Ion-channel regulation by G proteins.

Ion channels are end-targets (effectors) in a large number of regulatory pathways that are initiated by G-protein-coupled neurotransmitters and hormones. Modulation of ion channels by G proteins can be indirect (via second messengers and protein kinases) or direct, via physical interactions between G-protein subunits and the channel protein. These direct physical interactions are the focus of this review. A direct regulation has been firmly established for several voltage-dependent Ca(2+) channels and the G-protein-activated K(+) channels. In these ion-channel families, the G-protein beta gamma subunits (G beta gamma) are the active regulators, whereas the role of the alpha subunits (G alpha) remains poorly understood. Accumulating evidence suggests that intricate relationships between the receptor, G alpha, G beta gamma and the ion channel play a major role in determining the specificity and magnitude of the overall regulation.

Expression levels of RGS7 and RGS4 proteins determine the mode of regulation of the G protein-activated K(+) channel and control regulation of RGS7 by G beta 5.

Regulators of G protein signaling RGS4 and RGS7 accelerate the kinetics of K(+) channels (GIRKs) in the Xenopus oocyte system. Here, via quantitative analysis of RGS expression, we reveal biphasic effects of RGSs on GIRK regulation. At low concentrations, RGS4 inhibited basal GIRK activity, but stimulated it at high concentrations. RGS7, which is associated with the G protein subunit G beta 5, is regulated by G beta 5 by two distinct mechanisms. First, G beta 5 augments RGS7 activity, and second, it increases its expression. These dual effects resolve previous controversies regarding RGS4 and RGS7 function and indicate that they modulate signaling by mechanisms supplementary to their GTPase-activating protein activity.

Voltage clamp recordings from Xenopus oocytes.

Xenopus oocytes serve as a standard heterologous expression system for the study of cloned ion channels. The large size of these cells allows for relatively easy expression and recording of activity of exogenous ion channels (together with neurotransmitter receptors and/or various regulatory proteins) using the whole-cell two-electrode voltage clamp (TEVC) technique, as well as standard single-channel patch clamp recordings. Although usually advantageous, the cell size also dictates certain limits on the accuracy of recordings and requires specific modifications of recording methods. However, combining the advantages of the system with available recording methods enables the use of Xenopus oocytes for sophisticated multidisciplinary studies of ion channels.

Modulation of L-type Ca2+ channels by gbeta gamma and calmodulin via interactions with N and C termini of alpha 1C.

Neuronal voltage-dependent Ca(2+) channels of the N (alpha(1B)) and P/Q (alpha(1A)) type are inhibited by neurotransmitters that activate G(i/o) G proteins; a major part of the inhibition is voltage-dependent, relieved by depolarization, and results from a direct binding of Gbetagamma subunit of G proteins to the channel. Since cardiac and neuronal L-type (alpha(1C)) voltage-dependent Ca(2+) channels are not modulated in this way, they are presumed to lack interaction with Gbetagamma. However, here we demonstrate that both Gbetagamma and calmodulin directly bind to cytosolic N and C termini of the alpha(1C) subunit. Coexpression of Gbetagamma reduces the current via the L-type channels. The inhibition depends on the presence of calmodulin, occurs at basal cellular levels of Ca(2+), and is eliminated by EGTA. The N and C termini of alpha(1C) appear to serve as partially independent but interacting inhibitory gates. Deletion of the N terminus or of the distal half of the C terminus eliminates the inhibitory effect of Gbetagamma. Deletion of the N terminus profoundly impairs the Ca(2+)/calmodulin-dependent inactivation. We propose that Gbetagamma and calmodulin regulate the L-type Ca(2+) channel in a concerted manner via a molecular inhibitory scaffold formed by N and C termini of alpha(1C).

Imaging plasma membrane proteins in large membrane patches of Xenopus oocytes.

