Published online before print October 25, 2001, doi: 10.1161/hh2301.100815
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© 2001 American Heart Association, Inc.
Cellular Biology |
Ca2+ Elevation Evoked by Membrane Depolarization Regulates G Protein Cycle via RGS Proteins in the Heart
From the Department of Pharmacology II (M.I., A.I., S.F., Y.M., Y.K.), Graduate School of Medicine, Osaka University, Suita, Osaka, and Department of Nursing (Y.H.), Yamagata School of Health Science, Yamagata, Japan.
Correspondence to Yoshihisa Kurachi, MD, PhD, Department of Pharmacology II, Graduate School of Medicine, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan. E-mail ykurachi{at}pharma2.med.osaka-u.ac.jp
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Abstract |
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Regulators of G protein signaling (RGS), which act as GTPase activators, are a family of cytosolic proteins emerging rapidly as an important means of controlling G protein-mediated cell signals. The importance of RGS action has been verified in vitro for various kinds of cell function. Their in situ modes of action in intact cells are, however, poorly understood. Here we show that an increase in intracellular Ca2+ evoked by membrane depolarization controls the RGS action on G protein activation of muscarinic K+ (KG) channel in the heart. Acetylcholine-induced KG current exhibits a slow time-dependent increase during hyperpolarizing voltage steps, referred to as "relaxation." This reflects the relief from the decrease in available KG channel number induced by cell depolarization. This phenomenon is abolished when an increase in intracellular Ca2+ is prevented. It is also abolished when a calmodulin inhibitor or a mutant RGS4 is applied that can bind to calmodulin but that does not accelerate GTPase activity. Therefore, an increase in intracellular Ca2+ and the resultant formation of Ca2+/calmodulin facilitate GTPase activity of RGS and thus decrease the available channel number on depolarization. These results indicate a novel and probably general pathway that Ca2+-dependent signaling regulates the G protein cycle via RGS proteins.
Key Words: G protein-activated K+ channel • regulators of G protein signaling • Ca2+ • calmodulin • cell excitation
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Introduction |
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G protein-gated inward rectifier K+ (KG) channels, which are directly activated by the ß
subunits released from pertussis toxin-sensitive G proteins (designated GK), contribute to acetylcholine (ACh)-induced deceleration of heartbeat and neurotransmitter-evoked slow inhibitory postsynaptic potentials in different neurons.1–3 The cardiac KG channel is a heterotetramer composed of two kinds of inward rectifier K+ channel (Kir) subunits, GIRK1/Kir3.1 and GIRK4/Kir3.4,4 which can be reconstituted by expressing the Kir subunits and m2-muscarinic receptors in Xenopus oocytes. The reconstituted current, however, lacks several of the characteristic features of native KG currents. One of these features is an agonist concentration-dependent slow increase at hyperpolarized potentials, which is referred to as "relaxation."3,5 Since its first description in sinoatrial node cells,5 the molecular mechanism underlying this characteristic feature of the KG current has remained an enigma.
Recently a family of cytosolic proteins that act as regulators of G protein signaling (RGS) has been identified.6,7 These proteins accelerate GTP hydrolysis on 
subunits of Gi/o and/or Gq, and are supposed to play essential roles in the negative regulation of various G protein-mediated cell-signaling systems. In reconstituted systems, RGS proteins have been reported to accelerate the time course of activation and deactivation of KG currents induced by agonists.8–10 We have shown that one RGS protein, RGS4, restores the feature of relaxation to the reconstituted KG current11 and that this effect was mediated exclusively by the interaction of RGS domain with pertussis toxin-sensitive G subunit.12 The question of how the cytosolic RGS protein confers this membrane potential-dependent feature to KG channels, however, remained a mystery.
In this report, using native cardiac atrial myocytes, we show for the first time that this characteristic can be imputable to the voltage-dependent behavior of RGS proteins probably caused by depolarization-induced formation of the Ca2+/calmodulin (CaM) complex. This result not only reveals the molecular mechanism of the relaxation of native KG current, but also provides us with a novel concept that intracellular Ca2+-dependent pathways dynamically modulate G protein signaling via RGS proteins.
