca2+-dependent steps in KCa3.1 gating depended nonlinearly on Ca2+ concentration such that the Ca2+-binding rate constants could saturate at high Ca2+ concentrations. With this assumption, these authors were able to fit their entire data set (Fig. 20.6b). However, a further sensitivity analysis to evaluate the importance of each parameter in the model revealed that the data could be fit with a simpler four-state kinetic model that excluded the final C-C and C-O transitions shown in the box in Fig. 20.6a such that the data were fit with a single τ o and three τ c with the Ca2+-dependent rate constants saturating at high Ca2+. Finally, these authors reported unpublished observations suggesting that the gating behavior of KCa2.2 could also be described by a simpler four-state gating scheme with the Ca2+-dependent transitions being nonlinear. The nonlinear dependence of the opening transitions on Ca2+ observed by Bailey et al. (2010) compared to the linear dependence proposed by Hirschberg et al. (1998) may be based on the fact that Bailey et al. (2010) utilized 10 μM Ca2+ as a saturating level and this may begin to produce channel block (Ledoux et al. 2008). Whether this becomes important in physiological or pathophysiological situations is unclear.
5.2 KCa3.1 Pore Architecture and Allostery
The work of Simoes et al. (2002) was the first to examine the pore architecture of KCa3.1. These authors used the substituted cysteine accessibility method (SCAM) that combines site-directed mutagenesis and chemical modification with thiol-specific reagents to analyze the access of introduced cysteines (Falke et al. 1988; Karlin and Akabas 1998; Roberts et al. 1986; Akabas et al. 1992, 1994). Thus, by combining SCAM with the patch-clamp technique, gating-sensitive amino acids can be identified by alterations in channel gating (Holmgren et al. 1996). In this regard, Simoes et al. (2002) identified two regions (Val275–Val282 and Ala283–Ala286) along S6 of KCa3.1 that display functional differences to MTSET ([2-(trimethylammonium) ethyl] methanethiosulfonate bromide) binding. That is, in the open state, the addition of MTSET caused inhibition at positions Val275, Thr278, and Val282, implying that these residues line the lumen of the pore. In both channel open and closed configurations, the Ala283–Ala286 region is accessible to MTSET, suggesting this region is not embedded in the membrane and participates in the conformational changes in CaM/CaMBD to open the KCa3.1 channel’s pore.
Additionally, Klein et al. (2007) studied the KCa3.1 channel pore structure in the closed configuration and found that the modification rates of MTSEA (2-aminoethyl methanethiosulfonate hydrobromide), with a diameter 4.6 Å, for a residue located in the central cavity (Val275Cys) compared to a residue located at the C-terminal end of S6 (Ala286Cys) were found to differ by less than sevenfold, whereas experiments performed with MTSET, with a diameter of 5.8 Å, resulted in a modification rate 103–104 faster for the cysteine located in the C-terminal end compared to the cysteine in the central cavity. Modification rates of Val275Cys using Et-Hg+ (4.1 Å diameter) and Ag+ (2.55 Å diameter), which are smaller than MTSET, were found to be closed/open state independent, while modification rates for MTSET were 103 times faster for the open compared to the closed state. Based on these results, the authors proposed that the closed structure of KCa3.1 can be represented by a narrow passage centered at Val282, connecting the channel inner cavity to the cytosolic medium, rather than to the inverted teepee-like structure as described for KcsA channel (Doyle et al. 1998) (Fig. 20.7). As passage through the pore would not be restrictive to the diffusion of small reagents such as MTSEA, Et-Hg+, and Ag+, similar in size to the K+ ion, these data suggest that in the closed conformation, the cytoplasmic ends of S6 do not function as an obstructive barrier to the movement of K+ ions (Klein et al. 2007).
The substitution of Val188 (Val282 in KCa3.1) by less hydrophobic residues in the GIRK2 channel (Yi et al. 2001) or Pro475 (Ala283 in KCa3.1) by a more hydrophilic residue in the Shaker channel (Sukhareva et al. 2003) resulted in GIRK2 channels that were constitutively active or in Shaker channels that were unstable in the closed configuration. Using a glycine (Gly) scan analysis , Garneau et al. (2009) observed that the substitutions Ala279Gly and Val282Gly in S6 of KCa3.1 resulted in the channel being constitutively active in zero Ca2+. In contrast, when residues between Cys276 and Ala286, as well as Ala279 to Val282, were substituted with Gly, constitutive activation of KCa3.1 was not observed. These results demonstrate that hydrophobic interactions involving Val282 are key determinants of KCa3.1 gating (Fig. 20.7). The authors further showed that Ag+ enjoyed free access to the c hannel cavity in both an ion-conducting mutant, Val275Cys/Val282Gly, and the closed channel mutant, Val275Cys, further arguing against the activation gate of KCa3.1 being localized to the distal end of S6 (Garneau et al. 2009).
Although KCa3.1 has four cysteines in S6 (Cys267, 269, 276, 277), Simoes et al. (2002) demonstrated that MTSET only transiently inhibited KCa3.1 current, indicative of these cysteines being inaccessible for modification, i.e., these cysteines face away from the pore lumen. Subsequently, Bailey et al. (2010) evaluated the effect of another cysteine-modifying agent , parachloromercuribenze sulfonate (PCMBS) on KCa3.1 channel activity. In contrast to the MTS reagent, PCMBS activated KCa3.1 by inducing both an increase in current at saturating Ca2+ concentrations and a leftward shift in the Ca2+ activation curve to higher affinity. These results led the authors to conclude that PCMBS activated KCa3.1 via both Ca2+-independent and Ca2+-dependent pathways as reflected in the changes in both P o(max) and apparent Ca2+ affinity, respectively. Mutational analysis demonstrated that Cys276 was required for the PCMBS-mediated activation of KCa3.1. These authors further carried out a partial tryptophan scan of S6 and showed that both Leu281Trp and Val282Trp substitutions resulted in dramatic shifts in apparent Ca2+ affinity, similar to what was observed with PCMBS (Bailey et al. 2010). As crystallographic data h ave shown that PCMBS modification of cysteines can result in downstream amino acid side-chain perturbations (Ackerman et al. 2002), it was posited that the binding of PCMBS to Cys276 altered the neighboring Leu281 and Val282 residues (Bailey et al. 2010) (Fig. 20.7). In total, the results from Bailey et al. (2010) and Sauvé and colleagues (Simoes et al. 2002; Klein et al. 2007; Garneau et al. 2009) demonstrate that both luminal and non-luminal amino acids along S6 participate in the activation mechanism of KCa3.1 and that the C-terminal region of S6 must be allosterically coupled to the activation gate (Fig. 20.7).
An interesting characteristic of KCa3.1 gating is that the P o(max), at saturating Ca2+ concentrations, is ~0.1–0.2 compared with a P o(max) of ~0.8 for the KCa2.2 channel (Hirschberg et al. 1998). As noted above, binding of Ca2+ to the CaM N-lobe results in CaM associating with the CaMBD of adjacent KCa2.2 subunits, within the tetramer (Schumacher et al. 2001, 2004), and it is presumed by homology that this holds true for KCa3.1. Thus, Morales et al. (2013) undertook a series of mutagenesis studies designed to identify amino acids in the CaMBD that are critical to determining KCa3.1 P o(max). In this regard, these authors demonstrated that hydrophobic effects at Ser367 contribute to P o(max), whereas electrostatic interactions involving Arg362 and Glu363 regulate channel activation rate (τ on). That is, the Ser367Cys mutation resulted in an increase in P o(max) from 0.22 to 0.62 coupled with an increase in channel open time and a decrease in channel closed time. Additional substitutions with nonpolar amino acids of greater surface area (Thr, Leu, Trp) also increased P o(max), whereas substitution with an amino acid of similar surface area (Ala) had no effect. It was also demonstrated that neutralizing the charged amino acids at positions Arg362 and Glu363 resulted in dramatic shifts in τ on, indicative of amino acids not involved in KCa3.1-CaM binding but important in channel activation.
5.3 Calmodulin and KCa3.1 Gating
Following the cloning of KCa2.x, and subsequently KCa3.1, channels that exhibited Ca2+-dependent gating characteristics similar to what had been historically noted for the small and intermediate conductance, Ca2+-activated K+ channels, it raised the question of how Ca2+ mechanistically regulated these channels. It was noted by Kohler et al. (1996) for KCa2.x channels, as well as by both Joiner et al. (1997) and Ishii et al. (1997) for KCa3.1 channels, that these channels did not share homology with the maxi-K channels and did not possess motifs similar to an E-F hand capable of binding Ca2+ in order to induce gating. Shortly after the cloning of these channels, in a series of elegant studies, Adelman and colleagues (Xia et al. 1998; Keen et al. 1999) demonstrated that calmodulin bound constitutively to the proximal C-terminus of the KCa2.x channels at the CaMBD just distal to the sixth transmembrane domain. These authors further demonstrated that one calmodulin was bound to each of the four subunits of the tetrameric channel and that it was specifically the binding of Ca2+ to the N-lobe E-F hands of calmodulin that induced channel gating via a conformational change in calmodulin that was transmitted to the KCa2.x channel α-subunit. In contrast, the C-lobe E-F hands of calmodulin did not bind Ca2+ when it was associated with KCa2.x channels. Fanger et al. (1999) similarly demonstrated that KCa3.1 channels were constitutively bound to calmodulin that resulted in their Ca2+-dependent gating.
A significant breakthrough in understanding how the binding of Ca2+ to calmodulin resulted in channel gating came from the work of Schumacher et al. (2001, 2004). These authors solved the crystal structure for calmodulin bound to the CaMBD of KCa2.2 in both the apo and calcified states, thereby providing a structural mechanism for Ca2+ binding and the subsequent conformational changes leading to KCa2.2 gating. Based on these studies, it was proposed that the binding of Ca2+ to the N-lobes of CaM in KCa2.2 causes the formation of a dimeric complex between two adjacent CaMBD, while initiating a >90° rotation of the S6 helices leading to the opening of the channel pore. Because there is a high degree of homology in the calmodulin/CaMBD between KCa3.1 and KCa2.2, the gating mechanism proposed in KCa2.2 likely applies to KCa3.1. Therefore, the C-terminal portion of the S6 in KCa3.1 is thought to be involved in the transformation of energy from the calmodulin/CMBD into a mechanical force, modifying the confor mational state of the pore (Schumacher et al. 2001, 2004; Simoes et al. 2002; Klein et al. 2007; Garneau et al. 2009). Unfortunately, the linker between S6 and the CaMBD could not be resolved in these crystal structures and so the exact mechanism by which this force is exerted remains to be determined.
5.4 Role of KCa3.1 in Gating of Maxi-K (KCa1.1)
Interestingly, the gating of KCa3.1 has also been shown to directly regulate the gating of maxi-K (KCa1.1) channels. That is, Thompson and Begenisich (2006) showed that activation of KCa3.1 channels inhibits maxi-K channels in acinar cells, where the two channels are endogenously expressed, and in heterologous expression systems. Similar reports were described in human parotid (Nakamoto et al. 2007) and in mouse submandibular glands (Romanenko et al. 2007). The inhibition of maxi-K channels occurred whether the KCa3.1 channels were activated by direct perfusion of the cell with elevated Ca2+, by muscarinic receptor stimulation, or by the exogenous chemical activator DCEBIO at low Ca2+ concentration. In addition, the inhibition of maxi-K currents was blocked by TRAM-34, an inhibitor of KCa3.1 (Thompson and Begenisich 2006; Romanenko et al. 2009). Quantitative analysis led these authors to postulate that each maxi-K channel may be surrounded by four KCa3.1 channels and it would be inhibited if any one of these KCa3.1 channels opens (Thompson and Begenisich 2006). Further, since KCa3.1-induced inhibition of maxi-K channels takes place in excised membrane patches, the authors proposed a close interaction between the two channels without any intermediaries such that the block of the ion flow through the pore in the maxi-K channel may occur by inserting the cytoplasmic N-terminal domains of KCa3.1 (Thompson and Begenisich 2006), as was described for two maxi-K channel β-subunits (β2 and β3) (Wallner et al. 1999; Xia et al. 1999; Zhang et al. 2006; Li et al. 2007). In this regard, Thompson and Begenisich (2009) observed that the N-terminus of KCa3.1 shares general similarity with the N-terminal regions of these two maxi-K β-subunits, the N-terminal region peptide of KCa3.1 blocked maxi-K channels, and the activation of KCa3.1 competed with the N-terminus peptide for blocking of maxi-K channels. In total, these data resulted in these authors proposing a model where KCa3.1 channels are located sufficiently close to the maxi-K channels such that the activation of KCa3.1 results in a conformational change in their N-termini, which inserts through a cytoplasmic side portal in the BK channel, thereby inhibiting permeation through the maxi-K pore.
