The maxi-anion channel: a classical channel playing novel roles through an unidentified molecular entity
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- Sabirov, R.Z. & Okada, Y. J Physiol Sci (2009) 59: 3. doi:10.1007/s12576-008-0008-4
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The maxi-anion channel is widely expressed and found in almost every part of the body. The channel is activated in response to osmotic cell swelling, to excision of the membrane patch, and also to some other physiologically and pathophysiologically relevant stimuli, such as salt stress in kidney macula densa as well as ischemia/hypoxia in heart and brain. Biophysically, the maxi-anion channel is characterized by a large single-channel conductance of 300–400 pS, which saturates at 580–640 pS with increasing the Cl− concentration. The channel discriminates well between Na+ and Cl−, but is poorly selective to other halides exhibiting weak electric-field selectivity with an Eisenman’s selectivity sequence I. The maxi-anion channel has a wide pore with an effective radius of ~1.3 nm and permits passage not only of Cl− but also of some intracellular large organic anions, thereby releasing major extracellular signals and gliotransmitters such as glutamate− and ATP4−. The channel-mediated efflux of these signaling molecules is associated with kidney tubuloglomerular feedback, cardiac ischemia/hypoxia, as well as brain ischemia/hypoxia and excitotoxic neurodegeneration. Despite the ubiquitous expression, well-defined properties and physiological/pathophysiological significance of this classical channel, the molecular entity has not been identified. Molecular identification of the maxi-anion channel is an urgent task that would greatly promote investigation in the fields not only of anion channel but also of physiological/pathophysiological signaling in the brain, heart and kidney.
KeywordsMaxi-anion channelVolume-sensitive chloride channelPurinergic signalingATP releaseGlutamate release
In our previous review article , we highlighted a newly discovered physiological role of maxi-anion channel in ATP release in response to osmotic stress in mammary C127 cells and in response to salt stress in tubuloglomerular feedback in kidney macula densa. Since then, we have carried out extensive studies on the maxi-anion channel along the following four lines: (1) its biophysical properties including the pore size , (2) its role in ATP release from astrocytes [6, 7] and cardiomyocytes [2, 12] under ischemic or osmotic stress, (3) its role in glutamate release from astrocytes under ischemic or osmotic stress , and (4) its molecular identification [13, 14]. The present review article, thus, gives an updated account of the biophysical properties, the roles in release of ATP and glutamate under pathophysiological conditions, and the molecular identification of the maxi-anion channel. Also, we intended to provide a comprehensive account of physiological and pharmacological characteristics of the maxi-anion channel in the present review, because it passed over a decade since a most recent comprehensive review on this channel was published by Strange et al. .
Expression pattern of the maxi-anion channel
The volume-activated large-conductance anion channels exhibiting properties typical of the maxi-anion channels have been found in different types of cell preparations. The single-channel event of large conductance was first described by Blatz and Magleby  in the plasma membrane patches excised from rat skeletal muscles in primary culture. About the same time, large-conductance channels with anion selectivity and bell-shaped voltage dependency were described in myotubes obtained from chicken embryos and in mouse peritoneal macrophages , in Schwann cells from 1 to 2 days old rats in primary culture  and in A6 Xenopus kidney epithelial cells . Later, when a broader range of cell types was studied by patch-clamp technique, the activity of maxi-anion channels with a unitary conductance of 300–400 pS has been reported in almost every part of the whole organism. Maxi-anion channel activities have been found in freshly isolated frog skeletal muscles [20, 21], somatic muscles of Ascaris suum , as well as in cultured L6 rat muscle cells [23–26] and BC3H1 myoblasts . Smooth muscles from uterus  and colon [29, 