We describe the preparation of a Xenopus oocyte plasma membrane patch attached to a cover-slip with its intracellular face exposed to the bath solution. The proteins attached to the plasma membrane were visualized by confocal microscopy after fluorescence labelling. Since cortical microfilament elements were detected in these plasma membrane preparations we termed the patches plasma membrane-cortex patches. The way these patches are formed and the low concentration of proteins needed for cytochemical detection make the membrane-cortex patches similar to electrophysiological membrane patches and therefore allow the cytochemical study of ion channels to be correlated with electrophysiological experiments. Furthermore, the described patch is similar to manually isolated plasma membranes used for biochemical analysis by sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Cytochemical analysis of membrane-cortex patches also enables the detection of the two-dimensional pattern of organization of membrane proteins (clustered or non-clustered forms). In addition, patch preparations enable cytochemical study of the relative localization of membrane proteins. The methodology enables integration of electrophysiological, biochemical and cytochemical studies of ion channels, giving a comprehensive perspective on ion channel function.

Expression cloning of KCRF, a potassium channel regulatory factor.

By functional coexpression screening of a rat cDNA library in Xenopus oocytes, we have cloned a protein (KCRF: K Channel Regulatory Factor) that reduces currents of several K(+) channels: G protein-activated GIRK1/4 (K(ir)3.1/K(ir)3.4), inward rectifier IRK1 (K(ir)2.1), and voltage-dependent K(V)1.1/K(V)beta1.1. KCRF did not modulate two other K(+) channels: ROMK1 (K(ir)1.1) and GIRK1/2 (K(ir)3.1/K(ir)3.2) and the voltage-dependent L-type Ca(2+) channels. Western blot analysis showed that KCRF is ubiquitous in rat tissues. Biochemical and electrophysiological experiments revealed that coexpression of KCRF causes a decrease in the level of expression of IRK1 and K(V)1.1/K(V)beta1.1 proteins in the oocytes.

Slow modal gating of single G protein-activated K+ channels expressed in Xenopus oocytes.

The slow kinetics of G protein-activated K+ (GIRK) channels expressed in Xenopus oocytes were studied in single-channel, inside-out membrane patches. Channels formed by GIRK1 plus GIRK4 subunits, which are known to form the cardiac acetylcholine (ACh)-activated GIRK channel (KACh), were activated by a near-saturating dose of G protein betagamma subunits (Gbetagamma; 20 nM). The kinetic parameters of the expressed GIRK1/4 channels were similar to those of cardiac KACh. GIRK1/4 channels differed significantly from channels formed by GIRK1 with the endogenous oocyte subunit GIRK5 (GIRK1/5) in some of their kinetic parameters and in a 3-fold lower open probability, Po. The unexpectedly low Po (0.025) of GIRK1/4 was due to the presence of closures of hundreds of milliseconds; the channel spent approximately 90 % of the time in the long closed states. GIRK1/4 channels displayed a clear modal behaviour: on a time scale of tens of seconds, the Gbetagamma-activated channels cycled between a low-Po mode (Po of about 0.0034) and a bursting mode characterized by an approximately 30-fold higher Po and a different set of kinetic constants (and, therefore, a different set of channel conformations). The available evidence indicates that the slow modal transitions are not driven by binding and unbinding of Gbetagamma. The GTPgammaS-activated Galphai1 subunit, previously shown to inhibit GIRK channels, substantially increased the time spent in closed states and apparently shifted the channel to a mode similar, but not identical, to the low-Po mode. This is the first demonstration of slow modal transitions in GIRK channels. The detailed description of the slow gating kinetics of GIRK1/4 may help in future analysis of mechanisms of GIRK gating.

Heterologous facilitation of G protein-activated K(+) channels by beta-adrenergic stimulation via cAMP-dependent protein kinase.