6 Regulation of KCa3.1
As detailed above, the main regulator of KCa3.1 gating is Ca2+ binding to calmodulin constitutively associated with the CaMBD. However, shortly after the mechanism by which Ca2+-dependent gating was unraveled, it was shown that phosphorylation modified this Ca2+-dependent gating process. That is, Gerlach et al. (2000) first demonstrated a role for kinases in the regulation of KCa3.1 by showing that hydrolyzable ATP analogues, but not other nucleoside triphosphates, activated the channel via an increase in P o(max) without changing the apparent Ca2+ sensitivity. Addition of alkaline phosphatase eliminated this ATP-dependent activation of KCa3.1, indicative of a phosphorylation event. Indeed, this ATP-dependent activation was shown to be mediated through PKA when KCa3.1 was heterologously expressed in Xenopus oocytes or when endogenously expressed in T84 cells, but was independent of PKA when heterologously expressed in HEK cells, suggesting additional kinases may also regulate KCa3.1. In contrast to KCa3.1, KCa2.3 was not activated by ATP under similar conditions. Thus, using a series of KCa3.1/KCa2.3 chimeras, these authors further demonstrated that the kinase-dependent activation of KCa3.1 could be localized to a 14 amino acid domain w ithin the C-terminus (Arg355–Met368), distal to the CaMBD (Gerlach et al. 2001). Additional studies by Jones et al. (2007) indicated that the ATP-dependent activation of KCa3.1 also depended upon an N-terminal RKR motif. That is, mutation of 15RKR17 to alanines resulted in a channel that was no longer sensitive to ATP and alkaline phosphatase no longer reduced channel activity. Finally, the open probability (P o) of these mutated channels was reduced fourfold compared to WT KCa3.1, suggesting that the ATP-/kinase-dependent regulation of KCa3.1 observed by these authors is dependent upon a close N-/C-terminal association. In this regard, it has similarly been demonstrated that KCa2.2 associates with both protein kinase CK2 and protein phosphatase 2A (PP2A), in a manner that brings the N- and C-termini into close proximity, such that they phosphorylate and dephosphorylate the associated calmodulin, respectively, resulting in a shift in the apparent Ca2+-sensitivity of the channel (Bildl et al. 2004; Allen et al. 2007).
Additional studies have shown that PKC regulates KCa3.1 function (Wulf and Schwab 2002), although this appears to be an indirect effect as mutation of the four conserved PKC phosphorylation sites has no effect on activation (Gerlach et al. 2000). Also, AMP-activated protein kinase has been shown to directly interact with the distal C-terminus of KCa3.1 between Asp380 and Ala400, where activation of AMP-activated protein kinase results in an inhibition of KCa3.1 function and subsequent inhibition of transepithelial Cl− secretion (Klein et al. 2009). In additional studies, Skolnik and colleagues (Srivastava et al. 2005) utilized a yeast two-hybrid approach to identify the myotubularin, MTMR6, a lipid (PI(3)P) phosphatase, as an interacting protein with a C. elegans KCa2.x (KCNL) family member. This interaction occurred via the channel coiled-coil domain, a region Syme et al. (2003) had previously shown to be required for proper channel assembly and trafficking (see below). Srivastava et al. (2006) further demonstrated that this MTMR6-dependent regulation required the same 14 amino acid region of KCa3.1 previously shown to be required for ATP-/kinase-dependent activation (Gerlach et al. 2001) (Fig. 20.2). In an exciting next step, Skolnik and colleagues demonstrated that nucleoside diphosphate kinase B (NDPK-B) (Fig. 20.2), a histidine kinase, directly binds and activates KCa3.1 by phosphorylating His358 and that this is reversed by protein histidine phosphatase (Srivastava et al. 2008). Further, NDPK-B knockout mice exhibit a reduced KCa3.1 activity (Di et al. 2010), indicative of this pathway being functional in vivo, as well. Finally, it has also been demonstrated that the nitric oxide (NO) donor, sodium nitroprusside, activated KCa3.1 channels in interstitial cells of Cajal (Zhu et al. 2007). It was subsequently shown that NO activates KCa3.1 via the PKG pathway in human dermal fibroblasts (Bae et al. 2014). In total, these results suggest that a kinase/phosphatase protein regulatory complex exists for the careful regulation of KCa3.1.
Devor and Frizzell (1998) demonstrated that IK channels in the T84 epithelial cell line were inhibited by numerous fatty acids, including arachidonic acid with an apparent K i of 425 nM. Indeed, in intact epithelial monolayers, inhibition of cytosolic phospholipase A2 resulted in a significant increase in Ca2+-mediated agonist transepithelial Cl− secretion, suggesting that agonist-mediated generation of arachidonic acid may blunt the response to these agonists. Subsequently, Hamilton et al. (2003) identified two amino acids in the pore of KCa3.1 that were responsible for the arachidonic-mediated inhibition of the channel and further demonstrated that introduction of these amino acids into KCa2.2 resulted in this channel being sensitive to arachidonic acid block. Interestingly, these two amino acids, Thr250 and Val275, had previously been shown to be responsible for the clotrimazole-dependent inhibit ion of KCa3.1 (Wulff et al. 2001) (Fig. 20.7).
7 Trafficking of KCa3.1
The physiological response of an ion channel is dependent upon both its gating, i.e., the likelihood that the channel is in open or closed state (P o), and the number of channels (N) at the cell surface. The N of channels is in turn determined by the relative rates of trafficking along the anterograde and retrograde pathways . Studies over the past 15 years on the trafficking of KCa3.1 channels have revealed key molecular components in the assembly, delivery to the plasma membrane, and subsequent internalization and degradation of these channels. A schematic representation of the known intracellular trafficking pathways for KCa3.1 is presented in Fig. 20.8.
7.1 Anterograde Trafficking of KCa3.1
Prior to being delivered to the plasma membrane via the biosynthetic route, KCa3.1 must be correctly folded and assembled into tetramers in the endoplasmic reticulum (ER; Ellgaard et al. 1999; Papazian 1999; Deutsch 2002). Since the cloning of the KCa2.x/KCa3.1 channels, numerous investigators have identified motifs or regions within the N- and C-termini of KCa3.1 that are required for proper subunit folding and association. In an initial series of studies, Kaczmarek and colleagues (Joiner et al. 2001) and Adelman and colleagues (Lee et al. 2003) demonstrated that, apart from its role in channel gating , constitutive association of calmodulin with the proximal C-terminal CaMBD of KCa3.1 and KCa2.x channels regulates the surface expression of these channels by regulating their multimerization. In addition to the CaMBD , the distal C-termini of KCa3.1 and KCa2.x channels contain a leucine zipper motif that was first identified by Khanna et al. (1999) as being required for the expression of a hyperpolarizing K+ conductance. Devor and colleagues (Syme et al. 2003) further demonstrated that although this C-terminal leucine zipper is conserved in KCa3.1 and KCa2.3, only KCa3.1 expression was affected by mutations in the leucine zipper. These results suggest a clear divergence in the structural requirements for the folding and trafficking of KCa family members. These authors further demonstrated that the C-terminus of KCa3.1 was capable of co-assembly and that this was abrogated by mutations in the leucine zipper. However, these mutations did not preclude KCa3.1 from self-assembly (Syme et al. 2003). Thus, while mutations of the leucine zipper do not compromise multimer formation, they do alter the assembly of the distal C-terminal tail of KCa3.1, affecting the proper folding of the channel. As leucine zippers are known to be involved in protein-protein interactions (Kobe and Deisenhofer 1994), this suggests that the C-terminal leucine zipper in KCa3.1 may be required for interactions with regulatory proteins important for channel trafficking. As mentioned above, the PI(3)P phosphatase, MTMR6, interacts with KCa3.1 via the coiled-coil domains on both proteins (Srivastava et al. 2005). Whether similar interactions take place early in the anterograde pathway that regulates KCa3.1 trafficking remains to be determined.
While initial studies on the assembly and trafficking of KCa3.1 focused on the C-terminus, numerous studies have also identified a role for the N-terminus in anterograde trafficking. Jones et al. (2004) demonstrated that the N-terminus of KCa3.1 contains overlapping leucine zipper (Leu18/Leu25/Leu32/Leu39) and dileucine (Leu18/Leu19) motifs essential for channel tetramerization and targeting to the plasma membrane. These authors further demonstrated a critical role for a lysine in the cytosolic S4–S5 linker of KCa3.1 (Lys197) and KCa2.3 (Lys453) in protein trafficking (Jones et al. 2005). Mutation of these lysines resulted in these KCa channels failing to exit the ER, suggesting that critical associations required for channel trafficking are disrupted (Fig. 20.8). Additional studies demonstrated that misfolded KCa3.1 and KCa2.3 channels are polyubiquitylated prior to degradation (Gao et al. 2008). These authors further demonstrated a role for the ER integral membrane protein , Derlin-1, and the cytosolic chaperone AAA-ATPase p97 in the ER translocation, protein dislocation, and targeting of misfolded and polyubiquitylated KCa3.1 and KCa2.3 channels for proteasomal degradation (Fig. 20.8).
The anterograde trafficking of KCa channels requires that they be properly assembled into tetramers . It is generally believed that the 6-TMD KCNN gene family members (KCa2.x, KCa3.1) are evolutionarily older than the voltage-gated K+ channels (Kv) (Anderson and Greenberg 2001), and from the crystal structure data of Kv channels, it is clear that the additional four TM domains (S1–S4 ), which were appended to the central pore (S5-pore-S6) to form the 6-TMD channels, form a unique structural domain (Lee et al. 2005). A fundamental difference between the Kv and KCa3.1/KCa2.x channels is that the KCa channels exhibit no voltage dependence to their gating. The voltage dependence of Kv channels is conferred by a series of 4–7 positively charged amino acids in S4 as well as critical negatively charged amino acids in S2 and S3 (Aggarwal and MacKinnon 1996; Seoh et al. 1996). In addition to conferring voltage dependence , Papazian and colleagues (Papazian et al. 1995; Tiwari-Woodruff et al. 1997, 2000; Myers et al. 2004) demonstrated that these charged amino acids are essential for channel biogenesis. That is, key salt bridges are required to maintain the fundamental fold of this S1–S4 domain. Indeed, it has been shown that these electrostatic interactions are required at the initial folding process as the nascent polypeptide chain is being formed prior to entering th e ER (Sato et al. 2002, 2003). Although KCa3.1 and KCa2.x channels are voltage independent, these channels conserve two critical arginines in S4 as well as a glutamic acid in S3. Devor and colleagues (Gao et al. 2008) demonstrated that mutation of these charged amino acids in S3 or S4 resulted in the rapid degradation of KCa3.1, KCa2.3, and KCa2.2 suggesting that, similar to Kv channels, key electrostatic interactions required for folding of the S1–S4 domain are abrogated, resulting in channel degradation. The observation that S4 arginines, as well as an S3 glutamic acid, share a conserved function between Kv channels and the ligand-gated KCa channels led the authors to speculate that the role of these charged amino acids in establishing the proper folding of the S1–S4 domain evolved prior to the establishment of voltage dependence and that the basic structure of the S1–S4 domain is conserved between the Kv and KCa channels.
As noted, the splice variant KCNN4c is missing 29 amino acids, including the entire second transmembrane domain (S2) and is only expressed at the cell surface in the presence of the maxi-K β1-subunit (Barmeyer et al. 2010) (Fig. 20.8). Thus, a key remaining question is what is the structural topology of KCNN4c? That is, does the S1 segment of KCNN4c still act as a TMD or is it perhaps cytosolic in the final folded KCNN4c structure? In this way, the two TMDs of the β1-subunit may substitute for S1–S2 in KCNN4c. In the case of the KAT1, Shaker-type K+ channel, S1 and S2 exhibited typical topogenic properties in which S1 is inserted into the ER membrane followed by the insertion of S2 (Sato et al. 2002). In contrast, in Kv1.3, the S2 segment appears to function as the signal sequence to establish topology such that S1 remains cytosolic and translocation into the Sec61 translocon and the ER membrane is initiated by S2 (Tu et al. 2000). Similarly, in the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel, the second TMD initiates translocation rather than TMD1 as a means of establishing proper channel topology (Lu et al. 1998; Sadlish and Skach 2004). It has also been demonstrated in the KAT1 channel that S3 and S4 are posttranslationally inserted into the ER membrane following release of the positively charged S4 from the ribosome (Sato et al. 2002). Indeed, this was shown to be dependent upon the known S3–S4 salt-bridge interactions in this channel (Sato et al. 2002). As noted, we have shown that these S3–S4 interactions exist in KCa3.1, although this channel is voltage independent (Gao et al. 2008). Thus, interactions between S3 and S4 may similarly be required for the insertion of this domain into the membrane in KCa3.1. Finally, it has been shown that the S5-P-S6 domain can be integrated independent of the S1–S4 TMDs of KAT1 and Kv1.3, consistent with these regions of the channels being unique structural domains (Tu et al. 2000; Sato et al. 2002). Based on these previous studies, it is interesting to speculate that S1 of KCNN4c remains in the cytosol as it is typically inserted into the translocon and ER membrane together with S2. The subsequent translation of S3 and S4 results in salt-bridge pairings such that these TMDs are inserted into the membrane, thereby establishing the proper topology of the channel, with the S5-P-S6 region being inserted as in the full-length channel. Future studies will undoubtedly answer these interesting questions concerning KCNN4c biogenesis in addition to defining how and where the β1-subunit associates with KCNN4c to divert the channel from the degradative pathway along the biosynthetic route.