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Materials and Methods |
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Preparation of Isolated Atrial Myocytes
Single atrial myocytes were enzymatically isolated from hearts removed from adult male Wistar-Kyoto rats as described elsewhere.13 Briefly, rats were deeply anesthetized by intraperitoneal injection of pentobarbital. A cannula was inserted into the aorta, and the heart was perfused in a retrograde manner through the coronary arteries. The heart was digested by collagenase (Boehringer Mannheim) in nominally Ca2+-free solution at 37°C for 10 minutes. Dissociated myocytes were seeded on glass coverslips (15 mm in diameter) that had been coated with poly-D-lysine (Sigma), kept in a humidified environment of 0.5% CO2 at 37°C, and fed with medium M199 (PAA Laboratories) containing gentamycin and kanamycin (25 mg/L each).
Electrophysiological Recordings
Whole-cell currents were measured at room temperature by a patch-clamp amplifier (Axon 200A, Axon Instruments) and recorded on videocassette tape with a PCM converter system (VR-10B, Instrutech). For analysis, data were reproduced, low-pass-filtered at 1 kHz (-3 dB) by an eight-pole Bessel filter, sampled at 5 kHz, and analyzed offline on a computer with commercially available software (Patch Analyst Pro, MT Corp). The control bathing solution contained the following (in mmol/L): 115 NaCl, 20 KCl, 1.8 CaCl2, 0.53 MgCl2, 5.5 glucose, and 5.5 HEPES-NaOH (pH 7.4). Ca2+-free bathing solution contained the following (in mmol/L): 115 NaCl, 20 KCl, 5 EGTA, 0.53 MgCl2, 5.5 glucose, and 5.5 HEPES-NaOH (pH 7.4). The pipette (internal) solution contained the following (in mmol/L): 150 KCl, 5 EGTA, 1 MgCl2, 3 K2ATP, 0.1 Na2GTP, and 5 HEPES-KOH (pH 7.3). In some experiments, BAPTA replaced EGTA in the pipette solution. The ACh-induced muscarinic K+ (KG) currents were obtained by digitally subtracting currents recorded under control conditions from those recorded in the presence of ACh. Rapid superfusion of cells was achieved using microcapillary and solution exchange instruments. In this system, alterations of solutions surrounding myocytes measured by perfusing high K+-bathing solution occurred within 10 ms. Results are shown as mean values obtained from n myocytes, and error bars represent SEM. Statistical differences were evaluated by the Student unpaired t test. Statistical probability of P<0.05 was taken as a significant difference.
In Vitro Binding Assay
Glutathione-S-transferase (GST)-fusioned RGS4 was prepared by previously described methods.11,12 CaM covalently attached to agarose beads (CaM-agarose) was purchased from Sigma. Binding assays of GST-RGS4 and CaM-agarose were basically performed as described by Popov et al.14 In brief, purified GST-RGS4 (wild-type and N128H mutant) and GST (0.2 nmol each) were each incubated with CaM-agarose beads (20 µg of CaM) at 4°C for 1 hour in a binding buffer (in mmol/L, 150 NaCl, 1 DTT, 10 HEPES-NaOH [pH 7.4], EGTA, and CaCl2). EGTA and CaCl2 were added to the binding buffer to adjust its free Ca2+ concentration calculated to be 
10 µmol/L or <0.5 nmol/L. CaM kinase II (290 to 309) CaM antagonist (Biomol Research Laboratories) was added to make a final concentration of 1 µmol/L. The beads were then extensively washed and incubated with SDS-PAGE loading buffer for 5 minutes at 95°C. The bound protein was separated by SDS-PAGE and visualized by Coomassie brilliant blue R-250 staining.
