Activation of the Ca2+-sensing receptors increases currents through inward rectifier K+ channels via activation of phosphatidylinositol 4-kinase
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Inward rectifier K+ channels are important for maintaining normal electrical function in many cell types. The proper function of these channels requires the presence of membrane phosphoinositide 4,5-bisphosphate (PIP2). Stimulation of the Ca2+-sensing receptor CaR, a pleiotropic G protein-coupled receptor, activates both Gq/11, which decreases PIP2, and phosphatidylinositol 4-kinase (PI-4-K), which, conversely, increases PIP2. How membrane PIP2 levels are regulated by CaR activation and whether these changes modulate inward rectifier K+ are unknown. In this study, we found that activation of CaR by the allosteric agonist, NPSR568, increased inward rectifier K+ current (I K1) in guinea pig ventricular myocytes and currents mediated by Kir2.1 channels exogenously expressed in HEK293T cells with a similar sensitivity. Moreover, using the fluorescent PIP2 reporter tubby-R332H-cYFP to monitor PIP2 levels, we found that CaR activation in HEK293T cells increased membrane PIP2 concentrations. Pharmacological studies showed that both phospholipase C (PLC) and PI-4-K are activated by CaR stimulation with the latter played a dominant role in regulating membrane PIP2 and, thus, Kir currents. These results provide the first direct evidence that CaR activation upregulates currents through inward rectifier K+ channels by accelerating PIP2 synthesis. The regulation of I K1 plays a critical role in the stability of the electrical properties of many excitable cells, including cardiac myocytes and neurons. Further, synthetic allosteric modulators that increase CaR activity have been used to treat hyperparathyroidism, and negative CaR modulators are of potential importance in the treatment of osteoporosis. Thus, our results provide further insight into the roles played by CaR in the cardiovascular system and are potentially valuable for heart disease treatment and drug safety.
KeywordsCalcium-sensing receptors Inward rectifier K+ channels PIP2 Membrane excitability
Kir2.x channels are inward rectifier K+ channels that play an important role in maintaining stable resting membrane potentials, controlling excitability, and shaping the initial depolarization and final repolarization of ventricular myocytes [13, 19, 21, 27]. Gain and loss of function of Kir2.x channels, which mediate cardiac inwardly rectifying currents (I K1), can cause reentry and arrhythmia, respectively . Reentry is facilitated by shortening of the action potential duration (APD), which abbreviates refractoriness. On the other hand, excessive APD prolongation may cause torsades de pointes arrhythmia and sudden cardiac death .
We previously demonstrated that extracellular spermine inhibits the outward current through Kir2.1 channels expressed in oocytes and the outward I K1 of myocytes, but the effect was much greater in oocytes than in cardiac myocytes . However, why the effects of extracellular spermine are quantitatively different in oocytes and guinea pig myocytes is unclear . It may be simply attributable to the fact that the effects of spermine on Kir channels in different cell types vary. Alternatively, the actions of extracellular spermine may be more diverse in complex cell types such as cardiac myocytes. For example, extracellular spermine can activate the calcium-sensing receptor, CaR . This receptor was first discovered in the parathyroid gland, where its activation by extracellular Ca2+ was shown to decrease the release of parathyroid hormone . CaR is also highly expressed in the kidney, bone, blood vessels, brain, and heart . CaR is a pleiotropic G protein-coupled receptor and thus couples to more than one type of G protein . Notably, several signaling pathways activated by stimulation of CaR regulate I K1. For example, increases in intracellular Ca2+ concentration ([Ca2+] i ) and activation of protein kinase C (PKC) inhibit this current [5, 6, 15], whereas a rise in phosphoinositide 4,5-bisphosphate (PIP2) levels enhances the current . PIP2, acting as a second messenger, plays an important role in modulating several ion channels and transporters [8, 30]. Stimulation of CaR activates both Gq/11, which decreases PIP2, and phosphatidylinositol 4-kinase (PI-4-K), which increases PIP2. But how membrane PIP2 levels are regulated by CaR activation and whether the resulting changes regulate inward rectifier K+ channels are unknown.
In this study, we monitored PIP2 levels using the fluorescent PIP2 probe tubby-R332H-cYFP  and investigated how this regulation affects Kir2.1 channels expressed in HEK293T and I K1 in guinea pig ventricular myocytes.