7.2 Retrograde Trafficking of KCa3.1
Compared to the extensive studies on the KCa channels and anterograde trafficking, the mechanisms of endocytosis and downstream sorting of these channels have only recently begun to be unraveled. To begin to elucidate these mechanisms, Devor and colleagues (Gao et al. 2010) developed a strategy for monitoring the endocytic routes of KCa3.1 based on the work of Ting and colleagues (Howarth et al. 2005). For these studies, the biotin ligase acceptor peptide (BLAP) sequence was inserted into the second extracellular loop of KCa3.1, followed by a fast and specific biotinylation of the channels at the cell surface using recombinant biotin ligase (BirA) (Gao et al. 2010; Balut et al. 2010a). Addition of fluorophore-conjugated streptavidin allows the fate of the endocytosed channels to be addressed using a combination of biochemical and imaging techniques. Using these approaches, Balut et al. (2010a) demonstrated that KCa3.1 is endocytosed from the plasma membrane within 60–90 min. This endocytic step of KCa3.1 has been proposed to be clathrin dependent by Schwab et al. (2012) in migrating MDCK cells. In addition, a dileucine motif present in the C-terminus of KCa3.1 (Leu344/Leu345) appears to be important for channel internalization (Schwab et al. 2012), consistent with adaptor protein binding and clathrin-dependent endocytosis. Subsequent to endocytosis, KCa3.1 is targeted for lysosomal degradation via a Rab7- and MVB (multivesicular body)/ESCRT (endosomal sorting complex required for transport)-dependent pathway (Balut et al. 2010a) (Fig. 20.8). By combining BLAP-tagged KCa3.1 with TUBEs (tandem ubiquitin-binding entities) and deubiquitylase Chip methodologies, Balut et al. (2011) further demonstrated that polyubiquitylation mediates the targeting of internalized KCa3.1 to the lysosomes and also that USP8 (ubiquitin-specific protease) regulates the rate of KCa3.1 degradation by deubiquitylating KCa3.1 prior to lysosomal delivery (Fig. 20.8). Importantly, it has been shown that KCa3.1 endocytosis can be reduced by blocking the ubiquitin-activating enzyme E1 (Balut et al. 2010b, 2011), suggesting that ubiquitylation of either the channel itself or associated adaptors functions as a signal for KCa3.1 endocytosis. In contrast to our data on the KCNN family member KCa2.3, which rapidly recycles back to the plasma membrane following endocytosis (Gao et al. 2010), we did not observe recycling of KCa3.1 following endocytosis suggesting this channel is targeted for degradation following endocytosis. The plausibility of a ubiquitin-dependent pathway is supported by data showing that in CD4 T cells the tripartite motif containing protein 27 (TRIM27)-dependent ubiquitylation of the class II PI3K-C2β kinase acts as a negative regulator of KCa3.1 activation (Cai et al. 2011). Thus, TRIM27 functions as an E3 ligase and mediates lysine 48 polyubiquitylation of PI3K-C2β kinase, leading to an inhibition of the PI3K enzyme activity. This leads to decreased levels of PI(3)P which, as discussed above, results in a decreased histidine phosphorylation and activation of KCa3.1 by NDPK-B (Cai et al. 2011).
7.3 Plasma Membrane Targeting of KCa3.1 in Polarized Cells
In contrast to the studies above, the trafficking of KCa3.1 in polarized cells has only just begun to be investigated. In T lymphocytes it has been shown that KCa3.1 is evenly distributed on the plasma membrane but rapidly translocates to the immunological synapse upon antigen stimulation as part of the signaling complex that facilitates T cell activation (Nicolaou et al. 2007). It has also been shown that a KCa3.1 isoform lacking the N-terminus is retained within the cytosol and acts as a dominant negative on the full-length channel in human lymphoid tissues (Ohya et al. 2011). This is consistent with the previously published role of the N-terminus in KCa3.1 trafficking (Jones et al. 2004). Additionally, it was demonstrated that KCa3.1 localizes with F-actin to the immunological synapse, emphasizing the role of cytoskeleton in providing directionality for channel movement (Nicolaou et al. 2007). An interesting observation is that signaling molecules such as PKA and PKC, which are known regulators of KCa3.1 function, as discussed above, accumulate in the immunological synapse on T cell activation, as well (Bi et al. 2001; Skalhegg et al. 1994; Zhou et al. 2004), and this close proximity could provide a regulatory mechanism for KCa3.1 function at the synapse.
To date, very few studies have explored the trafficking of KCa3.1 in polarized epithelia. While it has been shown that in migrating MDCK cells KCa3.1 displays a polarized distribution by accumulating at the leading edge of the lamellipodium (Schwab et al. 2006), these cells do not demonstrate classic apical-to-basolateral polarization. We recently utilized polarized MDCK, Caco-2, and FRT cells, grown on semipermeable Transwell® supports, to investigate the anterograde and retrograde trafficking of full-length KCa3.1 in polarized epithelia (Bertuccio et al. 2014). Given that Rabs 1, 2, and 6 have been shown to be involved in the trafficking of proteins from the ER to Golgi, while Rabs 8 and 10 are known to be involved in the trafficking of proteins from the Golgi to the plasma membrane (Martinez and Goud 1998; Babbey et al. 2006; Zhang et al. 2009; Dong et al. 2010), we determined whether any of these Rabs played a crucial role in the anterograde trafficking of KCa3.1 in polarized epithelia. In this regard, we demonstrated that Rab1 and Rab8 are required for the ER/Golgi exit of KCa3.1 and subsequent trafficking to the basolateral membrane (Fig. 20.8), whereas Rabs 2, 6, and 10 did not play a role in this trafficking step (Bertuccio et al. 2014). In contrast, trafficking of KCa2.3 was unaffected by siRNA-mediated knockdown or overexpression of dominant negatives of these Rabs, thereby confirming the specificity of these Rabs within the gene family. It has also been shown that the sorting of numerous proteins to the basolateral domain of polarized epithelia is the result of an association with the μ1 subunit of the adaptor protein-1 complex (AP-1) (Ohno et al. 1998; Muth and Caplan 2003; Cancino et al. 2007). The AP-1 complex is the only clathrin-associated adaptor complex implicated in basolateral sorting in polarized epithelia (Futter et al. 1998). Importantly, while μ1B is expressed in MDCK cells, it is not expressed in LLC-PK1 cells, and the lack of the μ1B subunit in LLC-PK1 cells has been shown to result in the mistargeting of some basolateral proteins to the apical surface, while stable expression of μ1B restores proper basolateral targeting (Fölsch et al. 1999). However, we demonstrated correct basolateral targeting of KCa3.1 in LLC-PK1 cells, indicating μ1B is not required for basolateral sorting of KCa3.1. Finally, it has been shown that proteins destined for the basolateral membrane can traffic either (1) from the Golgi to the basolateral membrane directly or (2) from the Golgi through recycling endosomes to the basolateral membrane (Fuller et al. 1985; Orzech et al. 2000). To determine whether KCa3.1 traffics via recycling endosomes, we carried out a series of studies to block trafficking through the recycling endosomes. We demonstrated that KCa3.1 did not traffic to the basolateral membrane via transferrin- and RME-1-positive recycling endosomes, suggesting this channel traffics directly to the basolateral membrane from the Golgi (Bertuccio et al. 2014). Finally, we demonstrated that, similar to our studies in non-polarized cells, KCa3.1 is endocytosed from the basolateral membrane of polarized MDCK, Caco-2, and FRT cells in a ubiquitin-dependent manner and that the channels are then targeted to the lysosome and proteasome for degradation (Bertuccio et al. 2014).
Whether the correct apical targeting of KCNN4c similarly requires Rabs 1 and 8 and whether this splice variant of KCa3.1 is directly targeted to the apical membrane or via the apical recycling endosomal compartment have yet to be addressed (Fig. 20.8). As detailed above, apical KCNN4c expression is regulated by knockdown of Epac1 with KCNN4c being redistributed to subapical vesicles. Similarly, inhibition of RhoA and ROCK induced a redistribution of KCNN4c out of the apical membrane (Sheikh et al. 2013). Whether KCNN4c is capable of recycling back to the apical membrane from this vesicular pool and whether this involves ubiquitylation and deubiquitylation as well as the ultimate degradative pathway for KCNN4c from the apical membrane remain open questions yet to be addressed.
8 Role of KCa3.1 in the Cell Cycle and Cell Proliferation
During cell proliferation, a cell progresses through various phases of the cell cycle (G0, G1, S, G2, and M). It has been known, for some time, that intracellular Ca2+, [Ca2+]i, levels alter cell proliferation (Hazelton et al. 1979). Additionally, Nilius and Wohlrab (1992), using human melanoma cells , provided the first evidence linking changes in the electrochemical gradient for Ca2+ entry into the cell due to membrane hyperpolarization and K+ channels. Eventually, Wonderlin and colleagues (Strobl et al. 1995; Wonderlin et al. 1995; Wonderlin and Strobl 1996) suggested that Ca2+-activated K+ channels might play a significant role in the regulation of the cell through the cell cycle. The coupled action of the cellular hyperpolarization via K+ channels and increased [Ca2+]i has been demonstrated to enhance passing of cells through G0/G1 and into S phase, resulting in cell proliferation (Schreiber 2005; Wonderlin and Strobl 1996; Wonderlin et al. 1995).
The basis of our appreciation of the possible role of KCa3.1 in cell proliferation in epithelial cells has arisen from cell differentiation and blocker pharmacology studies of lymphocytes. Cahalan and co-workers (Decoursey et al. 1984) first provided evidence for the relationship between K+ channels and the proliferative processes of T lymphocytes . Originally, the characterization of KCa3.1 in cell activation /proliferation was demonstrated in erythroid cells, T-lymphoblasts, and T-lymphocytes. Alper and colleagues (Vandorpe et al. 1998) reported, in mouse embryonic stem cells (ES ), that mRNA for mIK1 (mouse KCa3.1) was maintained at low levels in uninduced ES cells; however, it exhibited sustained high mRNA levels during erythroid differentiation of ES cells. Further, they demonstrated that increasing concentrations of clotrimazole (even as low as 10 nM) inhibited cell proliferation of human peripheral blood stem cells, and thus, these studies provide strong evidence that KCa3.1 participates in cell differentiation and proliferation (Vandorpe et al. 1998).
The information available about the role of KCa3.1 and the cell cycle and cell proliferation in epithelial cells is limited. Strobl and colleagues (Wang et al. 1998), using quinidine (a generic K+ channel blocker), provided early evidence that K+ channels might participate in the G phase of the cell cycle of MCF-7 breast cancer epithelial cells. Afterward, Ouadid-Ahidouch and colleagues demonstrated that the current density of KCa3.1 increased in MCF-7 cells that had been synchronized at the end of G1 or S phase when compared to those cells in the early G1 phase of the cell cycle (Ouadid-Ahidouch et al. 2004). Additionally, they reported that the increase in current density was in congruence with increased mRNA levels of KCa3.1 and the enhanced negative membrane potential was observed. To examine whether KCa3.1 influenced cell progression by altering the [Ca2+]i, Ouadid-Ahidouch et al. (2004) examined whether there was a correlation between effects of [Ca2+]i on K+ channel activity. They reported that [Ca2+]i was lower in the early G1 phase (49 ± 6 nM) when the activity of KCa3.1 was low and the membrane potential was depolarized. Additionally, the [Ca2+]i level was higher (240 ± 23 nM) in cells arrested at the end of G1 and [Ca2+]i reached high levels as cells progress into the S phase (323 ± 20 nM) when the activity of KCa3.1 was high and the membrane was hyperpolarized. Further, Ouadid-Ahidouch et al. (2004) demonstrated that clotrimazole caused a depolarization of membrane potential of cells arrested at the end of G1 and during the S phase, providing evidence of active KCa3.1, while clotrimazole had no effect on the membrane potential of cells arrested in early G1 phase . Finally, the treatment of MCF-7 cells with clotrimazole and econazole (another inhibitor of KCa3.1) caused a dose-dependent reduction in cell proliferation, and these inhibitors resulted in an increase of cells in the G1 phase and decrease in the number of cells moving into the S phase (Ouadid-Ahidouch et al. 2004). In agreement, Parihar et al. (2003), using LNCaP and PC-3 (prostate cancer epithelial cells), reported that both 1-EBIO and riluzole (activators of KCa3.1) increased cell proliferation of both types of prostate cells and resulted in decreased cell proliferation when administered in the presence of charybdotoxin and clotrimazole.