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Results |
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Slow Increase of KG Current at Hyperpolarized Potentials (Relaxation) Depends on Voltage-Dependent Ca2+ Influx Across the Membrane
Figure 1A illustrates a current induced by 10-7 mol/L of ACh in a rat atrial myocyte. When the membrane potential is clamped to +40 mV for 1 second, little outward current flows through KG channels. On hyperpolarization to -100 mV the inward KG current instantaneously jumps to one level (Iins) and then slowly increases to a steady level (Imax). The immediate increase in current reflects the rapid relief from the blockade of outward KG current by intracellular Mg2+ and/or polyamines, which is a general feature in all inward rectifier (Kir) K+ channels. The slow increase is a unique characteristic of the KG current and reflects a time-dependent release from inhibition of KG channel gating that is associated with depolarization. Figure 1B shows that with 10-7 mol/L ACh, modulation of the prepulse potential influenced the amplitude of Iins without affecting Imax, an effect that was essentially abolished with 10-6 mol/L ACh. The ratio Iins/Imax shows the amount of KG current available during each prepulse relative to that at -100 mV (Figure 1C). Thus in 10-7 mol/L ACh, KG channel availability was reduced with depolarization such that at +40 mV this was
30% of that at -100 mV (Figure 1C, open circles), whereas with 10-6 mol/L of ACh KG channel availability at +40 mV had been increased to 80% (Figure 1C, closed circles). These results indicate that at low concentrations of ACh the KG channels are inhibited on cell depolarization and that the slow increase in current (referred to as relaxation5) on hyperpolarization reflects relief from this inhibition. Increasing KG channel activity with 10-6 mol/L ACh overcomes this inhibition, and channel activity then becomes practically voltage independent.
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) or 10-6 mol/L (
) of ACh (n=10). D and E, KG current evoked by 10-7 mol/L of ACh when either extracellular free Ca2+ was removed by EGTA (D) or intracellular Ca2+ was chelated by BAPTA (E). F, Relationship between prepulse voltage and Iins/Imax ratio with intracellular BAPTA when currents were elicited by 10-7 mol/L (We have previously shown that RGS4 confers the feature of relaxation to the KG current reconstituted in Xenopus oocytes.11,12 But the mechanism remained unknown. It has been reported that RGS4 can bind to the Ca2+/CaM complex.14 This result led us to hypothesize that the Ca2+/CaM complex might be involved in the effect of RGS on relaxation of the KG channel. We tested the effect of Ca2+ on KG currents in two ways (Figure 1D). First we examined the effect of extracellular Ca2+ (Figure 1D). Compared with the control condition (Figure 1B), Iins of the KG current activated by 10-7 mol/L ACh was prominently increased in Ca2+-free bathing solution and the voltage dependence of the Iins/Imax ratio was similar in 10-7 mol/L and 10-6 mol/L ACh (not shown). Similar results were obtained when the pipette solution contained 5 mmol/L BAPTA, which is a faster Ca2+ chelator than EGTA15 (Figure 1E). The voltage dependence of the Iins/Imax ratio with 10-7 mol/L and 10-6 mol/L ACh measured with the BAPTA internal solution was also identical (Figure 1F) and corresponded to that recorded with 10-6 mol/L ACh under control conditions (Figure 1B). It would therefore seem that an increase in intracellular Ca2+ due to a membrane depolarization is associated with the reduction of the channel availability at depolarized potentials and thus causes the time-dependent increase in KG current at 10-7 mol/L ACh on hyperpolarization.