Materials and methods
Cell culture and transfection procedures
HEK293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Sigma Chemical Co., St. Louis, MO, USA) containing 10% (v/v) fetal bovine serum (FBS; Life Technologies, Paisley, Scotland) and 1% penicillin-streptomycin at 37 °C in a humidified atmosphere containing 5% CO2. HEK293T cells plated on poly-l-lysine-coated no. 1 glass coverslips (35 mm) were transiently transfected with 2 μg of the expression constructs Kir2.1-cyan fluorescent protein(eCFP), CaR-green fluorescent protein (GFP) (OriGene Technologies Inc., MD, USA), and/or tubby-R332H-yellow fluorescent protein (cYFP) using Lipofectamine 2000 (Invitrogen Co., Carlsbad, CA, USA). Cells were used 1–2 days after transfection. The Kir2.1-eCFP construct was generated by subcloning Kir2.1 cDNA into an XhoI/HindIII-digested peCFP vector (Clontech Lab Inc., Mountain View, CA, USA).
Isolation of guinea pig cardiac myocytes
Guinea pig (Hartley) ventricular myocytes were isolated using an enzymatic procedure described previously . Briefly, guinea pigs were anesthetized with sodium pentobarbital (50 mg/kg, i.v.) and hearts were isolated and retrogradely perfused, first with a Ca2+-free Tyrode’s solution (5 mM HEPES pH 7.4, 145 mM NaCl, 5 mM KCl, 2 mM MgCl2, 1 mM CaCl2, 5.5 mM glucose) and then with the same solution containing 0.5 mg/ml collagenase, 0.25 mg/ml protease, 1 mg/ml albumin, and 50 μM CaC12. The heart was minced, and cells were dissociated by gentle agitation in the enzyme solution. Isolated cells were stored at room temperature in modified Tyrode’s solution (pH 7.4) containing 100 mg/ml albumin and 10 mM glucose.
Currents were recorded at room temperature (21–24 °C) using the patch-clamp technique  in the whole-cell configuration and an Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA, USA). The bath contained Tyrode’s solution (see composition previously mentioned), and the pipette solution (pH 7.2) contained 138 mM K-aspartate, 3 mM MgATP, 5 mM Na2-phosphocreatine, 1 mM MgCl2, 5 mM EGTA, 5 mM HEPES, and 2 mM creatine. For recordings of I K1 in myocytes, 5 μM nifedipine, 1 μM atropine, 10 μM glibenclamide, and 1 mM 4-aminopyridine were added in Tyrode’s solution.
Currents were sampled and filtered at frequencies of 20 and 5 kHz, respectively. Command voltages were controlled by and data were acquired using the pCLAMP10 software (Molecular Devices). Recordings in HEK293T cells and myocytes were corrected for the liquid junction potential (−15 mV).
Measurement of fluorescence signals
Total internal reflection fluorescence (TIRF) fluorescence images of HEK293T cells were obtained at 22 °C using a Nikon Eclipse microscope with a Ti-E TIRF module (PFS) (Nikon Co, Tokyo, Japan) and an iXON Ultra 897 EMCCD camera (Andor Technology Ltd., Belfast, UK). Cells were visualized using a ×100 1.45 oil immersion objective lens. YFP fluorescence was monitored by exciting with a 515-nm laser (Cobolt Laser, Sweden) and collecting the emission at 535/30 nm (Chroma, Rockingham, VT). Averaged fluorescence intensity F of a region of interest was selected to cover the majority of the cell with background subtraction.
Averaged data are presented as means ± SEMs. Student’s t test for independent samples was used to assess the statistical significance of differences. Asterisks *, **, and *** indicate p < 0.05, 0.01, and 0.005, respectively.
Effects of CaR activation on the inhibition of Kir2.1 channels by extracellular spermine in HEK293T cells
These results suggest that spermine can enhance Kir2.1 channel activity through activation of CaR in addition to its direct inhibitory effect on the channel.
CaR activation increases currents mediated by Kir2.1 channels expressed in HEK293T cells
CaR activation increases Kir2.1 currents and PIP2 via activation of PI-4-K
CaR signaling through the Gq/11 pathway inhibits Kir2.1 channel activity
These results suggest that activation of CaR decreases Kir2.1 channel-mediated currents through activation of PLC.