There have been advances in our understanding of the “link” between KCa3.1 and the Ca2+ entry step into cells in the cell proliferation process. Lallet-Daher et al. (2009) examined aspects of the control of Ca2+ entry in human prostate cancer cell proliferation using LNCaP and PC-3 prostate cells that has furthered our understanding of the role of KCa3.1 in cell proliferation. They examined the mechanism by which the inhibition of KCa3.1 prevented cell proliferation by examining the changes in expression of p21Cip1 (cyclin-dependent kinase inhibitor 1) that is known to participate in the G1 phase (Ghiani et al. 1999). Indeed, when KCa3.1 was inhibited by TRAM-34, the expression (mRNA) of p21Cip1 of both LNCaP and PC-3 cells was increased by two- to fourfold, and they also reported a similar fold increase in p21Cip1 when they knocked down KCa3.1 with siRNA. Further, Lallet-Daher et al. (2009) provided a role of the Ca2+ channel, TRPV6, in the signaling pathway, when they demonstrated an association of TRPV6 and KCa3.1 by co-immunoprecipitation. Further, they also reported a novel role of TRPV6 in maintaining the hyperpolarization-activated Ca2+ entry by using an siRNA approach to knockdown TRPV6 and Ca2+ entry was not stimulated by activation of KCa3.1. These data certainly suggested a “close” association of KCa3.1 and TRPV6 in the regulation of cell proliferation. Finally, Ouadid-Ahdouch and colleagues (Dhennin-Duthille et al. 2011) and others (Bolanz et al. 2008) have now identified TRPV6 in MCF-7 cells, while Dhennin-Duthille et al. (2011) reported highly expressed levels of TRPV6 in human breast ductal adenocarcinoma cells compared to adjacent non-tumoral tissue. Therefore, the link between KCa3.1, TRPV6, and cell proliferation is gaining strength.
There is an exciting caveat to the KCa3.1 and cell proliferation story as Bruce and co-workers (Millership et al. 2011) reported that both a nonfunctional pore mutant of KCa3.1 (KCa3.1GYG/AAA) and a non-trafficking mutant of KCa3.1 (KCa3.1L18A/L25A; Jones et al. 2004) resulted in enhanced cell proliferation using a heterologous expression system (human embryonic kidney cells, HEK). These results indicated that KCa3.1 induced cell proliferation, but did not require membrane localized KCa3.1.
It appears that the role of KCa3.1 in cell proliferation in epithelial cells is just at its infancy. No doubt, it will be very interesting to see how this story develops over the next 5 years.
9 Role of KCa3.1 in Epithelial Diseases
Although KCa3.1 is expressed in a wide variety of epithelial tissues, no human disease has been directly associated with mutations in this channel. However, given the critical role of KCa3.1 in maintaining transepithelial ion and fluid transport, it is not surprising that this channel has been suggested to play a role in the etiology of various diseases (Table 20.1). In the kidney, autosomal-dominant polycystic kidney disease (ADPKD) is characterized by the progressive development and enlargement of multiple bilateral fluid-filled cysts. Evidence indicates that cyst formation in ADPKD is produced by tubular cell proliferation, anomalies in the extracellular matrix, and a net transepithelial Cl− secretion toward the cyst lumen (Ye and Grantham 1993; Li et al. 2004; Wallace et al. 1996; Mangos et al. 2010). Thus, a clear objective in ADPKD treatment is to slow or stop the growth of the cysts (Grantham et al. 2006a, b). Based on the known role of KCa3.1 in maintaining transepithelial Cl− secretion, it was demonstrated that inhibition of KCa3.1 by TRAM-34 blocks Cl− secretion and ameliorates cyst formation and enlargement in vitro (Albaqumi et al. 2008). A second kidney disease in which epithelial KCa3.1 has been implicated is diabetic nephropathy, which is a chronic complication that affects ~30 % of patients with diabetes mellitus (Reddy et al. 2008). In this case, Huang et al. (2013) demonstrated that KCa3.1 protein expression was upregulated in proximal tubular cells from patients with diabetic nephropathy (Huang et al. 2013). These authors similarly observed an increase in KCa3.1 expression in mouse models of diabetes and found that administration of TRAM-34 significantly reversed the increased KCa3.1 expression in the proximal tubule. Thus, inhibition of KCa3.1 may provide a novel approach in the treatment of diabetic nephropathy.
As noted above, Ca2+-activated K+ channels are known to mediate the secretion of fluid and electrolytes in epithelial cells of pancreatic ducts (Nguyen and Moody 1998) and this channel has been identified as KCa3.1 (Jung et al. 2006; Hayashi et al. 2012). Whether KCa3.1 channels are essential in the dysfunction of pancreatic epithelial transport in cystic fibrosis remains unknown, however. In additional studies, it has been demonstrated that the mRNA levels of KCa3.1 are upregulated in patients with pancreas ductal adenocarcinoma and inhibitors of KCa3.1 block the proliferation of these pancreatic cells (Jager et al. 2004).
As detailed above, and in other chapters in this volume, the human colon absorbs Na+, Cl−, and water and simultaneously secretes K+ and HCO3 − (Sandle 1998). In the distal colon, the electrogenic absorption of Na+ generates a lumen-negative electrical potential difference (PDte), and it is known that the downregulation of Na+ absorption promotes the pathogenesis of diarrhea in patients with ulcerative colitis (UC) (Sandle et al. 1986; Hawker et al. 1980; Amasheh et al. 2004; Greig et al. 2004). As indicated above, basolateral KCa3.1 plays a critical role in maintaining transepithelial ion transport by maintaining this transepithelial PDte. Thus, Sandle et al. (1990) have shown that the changes in transepithelial PDte associated with the inflamed human colon are at least partially the result of a depolarized basolateral membrane. Indeed, a variant of the KCa3.1 gene, KCNN4, has been reported to be associated with ileal Crohn’s disease (Simms et al. 2010). In this regard, Al-Hazza et al. (2012) demonstrated that the reduced Na+ absorption in UC was associated with an ~75 % decrease in basolateral KCa3.1 channel expression and activity in patien ts with UC, whereas crypts from patients with Crohn’s collagenous and infective colitis showed identical levels of KCa3.1 channel protein to those of control patients.
Bridges and colleagues (Devor et al. 1999, 2000; Singh et al. 2001) demonstrated that pharmacological activation of basolateral KCa3.1 with 1-EBIO or DCEBIO in airway epithelia from both wild-type CFTR-expressing and CF airway stimulates transepithelial Cl− secretion. These results led the authors to propose pharmacological modulation of basolateral KCa3.1 as a potential therapeutic strategy in airway diseases, including CF and chronic obstructive pulmonary disease (COPD). Indeed, Roth et al. (2011) demonstrated that activation of basolateral KCa3.1 with 1-EBIO potentiated the Cl− secretory response to cAMP in rectal biopsy tissues from CF patients compared to age-matched controls, thereby supporting this proposal.
While KCa3.1 has also been shown to play a critical role in cells of the immune system and the targeting of this channel in T cells has shown promise for the treatment of various diseases associated with the lung, intestine, and kidney, these studies have not been discussed in this chapter due to our emphasis on epithelial function. However, we would direct the reader to several excelle nt rev iews on this subject that have been recently written (Wulff and Zhorov 2008; Wulff and Castle 2010; Huang et al. 2014).
10 Conclusions
Based on the above, it is clear that basolateral membrane localized KCa3.1 plays a critical role in maintaining the electrochemical driving force for transepithelial ion and fluid transport across a wide array of epithelial tissues. More recent evidence also points to the crucial role of this channel in maintaining a hyperpolarized apical membrane potential, although additional studies are required to fully understand its regulation, trafficking, and function in this membrane. The study of any ion channel is dramatically augmented by having both activators and inhibitors such that its physiology can be explored, and it is clear that the identification of both activators (1-EBIO, DCEBIO) and inhibitors (clotrimazole) prior to the cloning of KCa3.1 undoubtedly propelled our rapid understanding of the role of these channels in epithelial physiology. The identification of both higher specificity and affinity activators and inhibitors, as reviewed in Chap. 26, has further fostered this advance. Clearly, there is still much to be learned about how KCa3.1 can be targeted for therapeutic gain in a wide array of epithelia and this will surely be a goal of investigators over the next decade.
References
Ackerman SJ, Liu L, Kwatia MA, Savage MP, Leonidas DD, Swaminathan GJ, Acharya KR (2002) Charcot-Leyden crystal protein (galectin-10) is not a dual function galectin with lysophospholipase activity but binds a lysophospholipase inhibitor in a novel structural fashion. J Biol Chem 277:14859–14868
Adelman JP, Maylie J, Sah P (2012) Small-conductance Ca2+-activated K+ channels: form and function. Annu Rev Physiol 75:245–269
Aggarwal SK, MacKinnon R (1996) Contribution of the S4 segment to gating charge in the Shaker K+ channel. Neuron 16:1169–1177
Akabas MH, Stauffer DA, Xu M, Karlin A (1992) Acetylcholine receptor channel structure probed in cysteine-substitution mutants. Science 258:307–310
Akabas MH, Kaufmann C, Archdeacon P, Karlin A (1994) Identification of acetylcholine receptor channel-lining residues in the entire M2 segment of the alpha subunit. Neuron 13:919–927
Albaqumi M, Srivastava S, Li Z, Zhdnova O, Wulff H, Itani O, Wallace DP, Skolnik EY(2008) KCa3.1 potassium channels are critical for cAMP-dependent chloride secretion and cyst growth in autosomal-dominant polycystic kidney disease. Kidney Int 74:740–749
Al-Hazza A, Linley JE, Aziz Q, Maclennan KA, Hunter M, Sandle GI (2012) Potential role of reduced basolateral potassium (IKCa3.1) channel expression in the pathogenesis of diarrhoea in ulcerative colitis. J Pathol 226:463–470
Allen D, Fakler B, Maylie J, Adelman JP (2007) Organization and regulation of small conductance Ca2+-activated K+ channel multiprotein complexes. J Neurosci 27:2369–2376
Almassy J, Won JH, Begenisich TB, Yule DI (2012) Apical Ca2+-activated potassium channels in mouse parotid acinar cells. J Gen Physiol 139:121–133
Amasheh S, Barmeyer C, Koch CS, Tavalali S, Mankertz J, Epple HJ, Gehring MM, Florian P, Kroesen AJ, Zeitz M, Fromm M, Schulzke JD (2004) Cytokine-dependent transcriptional down regulation of epithelial sodium channel in ulcerative colitis. Gastroenterology 126:1711–1720
Anderson PA, Greenberg RM (2001) Phylogeny of ion channels: clues to structure and function. Comp Biochem Physiol B Biochem Mol Biol 129:17–28
Ashton N, Argent BE, Green R (1991) Characteristics of fluid secretion from isolated rat pancreatic ducts stimulated with secretin and bombesin. J Physiol (Lond) 435:533–546