30] as well as cultured vascular smooth muscle cells from rat thoracic aorta [31–33] were found to express the maxi-anion channel activity. In the heart, maxi-anion channels were described in neonatal cardiac myocytes in primary culture [2, 12, 34, 35] and freshly isolated adult ventricular cardiomyocytes . In the nervous system, the maxi-anion channel activity was detected in embryonic Xenopus spinal neurons , demyelinated Xenopus axons , in neuroblastoma cell lines [3, 38–44] and in a hippocampal cell line [45, 46]. In glia, maxi-anion channels were found in cultured Schwann cells from 1 to 2 day old rats  and adult humans  as well as in freshly dissected rat spinal root Schwann cells . These channels were similar to those observed in cultured cortical astrocytes from rats [4, 49, 50] and mice [5–7, 51] as well as in a rat astrocytic RGCN cell line . In epithelia, the urinary bladder , gastric , pancreatic [55–57], colonic [58–60], airway [61–63], choroid plexus , bile duct [65, 66], ciliary [67–69], renal [9, 19, 70–78], vestibular , placental [80–86], ruminal  and ovarian  epithelial cells were also found to express the maxi-anion channel with properties similar to those of excitable cells. Resembling maxi-anion channels were also found in fibroblasts [14, 66, 88–92] and endothelial cells [93–97]. In the immune system, the maxi-anion channel activity has been confirmed in B lymphocytes [98–101], in T lymphocytes [102–104] and in peritoneal macrophages [17, 105]. In other tissues, mast cells , keratinocytes , osteogenic cells , cultured glomus cells of the carotid body , PC12 pheochromocytoma cells , pavement cells from the gills of the trout  and mammary gland C127 cells [8, 11, 112] have also been shown to possess this channel. Patch-clamping the intracellular organelles revealed the maxi-anion channel activity in sarcoplasmic reticulum “sarcoballs” , whereas the presence of this channel in endoplasmic reticulum  and in Golgi complex  was demonstrated by reconstituting these membranes into liposomes and lipid bilayers, respectively. Thus, it is likely that the maxi-anion channel is widely expressed in almost every part of the body.
Basic biophysical properties of the maxi-anion channel
The maxi-anion channel exhibits roughly uniform behavior in different types of cells. Its very large single-channel conductance (300–400 pS) in physiological conditions distinguishes it from other chloride channels. Most authors noted that the maxi-anion channel has multiple subconductance states of various levels, such as ~15, ~50, ~100, ~150 and ~200 pS [2, 17, 29, 95]. When the ambient Cl− concentration varied, the single-channel conductance saturated at 640 pS with Km = 112 mM in L6 myoblasts , at 581 pS with Km = 120 mM in T lymphocytes  and at 617 pS with Km = 77 mM in frog skeletal muscle “sarcoballs” .
In many cell types, the maxi-anion channel can discriminate well for anions over cations. A high permeability ratio of chloride over sodium (PCl/PNa) was reported for maxi-anion channel in bovine pigmented ciliary epithelial cells (24 ), T lymphocytes (30 ), neuroblastoma cells (30 ), in neonatal rat cardiac myocytes (24.6 ) and in mammary C127 cells (21–26 ). On the other hand, somewhat lower PCl/PNa (6–11) was observed for human colonic HT-29 cells , freshly isolated guinea pig fetal type II alveolar epithelial cells , rat bile duct epithelial cells , L6 rat muscle cells , and colonic smooth muscle cells . Studying the maxi-anion channel of T lymphocytes, Schlichter et al.  found a decrease in anion selectivity from PCl/PNa = 30 measured under balanced osmolarity to PCl/PNa = 11 in the presence of osmotic gradient. These results indicate that the channel is highly selective to anions but the degree of anion selectivity may vary not only with cell types but also with the experimental conditions.
Selectivity sequence and permeability ratios Px/PCl (given in parenthesis) for maxi-anion channel of different types of cells
Rat cultured Schwann cells
I− (1.4) > Br− (1.2) > Cl− (1.0) > methyl-SO4− (0.72) > SO42− (0.61) > acetate− (0.39) = isethionate− (0.39) > aspartate− (<0.03), glutamate− (<0.03)
Gray et al. 
Rabbit urinary bladder epithelial cells
Cl− (1.0) ≈ Br− (1.0) ≈ I− (1.0) ≈ SCN− (1.0) ≈ NO3− (1.0) > F− (0.57) > acetate− (0.30) > gluconate− (0.07)
Hanrahan et al. 