Effect of Intracellular BAPTA, a Rapid Ca2+-Chelating Agent, on Rapid Deactivation of Native KG Current
In the context of the interaction between KG currents and RGS, it is now widely accepted that the deactivation of ACh-induced KG current on washout of the agonist is accelerated by RGS proteins.8–10 Therefore we examined the effect of membrane potential and intracellular BAPTA on the deactivation (off) time course of the KG current (Figure 2). The membrane potential was held at either 0 or -80 mV, and 10-7 mol/L ACh was applied using a rapid perfusion system. In our assay system, T1/2 of activation (on) time courses was estimated as 400 to 800 ms, which was almost compatible with the values reported in previous measurements.16–18 Figure 2A shows that, under control conditions, whereas the on time course of the current induced by ACh was nearly the same at 0 and at -80 mV, the off time course was significantly faster at 0 than at -80 mV (Figures 2C and 2D). When BAPTA was included in the pipette solution, the off time course was significantly prolonged at both voltages (Figure 2B), although more so at 0 than at -80 mV, such that the difference between them was reduced (Figures 2B and 2D). These results may suggest that the action of intrinsic RGS proteins on deactivation of KG channel seems to be more augmented at 0 than at -80 mV and that the increase in intracellular Ca2+ may be involved in this voltage-dependent phenomenon. On the other hand, difference was not evident during the on time courses in this system (Figure 2C). The effect of extracellular application of EGTA on activation and deactivation time course of KG was also examined, and these time courses were shown to be much slower (T1/2 on=40±10 seconds, T1/2 off=35±7 seconds; not shown in Figure). These extremely slow time courses could be explained by the low binding affinity between receptor (m2R) and agonist (ACh) in the absence of extracellular Ca2+.19
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CaM Antagonist and Mutant RGS (N128H) Attenuate Both Rapid Deactivation and Relaxation of KG Current
We next examined the effects of a CaM antagonist and a mutant RGS4. Figure 3A shows the off time courses of ACh-induced KG current at 0 mV under different conditions. Under control conditions, the T1/2 of the off time course was 6 seconds. When a specific CaM antagonist, the peptide corresponding to residues 290 to 309 of CaM-dependent protein kinase II,20 was included in the pipette solution, the off time course was prolonged to 17 seconds. Next, GST-fusioned RGS4 proteins (wild-type and mutant/N128H) were added to the pipette solution and pushed into myocytes by pressure. The wild-type RGS4 slightly accelerated the off time course, although the difference was not statistically significant. In contrast, the mutant RGS4/N128H prominently prolonged it. CaM antagonist and the mutant RGS4 also increased the Iins and reduced the time-dependent component of KG current (Figure 3B), whereas the wild-type RGS4 was without effect. These results suggest that the acceleration of deactivation and relaxation of KG channels may share a common molecular mechanism. Because neither rapid deactivation nor relaxation was affected by either 10 µmol/L of KN-93 (a blocker of CaM-dependent protein kinase) or 1 µmol/L of cyclosporin A (a blocker of calcineurin, a CaM-dependent protein phosphatase) (not shown), it was suggested that the major downstream effectors of Ca2+/CaM were not involved in these phenomena.
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In Figure 3C, we examined the binding properties of wild-type and mutant RGS4 proteins to CaM. Both proteins could bind to CaM in the presence of Ca2+ but not in the absence of Ca2+. The binding was inhibited by the CaM antagonist. Recent structural analyses of RGS4 and Gi121 indicate that residue N128 of RGS4 is located facing the switch II region of Gi1 and plays a crucial role in facilitating GTP hydrolysis,22 and the substitution of N128 into histidine completely abolishes its function.23 Thus, the mutant RGS4 may compete with intrinsic RGS proteins in binding to the Ca2+/CaM complex but cannot accelerate the GTPase activity of subunit of GK protein. Therefore, it is expected that the acceleration by intrinsic RGS proteins of GTP hydrolysis on GK is directly disturbed by the injected mutant RGS4 in a competitive manner.