Babbey CM, Ahktar N, Wang E, Chen CC, Grant BD, Dunn KW (2006) Rab10 regulates membrane transport through early endosomes of polarized Madin-Darby canine kidney cells. Mol Biol Cell 17:3156–3175
Bae H, Lee HJ, Kim K, Kim JH, Kim T, Ko JH, Bang H, Lim I (2014) The stimulating effects of nitric oxide on intermediate conductance Ca2+-activated K+ channels in human dermal fibroblasts through PKG pathways but not PKA pathways. Chin J Physiol 57:137–151
Bailey MA, Grabe M, Devor DC (2010) Characterization of the PCMBS-dependent modification of KCa3.1 channel gating. J Gen Physiol 136:367–387
Balut CM, Gao Y, Luke C, Devor DC (2010a) Immunofluorescence-based assay to identify modulators of the number of plasma membrane KCa3.1 channels. Future Med Chem 2:707–713
Balut CM, Gao Y, Murray SA, Thibodeau PH, Devor DC (2010b) ESCRT-dependent targeting of plasma membrane localized KCa3.1 to the lysosomes. Am J Physiol Cell Physiol 299:C1015–C1027
Balut CM, Loch CM, Devor DC (2011) Role of ubiquitylation and USP8-dependent deubiquitylation in the endocytosis and lysosomal targeting of plasma membrane KCa3.1. FASEB J 25:3938–3948
Barmeyer C, Rahner C, Yang Y, Sigworth FJ, Binder HJ, Rajendran VM (2010) Cloning and identification of tissue-specific expression of KCNN4 splice variants in rat colon. Am J Physiol Cell Physiol 299:C251–C263
Basalingappa KM, Rajendran VM, Wonderlin WF (2011) Characteristics of Kcnn4 channels in the apical membranes of an intestinal cell line. Am J Physiol Gastrointest Liver Physiol 301:G905–G911
Begenisich T, Nakamoto T, Ovitt CE, Nehrke K, Brugnara C, Alper SL, Melvin JE (2004) Physiological roles of the intermediate conductance, Ca2+-activated potassium channel KCNN4. J Biol Chem 46:47681–47687
Bernard K, Bogliolo O, Soriani J, Ehrenfeld J (2003) Modulation of chloride secretion by basolateral SK4-like channels in a human bronchial cell line. J Membr Biol 196:15–31
Bertuccio CA, Lee S-L, Wu G, Butterworth MB, Hamilton KL, Devor DC (2014) Anterograde trafficking of KCa3.1 in polarized epithelia is Rab1- and Rab8-dependent and recycling endosome-independent. PLoS One 9, e92013
Bi K, Tanaka Y, Coudronniere N, Sugie K, Hong S, van Stipdonk MJ, Altman A (2001) Antigen-induced translocation of PKC-theta to membrane rafts is required for T cell activation. Nat Immunol 2:556–563
Bildl W, Strassmaier T, Thurm H, Andersen J, Eble S, Oliver D, Knipper M, Mann M, Schulte U, Adelman JP, Fakler B (2004) Protein kinase CK2 is coassembled with small conductance Ca2+-activated K+ channels and regulates channel gating. Neuron 43:847–858
Bolanz KA, Hediger MA, Landowski CP (2008) The role of TRPV6 in breast carcinogenesis. Mol Cancer Ther 7:271–279
Browning JG, Hardcastle J, Hardcastle PT, Sanford PA (1977) The role of acetylcholine in the regulation of ion transport by rat colon mucosa. J Physiol 272:737–754
Burckhardt B-C, Gogelein H (1992) Small and maxi K+ channels in the basolateral membrane of isolated crypts from rat distal colon: single-channel and slow whole-cell recordings. Pflugers Arch 420:54–60
Cai X, Srivastava S, Sun Y, Li Z, Wu H, Zuvela-Jelaska L, Li J, Salamon RS, Backer JM, Skolnik EY (2011) Tripartite motif containing protein 27 negatively regulates CD4 T cells by ubiquitinating and inhibiting the class II PI3K-C2beta. Proc Natl Acad Sci U S A 108:20072–20077
Cancino J, Torrealba C, Soza A, Yuseff MI, Gravotta D, Henklein P, Rodriguez-Boulan E, Gonzalez A (2007) Antibody to AP1B adaptor blocks biosynthetic and recycling routes of basolateral proteins at recycling endosomes. Mol Biol Cell 18:4872–4884
Catalan MA, Pena-Munzenmayer G, Melvin JE (2014) Ca2+-dependent K+ channels in exocrine salivary glands. Cell Calcium 55:362–368
Chang D, Dawson DC (1988) Digitonin-permeabilized colonic cell layers: demonstration of calcium-activated basolateral K+ and Cl− conductances. J Gen Physiol 92:281–306
Cowley EA, Linsdell P (2002) Characterization of basolateral K+ channels underlying anion secretion in the human airway cell line Calu-3. J Physiol 538:747–757
Cuthbert AW (2001) Assessment of CFTR chloride channel openers in intact normal and cystic fibrosis murine epithelia. Br J Pharmacol 132:659–668
Cuthbert AW, Hickman ME, Thorn P, MacVinish LJ (1999) Activation of Ca2+- and cAMP-sensitive K+ channels in murine colonic epithelia by 1-ethyl-2-benzimidazolone. Am J Physiol Cell Physiol 277:C111–C120
DeCoursey TE, Chandy KG, Gupta S, Cahalan MD (1984) Voltage-gated K+ channels in human T lymphocytes: a role in mitogenesis? Nature 307:465–468
Deutsch C (2002) Potassium channel ontogeny. Annu Rev Physiol 64:19–46
Devor DC, Duffey ME (1992) Carbachol induces K+, Cl− and nonselective cation conductances in T84 cells: a perforated patch-clamp study. Am J Physiol Cell Physiol 263:C780–C787
Devor DC, Frizzell RA (1993) Calcium-mediated agonists activate an inwardly rectified K+ channel in colonic secretory cells. Am J Physiol Cell Physiol 265:C1271–C1280
Devor DC, Frizzell RA (1998) Modulation of K+ channels by arachidonic acid in T84 cells. I. Inhibition of the Ca2+-dependent K+ channel. Am J Physiol Cell Physiol 274:C138–C148
Devor DC, Simasko SM, Duffey ME (1990) Carbachol induces oscillations of membrane potassium conductance in a colonic cell line, T84. Am J Physiol Cell Physiol 258:C318–C326
Devor DC, Ahmed Z, Duffey ME (1991) Cholinergic stimulation produces oscillations of cytosolic Ca2+ in a secretory epithelial cell line, T84. Am J Physiol Cell Physiol 260:C598–C608
Devor DC, Sekar MC, Frizzell RA, Duffey ME (1993) Taurodeoxycholate activates K+ and Cl− conductances via an IP3-mediated release of Ca2+ from intracellular stores. J Clin Invest 92:2173–2181
Devor DC, Singh AK, Bridges RJ, Frizzell RA (1996a) Modulation of Cl- secretion by benzimidazolones. II. Coordinate regulation of apical GCl and basolateral GK. Am J Physiol Lung Cell Mol Physiol 271:L785–L795
Devor DC, Singh AK, Frizzell RA, Bridges RJ (1996b) Modulation of Cl− secretion by benzimidazolones. I. Direct activation of a Ca2+-dependent K+ channel. Am J Physiol Lung Cell Mol Physiol 271:L775–L784
Devor DC, Singh AK, Gerlach AC, Frizzell RA, Bridges RJ (1997) Inhibition of intestinal Cl− secretion by clotrimazole: direct effect on basolateral membrane K+ channels. Am J Physiol Cell Physiol 273:C531–C540
Devor DC, Singh AK, Lambert LC, DeLuca A, Frizzell RA, Bridges RJ (1999) Bicarbonate and chloride secretion in Calu-3 human airway epithelial cells. J Gen Physiol 113:743–760
Devor DC, Bridges RJ, Pilewski JM (2000) Pharmacological modulation of ion transport across wild-type and ΔF508 CFTR-expressing human bronchial epithelia. Am J Physiol Cell Physiol 279:C461–C479
Dharmsathaphorn K, Pandol SJ (1986) Mechanism of chloride secretion induced by carbachol in a colonic epithelial cell line. J Clin Invest 77:348–354
Dharmsathaphorn K, McRoberts JA, Mandel KG, Tisdale LD, Masui H (1984) A human colonic tumor cell line that maintains vectorial electrolyte transport. Am J Physiol Gastrointest Liver Physiol 246:G204–G208
Dharmsathaphorn K, Mandel KG, Masui H, McRoberts JA (1985) Vasoactive intestinal polypeptide-induced chloride secretion by a colonic epithelial cell line: direct participation of a basolaterally localized Na+, K+, Cl− cotransport system. J Clin Invest 75:462–471
Dharmsathaphorn K, Cohn J, Beuerlein G (1989) Multiple calcium-mediated effector mechanisms regulate chloride secretory responses in T84 cells. Am J Physiol Cell Physiol 256:C1224–C1230
Dhennin-Duthille I, Gautier M, Faouzi M, Guilbert A, Brevet M, Vaudry D, Ahidouch A, Sevestre H, Ouadid-Ahidouch H (2011) High expression of transient receptor potential channels in human breast cancer epithelial cells and tissues: correlation with pathological parameters. Cell Physiol Biochem 28:813–822
Di L, Srivastava S, Zhdanova O, Sun Y, Li Z, Skolnik EY (2010) Nucleoside diphosphate kinase B knock-out mice have impaired activation of the K+ channel KCa3.1, resulting in defective T cell activation. J Biol Chem 285:38765–38771
Dong C, Yang L, Zhang X, Gu H, Lam ML, Claycomb WC, Xia H, Wu G (2010) Rab8 interacts with distinct motifs in α2B- and β2-adrenergic receptors and differentially modulates their transport. J Biol Chem 285:20369–20380
Doyle DA, Cabral JM, Pfuetzner RA, Kuo A, Gulbis JM, Cohen SL, Chait BT, MacKinnon R (1998) The structure of the potassium channel: molecular basis of K+ conduction and selectivity. Science 280:69–77
Dunn PM (1998) The action of blocking agents applied to the inner face of Ca2+-activated K+ channels from human erythrocytes. J Membr Biol 165:133–143
Ellgaard L, Molinari M, Helenius A (1999) Setting the standards: quality control in the secretory pathway. Science 286:1882–1888
Falke JJ, Dernburg AF, Sternberg DA, Zalkin N, Milligan DL, Koshland DE Jr (1988) Structure of a bacterial sensory receptor. A site-directed sulfhydryl study. J Biol Chem 263:14850–14858
Fanger CM, Ghanshani S, Logsdon NJ, Rauer H, Kalman K, Zhou J, Beckingham K, Chandy KG, Cahalan MD, Aiyar J (1999) Calmodulin mediates calcium-dependent activation of the intermediate conductance KCa channel, IKCa1. J Biol Chem 274:5746–5754
Flores CA, Melvin JE, Figueroa CD, Sepulveda FV (2007) Abolition of Ca2+-mediated intestinal anion secretion and increased stool dehydration in mice lacking the intermediate conductance Ca2+-dependent K+ channel KCNN4. J Physiol (London) 583:705–717
Florey HW, Wright RD, Jennings MA (1941) The secretions of the intestine. Physiol Rev 21:36–69
Fölsch H, Ohno H, Bonifacino JS, Mellman I (1999) A novel clathrin adaptor complex mediates basolateral targeting in polarized epithelial cells. Cell 99:189–198
Foskett JK, Gunter-Smith PJ, Melvin JE, Turner RJ (1989) Physiological localization of an agonist-sensitive pool of Ca2+ in parotid acinar cells. Proc Natl Acad Sci U S A 86:161–171
Fuller SD, Bravo R, Simons K (1985) An enzymatic assay reveals that proteins destined for the apical or basolateral domains of an epithelial cell line share the same late Golgi compartments. EMBO J 4:297–307
Furness JB, Robbins HL, Selmer IS, Hunne B, Chen MX, Hicks GA, Moore S, Neylon CB (2003) Expression of intermediate conductance potassium channel immunoreactivity in neurons and epithelial cells of rat gastrointestinal tract. Cell Tissue Res 314:179–189
Futter CE, Gibson A, Allchin EH, Maxwell S, Ruddock LJ, Odorizzi G, Domingo D, Trowbridge IS, Hopkins CR (1998) In polarized MDCK cells basolateral vesicles arise from clathrin-gamma-adaptin-coated domains on endosomal tubules. J Cell Biol 141:611–623
Gao L, Yankaskas JR, Fuller CM, Sorscher EJ, Matalon S, Forman HJ, Venglarik CJ (2001) Chlorzoxazone or 1-EBIO increases Na+ absorption across cystic fibrosis airway epithelial cells. Am J Physiol Lung Cell Mol Physiol 281:L1123–L1129
Gao Y, Chotoo CK, Balut CM, Sun F, Bailey MA, Devor DC (2008) Role of S3 and S4 transmembrane domain charged amino acids in channel biogenesis and gating of KCa2.3 and KCa3.1. J Biol Chem 283:9049–9059