Rat cultured pulmonary alveolar (type II) cells
I− (1.5) > Br− (1.02) ≥ Cl− (1.00) > NO3− (0.9) > gluconate− (0.6)
Schneider et al. 
Rat cultured smooth muscle cells from embryonic aorta
I− (1.4) > Br− (1.3) > Cl− (1.0) > F− (0.7)
Soejima and Kokubun 
Rat cultured glomus cells of the carotid body
Cl− (1.0) > HCO3− (0.71) > SO42− (0.57) > glutamate− (0.14)
Stea and Nurse 
Mouse B lymphocyte-myeloma hybridoma cells
F− (1.25) > I− (1.18) > SCN− (1.10) > Br− (1.07) > Cl− (1.00) > glucuronate− (0.78) > NO3− (0.68) > aspartate− (0.62)
Human T lymphocytes
I− (1.38) > NO3− (1.14) > Br− (1.04) > Cl− (1.0) > F− (0.57) > SCN− (0.56) > HCO3− (0.56) > SO42− (0.49) > gluconate− (0.29) ≈ propionate− (0.30) > aspartate− (0.08)
Schlichter et al. 
Chick cultured embryonic osteogenic cells
Cl− (1.0) > methylsulfate− (0.71) > gluconate− (0.25) > glutamate− (0.17)
Ravesloot et al. 
Human colon carcinoma HT-29cl.19A cells
I− (1.2) > Br− (1.05) > Cl− (1.0) > F− (0.46) > gluconate− (0.24)
Bajnath et al. 
Ascaris suum muscle membrane vesicles
I− > Br− = NO3− > Cl− > F−
Dixon et al. 
Rat muscle L6 cells
Br− (1.18) > I− (1.15) > NO3− (1.13) > Cl− (1.0) > methanesulfonate− (0.60) > HCO3− (0.59) > propionate− (0.44) > SO42− (0.40) > glutamate− (0.1)
Hurnak and Zachar 
Guinea-pig parietal cells
I− > Br− > Cl− > F−
Kajita et al. 
Mouse mammary C127 cells
I− (1.31) > Br− (1.14) > Cl− (1.0) > F− (0.61) > phosphate2− (0.43) > aspartate− (0.23) ≈ glutamate− (0.22)
Sabirov et al. 
Mouse mammary C127 cells
Cl− (1.0) > formate− (0.66) > pyruvate− (0.52) > methanesulfonate− (0.51) > acetate− (0.50) > propionate− (0.39) > glucuronate− (0.19) > glucoheptonate− (0.18) > gluconate− (0.17) > glutamate− (0.16) > lactobionate− (0.13)
Sabirov and Okada 
The maxi-anion channel in gastric parietal cells was insensitive to extracellular pH 5–8 . In cultured glomus cells of the rat carotid body the channel was insensitive to the intracellular pH 6.5–8 in inside-out patches . However, high external pH 9 shifted voltage dependence of the maxi-anion channel to more negative values in frog skeletal muscles . For the tracheal maxi-anion channel reconstituted into giant liposomes, low bath pH reduced channel open probability in inside-out patches yielding an apparent pK of 6.09 . However, the channel current amplitude did not change with acidification suggesting that the protonation site is located far from the channel permeation pathway.
Pharmacological properties of the maxi-anion channel
Gadolinium ions are considered as a relatively selective inhibitor of the stimulated ATP release [123, 124]. Gd3+ at the concentration of 30–50 μM effectively shuts down the maxi-anion channels from the extracellular (Fig. 5), but not the cytosolic, side [2, 5, 6, 8, 12, 14]. This is in contrast to the VSOR which is insensitive to gadolinium ions [8, 125]. Zn2+ ions also effectively blocked the maxi-anion channel both from extra- and intracellular sides in different cell types [26, 31, 93, 103, 111].
In mouse N1E 115 neuroblastoma cells, a type II pyrethroid, deltamethrin, at micromolar concentrations inhibited the maxi-anion channel activity , whereas ivermectin (10−7 M) and pentobarbitone (10−6 M) significantly increased open channel probability .