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Discussion |
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In this study, we showed that depolarization-induced decrease in available KG channel number, which caused relaxation, depended on extracellular Ca2+ (blocked by extracellular EGTA), an increase in intracellular Ca2+ (blocked by intracellular BAPTA), formation of Ca2+/CaM complex (blocked by a CaM antagonist), and RGS action on accelerating GTP hydrolysis of GK
subunit (blocked by a mutant RGS4). It is widely accepted that the deactivation time course of KG current on washout of agonist reflects the GTPase activity of GK and is accelerated by RGS proteins.8–10 We showed that the deactivation was faster at 0 than at -80 mV, and that the rapid deactivation was also disturbed by intracellular BAPTA, the CaM antagonist, and the mutant RGS4. These results indicate that depolarization-induced decrease in available KG channel number is mainly due to GTPase-accelerating action of intrinsic RGS proteins at depolarized potentials, and intracellular Ca2+ elevation and formation of Ca2+/CaM are required for this RGS action. In the preliminary experiments, we observed that neither the internal solution containing BAPTA (5 mmol/L) nor that containing Ca2+/CaM (100 nmol/L) affected GTP, GTPS, or Gß activation of KG channels in the inside-out patches of atrial cell membrane. These data show that lowering Ca2+ or Ca2+/CaM does not directly modulate G protein activation of the KG channel. We also observed that inhibitors of CaM-dependent protein kinase and calcineurin did not affect the relaxation of the KG current in the whole-cell recording, which may suggest that phosphorylation or dephosphorylation of the KG channel is not involved in the phenomenon. There is, however, still one of two possibilities for the action of Ca2+/CaM, as follows: (1) Ca2+/CaM somehow activates RGS, which causes the acceleration of the GK protein cycle, or (2) Ca2+/CaM modulates the GK protein cycle to be more receptive to interaction with RGS. Because Ca2+/CaM can bind to RGS4 proteins in vitro14 (Figure 3C), we think the former is more likely, although we cannot completely exclude the latter possibility. On the basis of these possibilities, we propose the following signaling cascade in formation of relaxation gating of cardiac KG channel (Figure 4).
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When membrane potential is depolarized, intracellular Ca2+ beneath the plasma membrane of cardiac myocytes increases. Ca2+ binds to CaM and Ca2+/CaM complex leads to the facilitation of RGS action to accelerate hydrolysis of GK-GTP to GK-GDP. GK-GDP binds free Gß to form trimeric G proteins, which therefore decreases the number of free Gß and thus the number of available KG channels at depolarized potentials. The time-dependent increase in KG current on hyperpolarization can then be interpreted as the reflection of the reverse reactions of these events, ie, less Ca2+ influx, less Ca2+/CaM, lesser activity of RGS, and more available Gß.
There still remain, however, two major questions in this reaction scheme. The first one is how Ca2+/CaM facilitates RGS action. It was reported that Ca2+/CaM binds RGS but does not change its GTPase accelerating activity in vitro.14 Therefore, some mechanisms other than direct facilitating action of Ca2+/CaM on the GTPase-accelerating function of RGS should be involved. One possibility is that Ca2+/CaM might cause rapid translocation of RGS proteins to the plasma membranes.24 It might also be the case that some unidentified factor(s) may be needed for Ca2+/CaM to facilitate the GTPase-accelerating action of RGS proteins. Further studies are needed to examine these possibilities. This will lead us to reach the final conclusion on the role of Ca2+/CaM in the formation of apparent voltage-dependent behavior of RGS action.
The second remaining question is how membrane depolarization causes Ca2+ elevation beneath the cell membrane. Because removal of Ca2+ from the extracellular solution abolished the relaxation, one of the major mechanisms may be depolarization-induced Ca2+ influx across the cell membrane. The depolarization-induced Ca2+ influx in cardiac cell membrane is carried mainly through the voltage-gated (L-type) Ca2+ channel and/or the reverse mode of Na+/Ca2+ exchanger. The voltage-gated Ca2+ channel, however, cannot be mainly responsible for the Ca2+ influx, because (1) the KG channel availability decreased monotonically as the membrane was depolarized and did not show such a bell-shaped voltage dependence that is observed in the current flowing through L-type Ca2+ channel, and (2) nifedipine (100 nmol/L to 1 µmol/L) did not significantly affect the relaxation property, although it inhibited channel current amplitude directly (not shown). The Na+/Ca2+ exchanger may also not be mainly responsible, because (1) the relaxation was detected in nominally Na+-free pipette solution and a decrease in extracellular Na+ did not affect the relaxation property, and (2) KB-R794325 (a blocker of the reverse mode of Na+/Ca2+ exchanger) did not significantly affect the relaxation property (not shown). We may therefore need to consider the possibility that some other mechanisms mediate the depolarization-induced Ca2+ elevation beneath the cell membrane for control of RGS action. The candidates might include TRP-family Ca2+-permeable channels showing voltage-dependent gating property.26
In addition to depolarization-induced Ca2+ elevation, it should be noted that the basal level of Ca2+ seems to play a significant role in controlling RGS action. As depicted in Figure 2, even at -80 mV the deactivation (off) time course of KG current became slower with the BAPTA-containing pipette solution than with the EGTA-containing (control) one, although the effect was not prominent compared with that at 0 mV. This suggests that even a very small amount of basal Ca2+ at the restricted domain probably just beneath the sarcolemma may contribute to the control of RGS action. Because neither voltage-dependent Ca2+ channels nor Na+/Ca2+ exchanger is supposed to carry significant Ca2+ influx across the cell membrane at -80 mV, a leak Ca2+ influx through, eg, nonselective cation channels27,28 and/or a basal Ca2+ release from internal pool might be involved in maintaining the basal Ca2+ level at the restricted domain. The mechanisms for Ca2+ increase on depolarization and those for control of basal Ca2+ level are not necessarily the same. Further studies are needed to clarify both mechanisms.