Gao Y, Balut CM, Bailey MA, Patino-Lopez G, Shaw S, Devor DC (2010) Recycling of the Ca2+-activated K+ channel, KCa2.3, is dependent upon RME-1, Rab35/EPI64C, and an N-terminal domain. J Biol Chem 285:17938–17953
Garneau L, Klein H, Banderali U, Longpré-Lauzon A, Parent L, Sauvé R (2009) Hydrophobic interactions as key determinants to the KCa3.1 channel closed configuration. An analysis of KCa3.1 mutants constitutively active in zero Ca2+. J Biol Chem 284:389–403
Gerlach AC, Gangopadhyay NN, Devor DC (2000) Kinase-dependent regulation of the intermediate conductance, calcium-dependent potassium channel, hIK1. J Biol Chem 275:585–598
Gerlach AC, Syme CA, Giltinan L, Adelman JP, Devor DC (2001) ATP-dependent activation of the intermediate conductance, Ca2+-activated K+ channel, hIK1, is conferred by a C-terminal domain. J Biol Chem 276:10963–10970
Ghiani CA, Eisen A, Yuan X, DePinho RA, McBain CJ, Gallo V (1999) Neurotransmitter receptor activation triggers p27Kip1 and p21CIP1 accumulation and G1 cell cycle arrest in oligodendrocyte progenitors. Development 126:1077–1090
Grantham JJ, Chapman AB, Torres VE (2006a) Volume progression in autosomal dominant polycystic kidney disease: the major factor determining clinical outcomes. Clin J Am Soc Nephrol 1:148–157
Grantham JJ, Torres VE, Chapman AB, Guay-Woodford LM, Bae KT, King BF Jr, Wetzel LH, Baumgarten DA, Kenney PJ, Harris PC, Klahr S, Bennet WM, Hirscvhman GN, Meyers CM, Zhang X, Zhu F, Miller JP, Investigators CRISP (2006b) Volume progression in polycystic kidney disease. N Engl J Med 354:2122–2130
Gray MA, Pollard CE, Harris A, Coleman L, Greenwell JR, Argent BE (1990) Anion selectivity and block of the small conductance chloride channel on pancreatic duct cells. Am J Physiol Cell Physiol 259:C752–C761
Greger R, Schlatter E (1984a) Mechanism of NaCl secretion in the rectal gland of spiny dogfish (Squalus acanthias). I. Experiments in isolated in vitro perfused rectal gland tubules. Pflügers Arch 402:63–75
Greger R, Schlatter E (1984b) Mechanism of NaCl secretion in the rectal gland of spiny dogfish (Squalus acanthias). II. Effects of inhibitors. Pflugers Arch 402:364–375
Greger R, Schlatter E, Wang F, Forrest JN Jr (1984) Mechanism of NaCl secretion in rectal gland tubules of spiny dogfish (Squalus acanthias). III. Effects of stimulation of secretion by cyclic AMP. Pflugers Arch 402:376–384
Greig ER, Boot-Handford RP, Mani V, Sandle GI (2004) Decreased expression of apical Na+ channels and basolateral Na+, K+-ATPase in ulcerative colitis. J Pathol 204:84–92
Grygorczyk R, Schwarz W (1985) Ca2+-activated K+ permeability in human erythrocytes: modulation of single-channel events. Eur Biophys J 12:57–65
Gutman GA, Chandy KG, Adelman JP, Aiyar J, Bayliss DA, Clapham DE, Covarriubias M, Desir GV, Furuichi K, Ganetzky B, Garcia ML, Grissmer S, Jan LY, Karschin A, Kim D, Kuperschmidt S, Kurachi Y, Lazdunski M, Lesage F, Lester HA, McKinnon D, Nichols CG, O’Kelly I, Robbins J, Robertson GA, Rudy B, Sanguinetti M, Seino S, Stuehmer W, Tamkun MM, Vandenberg CA, Wei A, Wulff H, Wymore RS, International Union of Pharmacology (2003) International Union of Pharmacology. XLI. Compendium of voltage-gated ion channels: potassium channels. Pharmacol Rev 55:583–586
Hamilton KL, Meads L, Butt AG (1999) 1-EBIO stimulates Cl− secretion by activating a basolateral K+ channel in the mouse jejunum. Pflugers Arch 439:158–166
Hamilton KL, Syme CA, Devor DC (2003) Molecular localization of the inhibitory arachidonic acid binding site to the pore of hIK1. J Biol Chem 278:16690–16697
Hardcastle PT, Eggenton J (1973) The effect of acetylcholine on the electrical activity of intestinal epithelial cells. Biochim Biophys Acta 298:95–100
Hawker PC, McKay JS, Turnberg LA (1980) Electrolyte transport across colonic mucosa from patients with inflammatory bowel disease. Gastroenterology 79:508–511
Hayashi M, Wang J, Hede SE, Novak I (2012) An intermediate-conductance Ca2+-activated K+ channel is important for secretion in pancreatic duct cells. Am J Physiol Cell Physiol 303:C151–C159
Hazelton B, Mitchell B, Tupper J (1979) Calcium, magnesium, and growth control in the WI-38 human fibroblast cell. J Cell Biol 83:487–498
Hede SE, Amstrup J, Klaerke DA, Novak I (2005) P2Y2 and P2Y4 receptors regulate pancreatic Ca2+-activated K+ channel differently. Pflugers Arch 450:429–436
Hirschberg B, Maylie J, Adelman JP, Marrion NV (1998) Gating of recombinant small-conductance Ca-activated K+ channels by calcium. J Gen Physiol 111:565–581
Holmgren M, Liu Y, Xu Y, Yellen G (1996) On the use of thiol-modifying agents to determine channel topology. Neuropharmacology 35:797–804
Hoque KM, Woodward OM, van Rossum DB, Zachos NC, Chen L, Leung GP, Guggino WB, Guggino SE, Tse CM (2010) Epac1 mediates protein kinase A-independent mechanism of forskolin-activated intestinal chloride secretion. J Gen Physiol 135:43–58
Howarth M, Takao K, Hayashi Y, Ting AY (2005) Targeting quantum dots to surface proteins in living cells with biotin ligase. Proc Natl Acad Sci U S A 102:7583–7588
Huang C, Shen S, Ma Q, Chen J, Gill A, Pollock CA, Chen XM (2013) Blockade of KCa3.1 ameliorates renal fibrosis through the TGF-β1/Smad pathway in diabetic mice. Diabetes 62:2923–2934
Huang C, Pollock CA, Chen XM (2014) Role of potassium channel KCa3.1 in diabetic nephropathy. Clin Sci (Lond) 127:423–433
Hubel KA (1976) Intestinal ion transport: effect of norepinephrine, pilocarpine, and atropine. Am J Physiol 231:252–257
Hubel KA (1977) Effects of bethanechol on intestinal ion transport in the rat. Proc Soc Exp Bio Med 154:41–44
Isaacs PET, Corbett CL, Riley AK, Hawker PC, Turnberg LA (1976) In vitro behavior of human intestinal mucosa. J Clin Invest 58:535–542
Ishiguro H, Steward MC, Naruse S, Ko SBH, Goto H, Case RM, Kondo T, Yamamoto A (2009) CFTR functions as a bicarbonate channel in pancreatic duct cells. J Gen Physiol 133:315–326
Ishii TM, Silvia C, Hirschberg B, Bond CT, Adelman JP (1997) A human intermediate conductance calcium-activate potassium channel. Proc Natl Acad Sci U S A 94:11651–11656
Jacobowitz D (1965) Histochemical studies of the autonomic innervation of the gut. J Pharmacol Exp Ther 149:358–364
Jager H, Dreker T, Buck A, Giehl K, Gress T, Grissmer S (2004) Blockage of intermediate-conductance Ca2+ activated K+ channels inhibit human pancreatic cancer cell growth in vitro. Mol Pharmacol 65:630–638
Jensen BS, Strobaek D, Christophersen P, Jorgensen TD, Hansen C, Silahtaroglu A, Olesen SP, Ahring PK (1998) Characterization of the cloned human intermediate-conductance Ca2+-activated K+ channel. Am J Physiol Cell Physiol 275:C848–C856
Jensen BS, Strobaek D, Olesen SP, Christophersen P (2001) The Ca2+-activated K+ channel of intermediate conductance: a molecular target for novel treatments? Curr Drug Targets 2:401–422
Joiner WJ, Wang L-Y, Tang MD, Kaczmarek LK (1997) hSK4, a member of a novel subfamily of calcium-activated potassium channels. Proc Natl Acad Sci U S A 94:11013–11018
Joiner WJ, Khanna R, Schlichter LC, Kaczmarek LK (2001) Calmodulin regulates assembly and trafficking of SK4/IK1 Ca2+-activated K+ channels. J Biol Chem 276:37980–37985
Joiner WJ, Basavappa S, Vidyasagar S, Nehrke K, Krishnan S, Binder HJ, Boulpaep EL, Rajendran VM (2003) Active K+ secretion through multiple KCa-type channels and regulation by IKCa channels in rat proximal colon. Am J Physiol Liver Physiol 285:G185–G196
Jones HM, Hamilton KL, Papworth GD, Syme CA, Watkins SC, Bradbury NA, Devor DC (2004) Role of the NH2 terminus in the assembly and trafficking of the intermediate conductance Ca2+-activated K+ channel hIK1. J Biol Chem 279:15531–15540
Jones HM, Hamilton KL, Devor DC (2005) Role of an S4-S5 linker lysine in the trafficking of the Ca2+-activated K+ channels IK1 and SK3. J Biol Chem 280:37257–37265
Jones HM, Bailey MA, Baty CJ, MacGregor GG, Syme CA, Hamilton KL, Devor DC (2007) An NH2-terminal multi-basic RKR motif is required for the ATP-dependent regulation of hIK1. Channels (Austin) 2:80–91
Jung SR, Kim K, Hille B, Nguyen TD, Koh DS (2006) Pattern of Ca2+ increase determines the type of secretory mechanism activated in dog pancreatic duct epithelial cells. J Physiol 576:163–178
Karlin A, Akabas MH (1998) Substituted-cysteine accessibility method. Methods Enzymol 293:123–145
Keen JE, Khawaled R, Farrens DL, Neelands T, Rivard A, Bond CT, Janowsky A, Fakler B, Adelman JP, Maylie J (1999) Domains responsible for constitutive and Ca2+-dependent interactions between calmodulin and small conductance Ca2+-activated potassium channels. J Neurosci 19:8830–8838
Khanna R, Chang MC, Joiner WJ, Kaczmarek LK, Schlichter LC (1999) hSK4/hIK1, a calmodulin-binding KCa channel in human T lymphocytes. Roles in proliferation and volume regulation. J Biol Chem 274:14838–14849
Klein H, Garneau L, Banderali U, Simoes M, Parent L, Sauvé R (2007) Structural determinants of the closed KCa3.1 channel pore in relation to channel gating: results from a substituted cysteine accessibility analysis. J Gen Physiol 129:299–315
Klein H, Garneau L, Trinh NT, Prive A, Dionne F, Goupil E, Thuringer D, Parent L, Brochiero E, Sauve R (2009) Inhibition of the KCa3.1 channels by AMP-activated protein kinase in human airway epithelial cells. Am J Physiol Cell Physiol 296:C285–C295
Kobe B, Deisenhofer J (1994) The leucine-rich repeat: a versatile binding motif. Trends Biochem Sci 19:415–421
Kohler R, Hirschberg B, Bond CT, Kinzie JM, Marrion NV, Maylie J, Adelman JP (1996) Small-conductance, calcium-activated potassium channels from mammalian brain. Science 273:1709–1714
Lallet-Daher H, Roudbaraki M, Bavencoffe A, Mariot P, Gackière F, Bidaux G, Urbain R, Gosset P, Delcourt P, Fleurisse L, Slomianny C, Dewailly E, Mauroy B, Bonnal JL, Skryma R, Prevarskaya N (2009) Intermediate-conductance Ca2+-activated K+ channels (IKCa1) regulate human prostate cancer cell proliferation through a close control of calcium entry. Oncogene 28:1792–1806
Ledoux J, Bonex AD, Nelson MT (2008) Ca2+-activated K+ channels in murine endothelial cells: block by intracellular calcium and magnesium. J Gen Physiol 131:125–135
Lee WS, Ngo-Anh TJ, Bruening-Wright A, Maylie J, Adelman JP (2003) Small conductance Ca2+-activated K+ channels and calmodulin: cell surface expression and gating. J Biol Chem 278:25940–25946
Lee S-Y, Lee A, Chen J, MacKinnon R (2005) Structure of the KvAP voltage-dependent K+ channel and its dependence on the lipid domain. Proc Natl Acad Sci U S A 102:15441–15446
Leinders T, van Kleef RG, Vijverberg HP (1992) Single Ca2+-activated K+ channels in human erythrocytes: Ca2+ dependence of opening frequency but not of open lifetimes. Biochim Biophys Acta 1112:67–74
Li H, Findlay IA, Sheppard DN (2004) The relationship between cell proliferation, Cl− secretion, and renal cyst growth: a study using CFTR inhibitors. Kidney Int 66:1926–1938