Activation of the maxi-anion channel by physiological/pathophysiological stimuli
Obviously, a very strict regulatory system controls the channel physiological state, which provides a low basal activity of maxi-anion channel in resting conditions. The mechanism of the maxi-anion channel activation by cellular swelling remains poorly understood at present. Hypoosmotic stress is known to activate several signaling pathways [1, 126, 127], which are also involved in the maxi-anion channel regulation. These include protein phosphorylation, changes in the intracellular concentration of Ca2+ and cAMP, G proteins, membrane stretch and cytoskeleton, and so on. Intracellular regulatory pathways involved in maxi-anion channel regulation were studied in a number of cell types. Thus, intracellular signaling via PKA and PKC phosphorylation has been suggested to regulate the maxi-anion channel in rabbit cortical collecting duct cells , bovine aortic endothelial cells , pig aortic endothelial cells , and rat vascular smooth muscle cells [31, 32]. Meanwhile, in rabbit colonic smooth muscle cells, PKA and PKC inhibitors had no effect on the maxi-anion channel activity . The channel was shown to be regulated by G proteins: the channels were activated by GTPγS and inhibited by GDPβS and pertussis toxin (PTX) in rabbit renal cortical collecting duct cells [75, 76] and in rabbit colonic smooth muscle cells . However, an opposite type of regulation was observed in rat bile duct epithelial cells, where PTX and GDPβS activated, whereas GTPγS inhibited the maxi-anion channels [65, 66], suggesting a cell-specific channel regulation by G proteins.
Activation of maxi-anion channels upon patch excision may imply an involvement of the cytoskeleton in the maxi-anion channel regulation. Indeed, in cortical collecting duct cells, Schwiebert et al.  demonstrated that in inside-out patches, the maxi-anion channel could be activated by disruption of F-actin using dihydrocytochalasins. Short actin filaments activated the channels, whereas long actin filaments inhibited, and 1 mM ATP reversed effect of dihydrocytochalasin B. In addition, phalloidin abolished the channel activation by negative pressure . Mills et al.  proposed that swelling-induced membrane stretch activates the maxi-anion channel by a mechanism, which involves fragmentation and depolymerization of F-actin.
Although most authors found that maxi-anion channels are insensitive to changes in the cytosolic free Ca2+ concentration in excised inside-out patches, elevation of cytosolic Ca2+ by a Ca-ionophore, A23187, was found to increase the incidence of channels in the cell-attached mode in human colonic cells HT-29 , Swiss 3T3 fibroblasts , pig aortic endothelial cells  and embryonic Xenopus spinal neurons .
On balance, it must be stated that available data are still fragmentary to deduce the precise mechanisms of activation of the maxi-anion channel. Also, studies for the molecular mechanisms have been largely hampered by the lack of molecular identification of this channel.
Hypothesized roles of the maxi-anion channel in the Cl− transport
As an electrogenic chloride-transporting pathway, the maxi-anion channel has been implicated in a number of physiological functions, which involve Cl− movement. Thus, the channel was deemed to provide a rout for solute transport and/or bicarbonate secretion in pancreatic duct cells , in alveolar epithelium , in syncytiotrophoblasts of placenta [83, 129], in sheep ruminal epithelium  and in glomus cells of the rat carotid body . The channel is thought to participate in the Cl− efflux during cell volume regulation in cortical collecting duct epithelium [9, 75], pigmented ciliary epithelial cells , lymphocytes [102, 104], myoblasts , placenta , neuronal cells  and astrocytes . The maxi-anion channel is expressed in the ciliary epithelium, where it may participate in the aqueous humor secretion [67, 130], and in the hepatic bile duct, where it could be involved in bile formation [65, 66]. Efflux of Cl− ions during apoptotic cell shrinkage is also thought to occur via maxi-anion channels in neuronal cells . Chloride influx as a counter ion for potassium uptake is thought to be a function for the maxi-anion channel in Schwann cells [47, 48]. The maxi-anion channel may also be involved in regulating charge balance and membrane potential of the Golgi complex by providing a counter anion pathway for the H+-ATPase . Regulation by estrogens and antiestrogens links the maxi-anion channel to the intracellular signaling pathways of the cells expressing estrogen receptors [42, 89]. A possible role in signal transduction in B cell activation [99, 100] and in receptor-mediated initial cell depolarization in colonic smooth muscle cells  has also been proposed.