CaR activation increases I K1 in guinea pig ventricular myocytes
In summary, CaR stimulation activates both the PLC and PI-4-K pathways, but the effect on PI-4-K dominates, resulting in increases of Kir2.1 currents recorded from HEK293T cells transfected with Kir2.1 and I K1 in guinea pig ventricular myocytes.
CaR activation increases membrane PIP2 levels and Kir currents
The purpose of this study was to examine whether CaR activation regulates inward rectifier K+ channels by modulating the level of membrane PIP2. To achieve this goal, we investigated the effects of activating CaR on currents mediated by Kir2.1 channels expressed in HEK293T cells and on I K1 in guinea pig ventricular myocytes. We found that activation of CaR by an allosteric agonist (NPSR568) increased both endogenous I K1 and currents mediated by exogenously expressed Kir2.1 to a similar extent. Further, monitoring PIP2 levels using the PIP2-binding probe tubby-R332H-cYFP demonstrated that CaR activation increased PIP2 concentration at the plasma membrane in HEK293T cells. These effects were abolished by PIK-93, suggesting the involvement of PI-4-K. CaR activation facilitates the recovery of membrane PIP2 following PLC activation, further supporting this notion. Collectively, these results provide the first direct evidence that CaR activation increases inward rectifier K+ channel currents by accelerating PIP2 synthesis in HEK293T cells and possibly also in guinea pig ventricular myocytes.
PIP2 acts as a second messenger molecule to play an important role in modulating a number of ion channels and transporters [8, 9, 30]. Previous literature reports have enhanced our understanding of this topic, yet significant gaps remain. First, although it has been shown that CaR can regulate ion channels in cells , CaR regulation of ion channels through modulation of PIP2 is a mechanism that has not been explored. Second, most previous investigations have focused on single signaling pathways in PIP2 regulation and its association with channel activities. Yet, multiple pathways regulate membrane PIP2, and how they are integrated to regulate ion channels remains elusive. In the current study, we explored the involvement of two signaling pathways engaged by activation of CaR in the regulation of PIP2. Our data revealed that activation of CaR results in increases in membrane PIP2 and Kir currents through PI-4-K activation, although the activation of PLC also makes contribution to the regulation of Kir channels.
Third, most studies have focused on the effect of PIP2 depletion on ion channels expressed in heterologous systems [23, 24, 30]. PIP2 depletion has also been studied in native cells. For example, it has been shown that, in normal isolated Müller cells, activation of Gq/11-coupled metabotropic glutamate receptors (mGluRs) with the mGluR I agonist (S)-3,5-dihydroxyphenylglycine (DHPG) suppresses Kir currents through the intracellular Ca2+-dependent PLC/IP3-ryanodine/PKC signaling pathway . On the other hand, little is known about whether resynthesis of PIP2 regulates ion channels in heterologous expression systems or in native cells. PI-4-K is involved in the constitutive biosynthesis of PIP2 and in PIP2 resynthesis after its breakdown by PLC. Can physiological regulators that target PI-4-K affect PIP2 levels and modulate channel activities? A previous study has shown that resynthesis of PIP2 via PI-4-K mediates adaptation of caffeine responses in taste receptor cells by regulating Kir and KV channels . Our study provides additional evidence that increasing membrane PIP2 by stimulating PI-4-K via CaR activation can regulate ion channel function.
Fourth, it is unclear whether physiological fluctuations in the levels of PIP2, as may occur with the activation of intracellular signaling pathways, are sufficient in and of themselves to regulate ion channels . It has been hypothesized that some channels interact with lipid partners with high affinity such that the lipid-binding site of the channel remains saturated during most physiological changes in lipid levels. By contrast, other channels bind with low affinity; for these channels, variations in lipid concentration may act as physiological signals to regulate the computational properties of neurons and transport properties of secretory cells . Among Kir channels, the constitutively active Kir1.1 and Kir2.1 interact with PIP2 with high affinity , whereas Kir3 channels show weaker interactions with PIP2 . Application of exogenous PIP2 dynamically activates these channels in reverse order, namely Kir3.1/3.4 > Kir2.1 > Kir1.1 [10, 26, 36]. Whether Kir2.1 channels are sensitive to changes in membrane PIP2 level under physiological conditions remains unclear. Our study provides the first direct evidence that activation of CaR can increase I K1 via the PI-4-K pathway.