Li H, Yao J, Tong X, Guo Z, Wu Y, Sun L, Pan N, Wu H, Xu T, Ding J (2007) Interaction sites between the Slo1 pore and the NH2 terminus of the β2 subunit, probed with a three-residue sensor. J Biol Chem 282:17720–17728
Li W, Halling DB, Hall AW, Aldrich RW (2009) EF hands at the N-lobe of calmodulin are required for both SK channel gating and stable SK-calmodulin interaction. J Gen Physiol 134:281–293
Linsdell P, Tabcharani JA, Hanrahan JW (1997) Multi-ion mechanism for ion permeation and block in the cystic fibrosis transmembrane conductance regulator chloride channel. J Gen Physiol 110:365–377
Logsdon NJ, Kang J, Togo JA, Christian EP, Aiyar J (1997) A novel gene, hKCa4, encodes the calcium-activated potassium channel in human T lymphocytes. J Biol Chem 272:32723–32726
Lohrmann E, Burhoff I, Nitschke RB, Lang HJ, Mania D, Englert HC, Hropot M, Warth R, Rohm W, Bleich M, Greger R (1995) A new class of inhibitors of cAMP-mediated Cl secretion in rabbit colon, acting by the reduction of cAMP-activated K conductance. Pfluegers Arch 429:517–530
Loo DDF, Kaunitz JD (1989) Ca2+ and cAMP activate K+ channels in the basolateral membrane of crypt cells isolated from rabbit distal colon. J Membr Biol 110:19–28
Lu Y, Xiong X, Helm A, Kimani K, Bragin A, Skach WR (1998) Co- and posttranslational translocation mechanisms direct cystic fibrosis transmembrane conductance regulator N terminus transmembrane assembly. J Biol Chem 273:568–576
MacVinish LJ, Keogh J, Cuthbert AW (2001) EBIO, an agent causing maintained epithelial chloride secretion by co-ordinate actions at both apical and basolateral membranes. Pflugers Arch 443:S127–S131
Madara JL, Stafford J, Dharmsathaphorn K, Carlson S (1987) Structural analysis of a human intestinal epithelial cell line. Gastroenterology 92:1133–1145
Mall M, Gonska T, Thomas J, Schreiber R, Seyedwitz HH, Kuehr J, Brandis M, Kunzelmann K (2003) Modulation of Ca2+-activated Cl− secretion by basolateral K+ channels in human normal and cystic fibrosis airway epithelia. Pediatr Res 53:608–618
Mandell KG, Dharmsathaphorn K, McRoberts JA (1986) Characterization of a cAMP-activated Cl− transport pathway in the apical membrane of a human colonic epithelial cell line. J Biol Chem 261:704–712
Mangos S, Lam PY, Zhao A, Liu Y, Mudumana S, Vasilyev A, Liu A, Drummond IA (2010) The ADPKD genes pkd1a/b and pkd2 regulate extracellular matrix formation. Dis Model Mech 3:354–365
Martinez O, Goud B (1998) Rab proteins. Biochim Biophys Acta 1404:101–112
Maruyama Y, Petersen OH (1984) Control of K+ conductance by cholecystokinin and Ca2+ in single pancreatic acinar cells studied by the patch-clamp technique. J Membr Biol 79:293–300
Maruyama Y, Gallacher DV, Petersen OH (1983a) Voltage and Ca2+-activated K+ channel in basolateral acinar cell membranes of mammalian salivary glands. Nature 302:827–829
Maruyama Y, Petersen OH, Flanagan P, Pearson GT (1983b) Quantification of Ca2+-activated K+ channels under hormonal control in pig pancreas acinar cells. Nature 305:228–232
McCann JD, Welsh MJ (1990) Basolateral K+ channels in airway epithelia. II. Role in Cl− secretion and evidence for two types of K+ channel. Am J Physiol Lung Cell Mol Physiol 258:L343–L348
McCann JD, Matsuda J, Garcia M, Kaczorowski G, Welsh MJ (1990) Basolateral K+ channels in airway epithelia I. Regulation by Ca2+ and block by charybdotoxin. Am J Physiol Lung Cell Mol Physiol 258:L334–L342
Millership J, Devor DC, Hamilton KL, Balut CM, Bruce JIE, Fearson IM (2011) Calcium activated K+ channels increase cell proliferation independent of K+ conductance. Am J Physiol Cell Physiol 300:C792–C802
Morales P, Garneau L, Klein H, Lavoie MF, Parent L, Sauvé R (2013) Contribution of the KCa3.1 channel-calmodulin interactions to the regulation of the KCa3.1 gating process. J Gen Physiol 142:37–60
Morris AP, Gallacher DV, Lee JAC (1986) A large conductance, voltage-and calcium-activated K+ channel in the basolateral membrane of rat enterocytes. FEBS Lett 206:87–92
Muth TR, Caplan MJ (2003) Transport protein trafficking in polarized cells. Annu Rev Cell Dev Biol 19:333–366
Myers MP, Khanna R, Lee EJ, Papazian DM (2004) Voltage sensor mutations differentially target misfolded K+ channel subunits to proteasomal and non-proteasomal disposal pathways. FEBS Lett 568:110–116
Nakamoto T, Srivastava A, Romanenko VG, Ovitt CE, Perez-Cornejo P, Arreola J, Begenisich T, Melvin JE (2007) Functional and molecular characterization of the fluid secretion mechanism in human parotid acinar cells. Am J Physiol Regul Integr Comp Physiol 292:R2380–R2390
Nehrke K, Quinn CC, Begenisich T (2003) Molecular identification of Ca2+-activated K+ channels in parotid acinar cells. Am J Physiol Cell Physiol 284:C535–C546
Nguyen TD, Moody MW (1998) Calcium-activated potassium conductances on cultured nontransformed dog pancreatic duct epithelial cells. Pancreas 17:348–358
Nicolaou SA, Neumeier L, Peng Y, Devor DC, Conforti L (2007) The Ca2+-activated K+ channel KCa3.1 compartmentalizes in the immunological synapse of human T lymphocytes. Am J Physiol Cell Physiol 292:C1431–C1439
Nilius B, Wohlrab W (1992) Potassium channels and regulation of proliferation of human melanoma cells. J Physiol 445:537–548
O’Hara B, de la Rosa DA, Rajendra VM (2014) Multiple mineralocorticoid response elements localized in different introns regulate intermediate conductance K+ (KCNN4) channel expression in the rat distal colon. PLoS One 9, e98695
Ohno H, Aguilar RC, Yeh D, Taura D, Saito T (1998) The medium subunits of adaptor complexes recognize distinct but overlapping sets of tyrosine-based sorting signals. J Biol Chem 273:25915–25921
Ohya S, Niwa S, Yanagi A, Fukuyo Y, Yamamura H, Imaizumi Y (2011) Involvement of dominant-negative spliced variants of the intermediate conductance Ca2+-activated K+ channel, KCa3.1, in immune function of lymphoid cells. J Biol Chem 286:16940–16952
Orzech E, Cohen S, Weiss A, Aroeti B (2000) Interactions between the exocytic and endocytic pathways in polarized Madin–Darby canine kidney cells. J Biol Chem 275:15207–15219
Ouadid-Ahidouch H, Roudbaraki M, Delcourt P, Ahidouch A, Joury N, Prevarskaya N (2004) Functional and molecular identification of intermediate-conductance Ca2+-activated K+ channels in breast cancer cells: association with cell cycle progression. Am J Physiol Cell Physiol 287:C125–C134
Papazian DM (1999) Potassium channels: some assembly required. Neuron 23:7–10
Papazian DM, Shao XM, Seoh S-A, Mock AF, Huang Y, Wainstock DH (1995) Electrostatic interactions of S4 voltage sensor in Shaker K+ channel. Neuron 14:1293–1301
Parihar AS, Coghlan MJ, Gopalakrishnan M, Shieh C-C (2003) Effects of intermediate-conductance Ca2+-activated K+ channel modulators on human prostate cancer cell proliferation. Eur J Pharmacol 417:157–164
Pedersen KA, Schroder RL, Skaaning-Jensen B, Strobeak D, Olesen SP, Christophersen P (1999) Activation of the human intermediate-conductance Ca2+-activated K+ channel by 1-ethyl-2-benzimidazolinone is strongly Ca2+-dependent. Biochim Biophys Acta 1420:231–240
Reddy GR, Kotlyarevska K, Ransom RF, Menon RK (2008) The podocyte and diabetes mellitus: is the podocyte the key to the origins of diabetic nephropathy? Curr Opin Nephrol Hypertens 17:32–36
Reuss L, Reinach P, Weinman SA, Grady TP (1983) Intracellular ion activities and Cl− transport mechanisms in bullfrog corneal epithelium. Am J Physiol Cell Physiol 244:C336–C347
Rimele TJ, O’Dorisio MS, Gaginella TS (1981) Evidence for muscarinic receptors on rat colonic epithelial cells: binding of [3H]quinuclidinyl benzilate. J Pharmacol Exp Ther 218:426–434
Roberts DD, Lewis SD, Ballou DP, Olson ST, Shafer JA (1986) Reactivity of small thiolate anions and cysteine-25 in papain toward methyl methanethiosulfonate. Biochemistry 25:5595–5601
Romanenko V, Nakamoto T, Srivastava A, Melvin JE, Begenisich T (2006) Molecular identification and molecular roles of parotid acinar maxi-K channels. J Physiol 281:27964–27972
Romanenko VG, Nakamoto T, Srivastava A, Begenisich T, Melvin JE (2007) Regulation of membrane potential and fluid secretion by Ca2+-activated K+ channels in mouse submandibular glands. J Physiol 581:801–817
Romanenko VG, Roser KS, Melvin JE, Begenisich T (2009) The role of cholesterol and the cytoskeleton in the interaction between IK1 and maxi-K channels. Am J Physiol Cell Physiol 296:C878–C888
Roth EK, Hirtz S, Duerr J, Wenning D, Eichler I, Seydwitz HH, Amaral MD, Mall MA (2011) The K+ channel opener 1-EBIO potentiates residual function of mutant CFTR in rectal biopsies from cystic fibrosis patients. PLoS One 6, e24445
Rufo PA, Jiang L, Moe SJ, Brugnara C, Alper SL, Lencer WI (1996) The antifungal antibiotic, clotrimazole, inhibits Cl− secretion by polarized monolayers of human colonic epithelial cells. J Clin Invest 98:2066–2075
Rufo PA, Merlin D, Riegler M, Ferguson-Maltzman MH, Dickinson BL, Brugnara C, Alper SL, Lencer WI (1997) The antifungal antibiotic, clotrimazole, inhibits chloride secretion by human intestinal T84 cells via blockade of distinct basolateral K+ conductances. Demonstration of efficacy in intact rabbit colon and in an in vivo mouse model of cholera. J Clin Invest 100:3111–3120
Sadlish H, Skach WR (2004) Biogenesis of CFTR and other polytopic membrane proteins: new roles for the ribosome-translocon complex. J Membr Biol 202:115–126
Sandle GI (1998) Salt and water absorption in the human colon: a modern appraisal. Gut 43:294–299
Sandle GI, Wills NK, Alles W, Binder HJ (1986) Electrophysiology of the human colon: evidence of segmental heterogeneity. Gut 27:999–1005
Sandle GI, Higgs N, Crowe P, Marsh MM, Venkatesan S, Peters TJ (1990) Cellular basis for defective electrolyte transport in inflamed human colon. Gastroenterology 99:97–105
Sato Y, Sakaguchi M, Goshima S, Nakamura T, Uozumi N (2002) Integration of Shaker-type K+ channel, KAT1, into the endoplasmic reticulum membrane: synergistic insertion of voltage-sensing segments, S3-S4, and independent insertion of pore-forming segments, S5-P-S6. Proc Natl Acad Sci U S A 99:60–65
Sato Y, Sakaguchi M, Goshima S, Nakamura T, Uozumi N (2003) Molecular dissection of the contribution of negatively and positively charged residues in S2, S3, and S4 to the final membrane topology of the voltage sensor in the K+ channel, KAT1. J Biol Chem 278:13227–13234
Schreiber R (2005) Ca2+ signaling, intracellular pH and cell volume in cell proliferation. J Membr Biol 205:129–137
Schumacher MA, Rivard AF, Bachinger HP, Adelman JP (2001) Structure of the gating domain of a Ca2+-activated K+ channel complexed with Ca2+/calmodulin. Nature 410:1120–1124
Schumacher MA, Crum M, Miller MC (2004) Crystal structures of apocalmodulin and an apocalmodulin/SK potassium channel gating domain complex. Structure 12:849–860
Schwab A, Wulf A, Schulz C, Kessler W, Nechyporuk-Zloy V, Romer M, Reinhardt J, Weinhold D, Dieterich P, Stock C, Hebert SC (2006) Subcellular distribution of calcium-sensitive potassium channels (IK1) in migrating cells. J Cell Physiol 206:86–94