Nevertheless, this study in native cardiac myocytes provides the answer for the long-sought-after molecular mechanism underlying the characteristic voltage-dependent relaxation gating of KG channels. Also we show, in situ, the mode of action of negative regulators of the G protein cycle, which unexpectedly provides us with a novel principle, ie, that the cell signaling through Ca2+-dependent pathways can dynamically regulate G protein signaling via cytosolic RGS proteins. In this context, the KG channel can be regarded as an example of a G protein effector molecule through which we can detect the G protein cycle in real time with high temporal resolution. Therefore, the principle proposed here should not be limited only to the KG channel system but must also be applicable to other G protein signaling machinery controlling such target proteins as neuronal (N- and P/Q-type) Ca2+ channels, phospholipase C, and adenylyl cyclases. Adenylyl cyclases are often subject to dual regulation by stimulatory Gs (RGS-insensitive) and inhibitory Gi/o (RGS-sensitive) proteins,29,30 where Ca2+ elevation may then suppress exclusively Gi/o and result in a dynamic regulation of the stimulatory signal via Gs. To elucidate the relevance and significance of this novel principle, however, further studies are needed.
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Acknowledgments |
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This work was supported by a Grant-in-Aid for Specific Research on Priority Area B (12144207) (to Y.K.); by a Grant-in-Aid for Encouragement of Young Scientists (13770044) (to M.I.) from the Ministry of Education, Science, Sports and Culture of Japan; by a Grant-in-Aid from the Research for the Future Program of the Japan Society for the Promotion of Science (96L00302) (to Y.K.); and by a Grant-in-Aid from the Ueda Memorial Trust Fund for Research of Heart Diseases (to S.F.). We thank Dr Ian Findlay (Université de Tours, France) for critically reading this manuscript.
Received August 2, 2001; revision received October 15, 2001; accepted October 15, 2001.
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 P. Tosetti, N. Pathak, M. H. Jacob, and K. Dunlap RGS3 mediates a calcium-dependent termination of G protein signaling in sensory neurons PNAS, June 10, 2003; 100(12): 7337 - 7342. [Abstract] [Full Text] [PDF]
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 S. Hollinger and J. R. Hepler Cellular Regulation of RGS Proteins: Modulators and Integrators of G Protein Signaling Pharmacol. Rev., September 1, 2002; 54(3): 527 - 559. [Abstract] [Full Text] [PDF]
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M. Ishii, A. Inanobe, and Y. Kurachi PIP3 inhibition of RGS protein and its reversal by Ca2+/calmodulin mediate voltage-dependent control of the G protein cycle in a cardiac K+ channel PNAS, April 2, 2002; 99(7): 4325 - 4330. [Abstract] [Full Text] [PDF]
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M. Ishii, A. Inanobe, and Y. Kurachi PIP3 inhibition of RGS protein and its reversal by Ca2+/calmodulin mediate voltage-dependent control of the G protein cycle in a cardiac K+ channel PNAS, April 2, 2002; 99(7): 4325 - 4330. [Abstract] [Full Text] [PDF]
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