The modulation of Kir2.1 channels by PIP2 via CaR activation appears to be voltage dependent. The underlying mechanisms are unknown. It has been previously shown that polyamines bound to the Kir2.1 channel at positive driving force interact with the gating of Kir2.1 channels by PIP2 . It is possible that the voltage-dependent effect of PIP2 is related to the voltage-dependent polyamine block of the channel. Further, it is shown that PIP2 shifts the voltage dependence of conductance by promoting the opening of the ion conduction gate and by a negative surface charge in Slo1 BK channels . PIP2 may also exert similar effects on Kir, and thus, the effect appears to be different at different voltages.
In addition to regulating PIP2, CaR also modulates several cellular events including increases of [Ca2+] i , PKC, and arachidonic acids as well as a decrease in PKA [20, 33]. Several of these intracellular signaling molecules regulate Kir2.1 and/or I K1 [5, 17, 18, 40]. This study investigated only the effect of PIP2 on Kir2.1 upon CaR activation. How other intracellular signaling is involved in the regulation of Kir2.1/I K1 via CaR activation requires further studies. Further, besides I K1, the electrical properties of the membrane involve other ion channels. The overall physiological and pathological functions require further investigation when all the effects during CaR activation are taken into consideration.
In this study, we used PIK-93 to inhibit PI-4-K IIIβ. Because PIK-93 inhibits PI-3-K and PI-4-K with similar potency [2, 16], the contribution of PI-4-K-induced increases of Kir2.1 currents and I K1 upon CaR activation is underestimated. Our study provides the qualitative analysis of involvement of PI-4-K signaling in CaR activation. For more quantitative analysis, future studies with specific PI-4-K inhibitors are required.
Pathophysiological implications of CaR regulation of I K1
It has been previously shown that putrescine significantly reduces the arrhythmia associated with cardiac ischemia and reperfusion . Furthermore, ischemia/reperfusion results in depletion of the myocardial polyamine pool. Application of exogenous spermine restores the intracellular polyamine pool and reduces cardiac myocyte necrosis, suggesting that the loss of spermine might be involved in the cardiac injury produced by reperfusion . These cardioprotective effects have been attributed to the membrane-stabilizing and antioxidative effects of putrescine . However, because ischemia and reperfusion can lead to upregulation of CaR in the heart during a myocardial infarction  and because polyamines are potent agonists of CaR, it is possible that the cardioprotective effects of polyamines are the result of CaR activation. The results from our study suggest that CaR activation may stabilize the electrical properties of the cardiac membrane by increasing I K1. This action may play an important role in the cardioprotective effects of polyamines during ischemia/reperfusion injury, although further studies are required to confirm this hypothesis. CaR functions as an integrator of extracellular stimuli and is probably always activated to some extent . It remains to be determined whether CaR calcilytics are capable of decreasing I K1 and thus affect cardiac electrical properties.
Extracellular Ca2+ is a low-affinity agonist for CaR, and thus, under physiological conditions, CaR is subjected to the tonic stimulation of extracellular Ca2+. Investigations on this tonic activation of CaR may shed light on the physiological functions of CaR.
Our data show that increases of endogenous PIP2 in cardiac myocytes regulate I K1. This novel relationship supports a model in which autonomic stimulation of cardiac CaR may alter I K1-dependent repolarization and excitability and impact cardiac function. Our study explored important questions related to PIP2 modulation by CaR activation and showed that CaR can upregulate inward rectifier K+ channels in both native cells and a heterologous expression system. Given the increasing clinical use of calcimimetics in the treatment of hyperparathyroidism and the potential for using calcilytics in the treatment of osteoporosis, it is essential to understand the role of CaR in cardiac function .
We thank Dr. Andrew Tinker for generously providing the tubby-R332H-cYFP construct and Dr. Fang Liao for the stable CCR6 HEK cells. This work was supported by a grant from Academia Sinica and the MOST of Taiwan (102-2320-B-001-004).
Compliance with ethical standards
All applicable international and institutional guidelines for the care and use of animals were followed. All procedures performed in this study involving animals were in accordance with the ethical standards of and approved by Academia Sinica Institutional Animal Care and Utilization Committee.
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