Schwab A, Nechyporuk-Zloy V, Gassner B, Schulz C, Kessler W, Mally S, Romer M, Stock C (2012) Dynamic redistribution of calcium sensitive potassium channels (hKCa3.1) in migrating cells. J Cell Physiol 227:686–696
Seoh S-A, Sigg D, Papazian DM, Bezanilla F (1996) Voltage-sensing residues in the S2 and S4 segments of the Shaker K+ channel. Neuron 16:1159–1167
Sepulveda FV, Mason WT (1985) Single channel recordings obtained from basolateral membranes of isolated rabbit enterocytes. FEBS Lett 191:87–91
Sheikh IA, Koley H, Chakrabarti MJ, Hoque KM (2013) The EZpac1 signaling pathway regulates Cl− secretion via modulation of apical KCNN4c channels in diarrhea. J Biol Chem 288:20404–20415
Simms LA, Doecke JD, Roberts RL, Fowler EV, Zhao ZZ, McGuckin MA, Huang N, Hayward NK, Webb PM, Whiteman DC, Cavanaugh JA, McCallum R, Florin TH, Barclay ML, Gearry RB, Merriman TR, Montgomery GW, Radford-Smith GL (2010) KCNN4 gene variant is associated with ileal Crohn’s Disease in the Australian and New Zealand population. Am J Gastroenterol 105:2209–2217
Simoes M, Garneau L, Klein H, Banderali U, Hobeila F, Roux B, Parent L, Sauvé R (2002) Cysteine mutagenesis and computer modeling of the S6 region of an intermediate conductance IKCa channel. J Gen Physiol 120:99–116
Singh S, Syme CA, Singh AK, Devor DC, Bridges RJ (2001) Benzimidazolone activators of chloride secretion: potential therapeutics for cystic fibrosis and chronic obstructive pulmonary disease. J Pharmacol Exp Ther 296:600–611
Singh SK, O’Hara B, Talukder JR, Rajendran VM (2012) Aldosterone induces active K+ secretion by enhancing mucosal expression of Kcnn4c and Kcnma1 channels in rat distal colon. Am J Physiol Cell Physiol 302:C1353–C1360
Skalhegg BS, Tasken K, Hansson V, Huitfeldt HS, Jahnsen T, Lea T (1994) Location of cAMP-dependent protein kinase type I with the TCR-CD3 complex. Science 263:84–87
Smith PL, Frizzell RA (1984) Chloride secretion by canine tracheal epithelium IV. Basolateral membrane K permeability parallels secretion rate. J Membr Biol 77:187–199
Smith JJ, Welsh MJ (1992) cAMP stimulates bicarbonate secretion across normal, but not cystic fibrosis airway epithelia. J Clin Invest 89:1148–1153
Srivastava S, Li Z, Lin L, Liu G, Ko K, Coetzee WA, Skolnik EY (2005) The phosphatidylinositol 3-phosphate phosphatase myotubularin- related protein 6 (MTMR6) is a negative regulator of the Ca2+-activated K+ channel KCa3.1. Mol Cell Biol 25:3630–3638
Srivastava S, Choudhury P, Li Z, Liu G, Nadkarni V, Ko K, Coetzee WA, Skolnik EY (2006) Phosphatidylinositol 3-phosphate indirectly activates KCa3.1 via 14 amino acids in the carboxy terminus of KCa3.1. Mol Biol Cell 17:146–154
Srivastava S, Zhdanova O, Di L, Li Z, Albaqumi M, Wulff H, Skolnik EY (2008) Protein histidine phosphatase 1 negatively regulates CD4 T cells by inhibiting the K+ channel KCa3.1. Proc Natl Acad Sci U S A 105:14442–14446
Strobl JS, Wonderlin WF, Flynn DC (1995) Mitogenic signal transduction in human breast cancer cells. Gen Pharmacol 26:1643–1649
Sukhareva M, Hackos DH, Swartz KJ (2003) Constitutive activation of the Shaker Kv channel. J Gen Physiol 122:541–556
Suzuki K, Petersen OH (1985) The effects of Na+ and Cl− removal and of loop diuretics on acetylcholine-evoked membrane potential changes in mouse lacrimal acinar cells. Q J Exp Physiol 70:437–445
Syme CA, Gerlach AC, Singh AK, Devor DC (2000) Pharmacological activation of cloned intermediate- and small-conductance Ca2+-activated K+ channels. Am J Physiol Cell Physiol 278:C570–C581
Syme CA, Hamilton KL, Jones HM, Gerlach AC, Giltinan L, Papworth GD, Watkins SC, Bradbury NA, Devor DC (2003) Trafficking of the Ca2+-activated K+ channel, hIK1, is dependent upon a C-terminal leucine zipper. J Biol Chem 278:8476–8486
Thompson J, Begenisich T (2006) Membrane-delimited inhibition of maxi-K channel activity by the intermediate conductance Ca2+-activated K channel. J Gen Physiol 127:159–169
Thompson J, Begenisich T (2009) Mechanistic details of BK channel inhibition by the intermediate conductance, Ca2+-activated K channel. Channels 3:194–204
Tidball CS (1961) Active chloride transport during intestinal secretion. Am J Physiol 200:309–312
Tiwari-Woodruff SK, Schulteis CT, Mock AF, Papazian DM (1997) Electrostatic interactions between transmembrane segments mediate folding of Shaker K+ channel subunits. Biophys J 72:1489–1500
Tiwari-Woodruff SK, Lin MA, Schulteis CT, Papazian DM (2000) Voltage-dependent structural interactions in the Shaker K+ channel. J Gen Physiol 115:123–138
Trautmann A, Marty A (1984) Activation of Ca-dependent K channels by carbamylcholine in rat lacrimal glands. Proc Natl Acad Sci U S A 81:611–615
Tu L, Wang J, Helm A, Skach WR, Deutsch C (2000) Transmembrane biogenesis of Kv1.3. Biochemistry 39:824–836
Vandorpe DH, Shmukler BE, Jiang L, Lim B, Maylie J, Adelman JA, de Franceschi L, Cappellini MD, Brugnara C, Alper SL (1998) cDNA cloning and functional characterization of the mouse Ca2+-gated K+ channel, mIK1. Role in regulatory volume decrease and erythroid differentiation. J Biol Chem 273:21542–21553
Wallace DP, Grantham JJ, Sullivan LP (1996) Chloride and fluid secretion by cultured human polycystic kidney cells. Kidney Int 50:1327–1336
Wallner M, Meera P, Toro L (1999) Molecular basis of fast inactivation in voltage and Ca2+-activated K+ channels: a transmembrane β-subunit homolog. Proc Natl Acad Sci U S A 96:4137–4142
Wang S, Melkoumian Z, Woodfork KA, Cather C, Davidson AG, Wonderlin WF, Strobl SJ (1998) Evidence for an early G1 ionic event necessary for cell cycle progression and survival in the MCF-7 human breast carcinoma cell line. J Cell Physiol 176:456–464
Wang J, Kristian A, Novak I (2013) Purinergic regulation of CFTR and Ca2+-activated Cl− channels and K+ channels in human pancreatic duct epithelium. Am J Physiol Cell Physiol 304:C673–C684
Warth R, Hamm K, Bleich M, Kunzelmann K, von Hahn T, Schreiber R, Ullrich E, Mengel M, Trautmann N, Kindle P, Schwab A, Greger R (1999) Molecular and functional characterization of the small Ca2+-regulated K+ channel (rSK4) of colonic crypts. Pflugers Arch 438:437–444
Wasserman SI, Barrett KI, Huott PA, Beuerlein G, Kagnoff MF, Dharmsathaphorn K (1988) Immune-related intestinal Cl− secretion. I. Effect of histamine on the T84 cell line. Am J Physiol Cell Physiol 254:C53–C62
Welsh MJ, McCann JD (1985) Intracellular calcium regulates basolateral potassium channels in a chloride-secreting epithelium. Proc Natl Acad Sci U S A 82:8823–8826
Welsh MJ, Smith PL, Frizzell RA (1982) Chloride secretion by canine tracheal epithelium. II. The cellular electrical potential profile. J Membr Biol 70:227–238
Weymer A, Huott P, Liu W, McRoberts JA, Dharmsathaphorn K (1985) Chloride secretory mechanism induced by prostaglandin E1 in a colonic epithelial cell line. J Clin Invest 76:1828–1836
Wilson SM, Brown SG, McTavish N, McNeill RP, Husband EM, Inglis SK, Olver RE, Clunes MT (2006) Expression of intermediate-conductance, Ca2+-activated K+ channel (KCNN4) in H441 human distal airway epithelial cells. Am J Physiol Lung Cell Mol Physiol 291:L957–L965
Wonderlin WF, Strobl JS (1996) Potassium channels, proliferation and G1 progression. J Membr Biol 154:91–107
Wonderlin WF, Woodfork KA, Strobl JS (1995) Changes in membrane potential during the progression of MCF-7 human mammary tumor cells through the cell cycle. J Cell Physiol 165:177–185
Wright RD, Jennings MA, Florey HW, Lium R (1940) The influence of nerves and drugs on secretion by the small intestine and an investigation of the enzymes in intestinal juice. Q J Exp Physiol 30:73–120
Wu ZC, Kisslinger SD, Gaginella TS (1982) Functional evidence for the presence of cholinergic nerve endings in the colonic mucosa of the rat. J Pharmacol Exp Ther 221:664–669
Wulf A, Schwab A (2002) Regulation of a calcium-sensitive K+ channel (cIK1) by protein kinase C. J Membr Biol 187:71–79
Wulff H, Castle NA (2010) Therapeutic potential of KCa3.1 blockers: recent advances and promising trends. Expert Rev Clin Pharmacol 3:385–396
Wulff H, Zhorov BS (2008) K+ channel modulators for the treatment of neurological disorders and autoimmune diseases. Chem Rev 108:1744–1773
Wulff H, Miller MJ, Hansel W, Grissmer S, Cahalan MD, Chandy KG (2000) Design of a potent and selective inhibitor of the intermediate-conductance Ca2+-activated K+ channel: a potential immunosuppressant. Proc Natl Acad Sci U S A 97:8151–8156
Wulff H, Gutman GA, Cahalan MD, Chandy KG (2001) Delineation of the clotrimazole/TRAM-34 binding site on the intermediate conductance calcium-activated potassium channel, IKCa1. J Biol Chem 276:32040–32045
Xia XM, Fakler B, Rivard A, Wayman G, Johnson-Pais T, Keen JE, Ishii T, Hirschberg B, Bond CT, Lutsenko S, Maylie J, Adelman JP (1998) Mechanism of calcium gating in small-conductance calcium-activated potassium channels. Nature 395:503–507
Xia XM, Ding JP, Lingle CJ (1999) Molecular basis for the inactivation of Ca2+- and voltage-dependent BK channels in adrenal chromaffin cells and rat insulinoma tumor cells. J Neurosci 19:5255–5264
Ye M, Grantham JJ (1993) The secretion of fluid by renal cysts from patients with autosomal dominant polycystic kidney disease. N Engl J Med 329:310–311
Yi BA, Lin YF, Jan YN, Jan LY (2001) Yeast screen for constitutively active mutant G protein-activated potassium channels. Neuron 29:657–667
Zhang Z, Zhou Y, Ding JP, Xia XM, Lingle CJ (2006) A limited access compartment between the pore domain and cytosolic domain of the BK channel. J Neurosci 26:11833–11843
Zhang X, Wang G, Dupre DJ, Feng Y, Robitaille M, Lazartigues E, Feng YH, Hebert TE, Wu G (2009) Rab1 GTPase and dimerization in the cell surface expression of angiotensin II type 2 receptor. J Pharmacol Exp Ther 330:109–117
Zhou W, Vergara L, Konig R (2004) T cell receptor induced intracellular redistribution of type I protein kinase A. Immunology 113:453–459
Zhu Y, Ye J, Huizinga JD (2007) Clotrimazole-sensitive K+ currents regulate pacemaker activity in interstitial cells of Cajal. Am J Physiol Gastrointest Liver Physiol 292:G1715–G1725
Zimmerman TW, Binder HJ (1982) Muscarinic receptors on rat isolated colonic epithelial cells: a correlation between inhibition of [3H]quinuclidinyl benzilate binding and alteration in ion transport. Gastroenterology 83:1244–1251
Acknowledgments
The authors would like to thank Patrick Thibodeau (University of Pittsburgh) for the modeling of KCa3.1 (Fig. 20.7) and David Hess for the trafficking model (Fig. 20.8). DCD has been supported by National Institutes of Health grants (HL092157), and KLH has been supported by a UORG grant and an OSMS Strategic Research award from the University of Otago.
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Devor, D.C., Bertuccio, C.A., Hamilton, K.L. (2016). KCa3.1 in Epithelia. In: Hamilton, K., Devor, D. (eds) Ion Channels and Transporters of Epithelia in Health and Disease. Physiology in Health and Disease. Springer, New York, NY. https://doi.org/10.1007/978-1-4939-3366-2_20
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