It should however be noted that in most papers, the proposed physiological roles are mainly hypothetical without making critical comparison of pharmacology between the proposed functions and the maxi-anion channel.
Roles of the maxi-anion channel in stimulated release of ATP and glutamate
It is clear that the maxi-anion channel conducts glutamate−, ATP4− and MgATP2−. What is, however, the number of active channels that would be sufficient for the observed net release of glutamate and ATP? Previously our quantitative analyses of the channel’s conductance and the observed net release of these molecules suggested that brief opening of only a few maxi-anion channels would be sufficient to provide physiologically significant extracellular signals caused by the release of glutamate from cortical astrocytes  and that of ATP from cardiac myocytes .
The fact that the maxi-anion channel is blocked by ATP at millimolar range (see above) would suggest that some other intracellular anions (e.g. free amino acids or glycolysis intermediates) could also interact with the maxi-anion channel pore in vivo and interfere with the ionic fluxes. Although we did not detect any effect of glutamate on the channel amplitude up to 30 mM , we suppose that, in general, the fraction of the maxi-anion channels contributing to the release of ATP and/or glutamate could vary as a function of the concentrations of cytosolic constituents, which interact with the channel, depending on the intracellular state of the concerned cells.
Puzzle of the molecular identity of the maxi-anion channel
Comparison of the single-channel properties between the mitochondrial VDAC and the maxi-anion channel
PCl/PK (100/1,000 mM KCl)
13.5 ± 2.3 
0.58 ± 0.01 
[Cl−] dependence for single-channel conductance
Linear up to 10 nS with no saturation 
1.16 and 1.42 nm for different entrances 
Voltage-dependent “closed” state
Retains ~40% of initial conductance 
Plasmalemmal VDAC is not the only molecular candidate for the maxi-anion channel. Recently, Suzuki and Mizuno  have reported that a gene tweety found in Drosophila flightless locus has a structure similar to those of known channels. The human homologs of tweety (hTTYH1-3) have been suggested to provide a product, which represents a novel large-conductance Ca2+-activated chloride channel, while a related gene hTTYH1 gave rise to functional expression of the swelling-activated chloride channel. It has been hypothesized that hTTYH1 might be the large-conductance Ca2+-activated Cl− channel [176, 177]. We attempted to check this attractive hypothesis by transfecting two splice-variant clones of the TTYH1 gene (TTYH1-E and TTYH1-SV, kind gifts from Dr. M. Suzuki) into HEK293T cells and assaying the maxi-anion channel activity 1–5 days after transfection by patch excision. In these experiments, either control, TTYH1-E- or TTYH1-SV-transfected cells never showed the maxi-anion channel phenotype typical to C127 cells used as a positive control in these experiments . This result implies that the human homologs of tweety clones alone are unlikely to be molecular identity of the maxi-anion channel. We believe that more thorough tests of this attractive hypothesis are yet necessary. It might be meaningful to test some other variants of TTHY genes as well.
The maxi-anion channel in cardiomyocytes  and mammary C127 cells  was insensitive to octanol-1, suggesting that it is unrelated to connexins. A plasmalemmal subtype of the mitochondrial adenine nucleotide translocase (ANT), or ADP/ATP carrier (AAC), which mediates ATP/ADP exchange at the inner mitochondrial membrane, could also be ruled out based on the insensitivity of the maxi-anion channel to the potent and selective blockers of ANT, atractyloside and bongkrekic acid .
Taken together, we must summarize that the molecular entity of this classical channel has as yet been unidentified.
We are grateful to T. Okayasu for manuscript preparation. This work was supported by Grant-in-Aid for Scientific Research (A) and those for Scientific Research on Priority Areas to YO and that for Scientific Research (C) to RZS from the Japan Society for the Promotion of Science and the Ministry of Education, Culture, Sports, Science and Technology of Japan, as well as Grants-in-Aid from the Center for Science and Technology and Academy of Sciences of Uzbekistan to RZS.