To investigate possible effects of adrenergic stimulation on G protein-activated inwardly rectifying K(+) channels (GIRK), acetylcholine (ACh)-evoked K(+) current, I(KACh), was recorded from adult rat atrial cardiomyocytes using the whole cell patch clamp method and a fast perfusion system. The rise time of I(KACh ) was 0. 4 +/- 0.1 s. When isoproterenol (Iso) was applied simultaneously with ACh, an additional slow component (11.4 +/- 3.0 s) appeared, and the amplitude of the elicited I(KACh) was increased by 22.9 +/- 5.4%. Both the slow component of activation and the current increase caused by Iso were abolished by preincubation in 50 microM H89 (N-[2-((p -bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, a potent inhibitor of PKA). This heterologous facilitation of GIRK current by beta-adrenergic stimulation was further studied in Xenopus laevis oocytes coexpressing beta(2)-adrenergic receptors, m(2 )-receptors, and GIRK1/GIRK4 subunits. Both Iso and ACh elicited GIRK currents in these oocytes. Furthermore, Iso facilitated ACh currents in a way, similar to atrial cells. Cytosolic injection of 30-60 pmol cAMP, but not of Rp-cAMPS (a cAMP analogue that is inhibitory to PKA) mimicked the beta(2)-adrenergic effect. The possibility that the potentiation of GIRK currents was a result of the phosphorylation of the beta-adrenergic receptor (beta(2)AR) by PKA was excluded by using a mutant beta(2)AR in which the residues for PKA-mediated modulation were mutated. Overexpression of the alpha subunit of G proteins (Galpha(s)) led to an increase in basal as well as agonist-induced GIRK1/GIRK4 currents (inhibited by H89). At higher levels of expressed Galpha(s), GIRK currents were inhibited, presumably due to sequestration of the beta/gamma subunit dimer of G protein. GIRK1/GIRK5, GIRK1/GIRK2, and homomeric GIRK2 channels were also regulated by cAMP injections. Mutant GIRK1/GIRK4 channels in which the 40 COOH-terminal amino acids (which contain a strong PKA phosphorylation consensus site) were deleted were also modulated by cAMP injections. Hence, the structural determinant responsible is not located within this region. We conclude that, both in atrial myocytes and in Xenopus oocytes, beta-adrenergic stimulation potentiates the ACh-evoked GIRK channels via a pathway that involves PKA-catalyzed phosphorylation downstream from beta(2)AR.

Coupling of the muscarinic m2 receptor to G protein-activated K(+) channels via Galpha(z) and a receptor-Galpha(z) fusion protein. Fusion between the receptor and Galpha(z) eliminates catalytic (collision) coupling.

G protein-activated K(+) channel (GIRK), which is activated by the G(betagamma) subunit of heterotrimeric G proteins, and muscarinic m2 receptor (m2R) were coexpressed in Xenopus oocytes. Acetylcholine evoked a K(+) current, I(ACh), via the endogenous pertussis toxin (PTX)-sensitive G(i/o) proteins. Activation of I(ACh) was accelerated by increasing the expression of m2R, suggesting a collision coupling mechanism in which one receptor catalytically activates several G proteins. Coexpression of the alpha subunit of the PTX-insensitive G protein G(z), Galpha(z), induced a slowly activating PTX-insensitive I(ACh), whose activation kinetics were also compatible with the collision coupling mechanism. When GIRK was coexpressed with an m2R x Galpha(z) fusion protein (tandem), in which the C terminus of m2R was tethered to the N terminus of Galpha(z), part of I(ACh) was still eliminated by PTX. Thus, the m2R of the tandem activates the tethered Galpha(z) but also the nontethered G(i/o) proteins. After PTX treatment, the speed of activation of the m2R x Galpha(z)-mediated response did not depend on the expression level of m2R x Galpha(z) and was faster than when m2R and Galpha(z) were coexpressed as separate proteins. These results demonstrate that fusing the receptor and the Galpha strengthens their coupling, support the collision-coupling mechanism between m2R and the G proteins, and suggest a noncatalytic (stoichiometric) coupling between the G protein and GIRK in this model system.

The N terminus of the cardiac L-type Ca(2+) channel alpha(1C) subunit. The initial segment is ubiquitous and crucial for protein kinase C modulation, but is not directly phosphorylated.

The first 46 amino acids (aa) of the N terminus of the rabbit heart (RH) L-type cardiac Ca(2+) channel alpha(1C) subunit are crucial for the stimulating action of protein kinase C (PKC) and also hinder channel gating (Shistik, E., Ivanina, T., Blumenstein, Y., and Dascal, N. (1998) J. Biol. Chem. 273, 17901-17909). The mechanism of PKC action and the location of the PKC target site are not known. Moreover, uncertainties in the genomic sequence of the N-terminal region of alpha(1C) leave open the question of the presence of RH-type N terminus in L-type channels in mammalian tissues. Here, we demonstrate the presence of alpha(1C) protein containing an RH-type initial N-terminal segment in rat heart and brain by using a newly prepared polyclonal antibody. Using deletion mutants of alpha(1C) expressed in Xenopus oocytes, we further narrowed down the part of the N terminus crucial for both inhibitory gating and for PKC effect to the first 20 amino acid residues, and we identify the first 5 aa as an important determinant of PKC action and of N-terminal effect on gating. The absence of serines and threonines in the first 5 aa and the absence of phosphorylation by PKC of a glutathione S-transferase-fusion protein containing the initial segment suggest that the effect of PKC does not arise through a direct phosphorylation of this segment. We propose that PKC acts by attenuating the inhibitory action of the N terminus via phosphorylation of a remote site, in the channel or in an auxiliary protein, that interacts with the initial segment of the N terminus.

Regulation of cardiac L-type Ca2+ channel by coexpression of G(alpha s) in Xenopus oocytes.

Activation of G(alpha s) via beta-adrenergic receptors enhances the activity of cardiac voltage-dependent Ca2+ channels of the L-type, mainly via protein kinase A (PKA)-dependent phosphorylation. Contribution of a PKA-independent effect of G(alpha s) has been proposed but remains controversial. We demonstrate that, in Xenopus oocytes, antisense knockdown of endogenous G(alpha s) reduced, whereas coexpression of G(alpha s) enhanced, currents via expressed cardiac L-type channels, independently of the presence of the auxiliary subunits alpha2/delta or beta2A. Coexpression of G(alpha s) did not increase the amount of alpha1C protein in whole oocytes or in the plasma membrane (measured immunochemically). Activation of coexpressed beta2 adrenergic receptors did not cause a detectable enhancement of channel activity; rather, a small cAMP-dependent decrease was observed. We conclude that coexpression of G(alpha s), but not its acute activation via beta-adrenergic receptors, enhances the activity of the cardiac L-type Ca2+ channel via a PKA-independent effect on the alpha1C subunit.

Modal behavior of the Kv1.1 channel conferred by the Kvbeta1.1 subunit and its regulation by dephosphorylation of Kv1.1.

Modulation of fast-inactivating voltage-gated K+ channels can produce plastic changes in neuronal signaling. Previously, we showed that the voltage-dependent K+ channel composed of brain Kv1.1 and Kvbeta1.1 subunits (alpha(beta) channel) gives rise to a current that has a fast-inactivating and a sustained component; the proportion of the fast-inactivating component could be decreased by dephosphorylation of a basally phosphorylated Ser-446 on the alpha subunit. To account for our results we suggested a model that assumes a bimodal gating of the alpha(beta) channel. In this study, using single-channel analysis, we confirm this model. Two modes of gating were identified: (1) an inactivating mode characterized by low open probability and single openings early in the voltage step, and (2) a non-inactivating gating mode with bursts of openings. These two modes were non-randomly distributed, with spontaneous shifts between them. Each mode is characterized by a different set of open time constants (tau) and mean open times (t(0)). The non-inactivating mode is similar to the gating mode of a homomultimeric alpha channel. The phosphorylation-deficient alphaS446Abeta channel has the same two gating modes. Furthermore, alkaline phosphatase promoted the transition to the non-inactivating mode. This is the first report of modal behavior of a fast-inactivating K+ channel; furthermore, it substantiates the notion that direct phosphorylation is one mechanism that regulates the equilibrium between the two modes and thereby regulates the extent of macroscopic fast inactivation of a brain K+ channel.

Crucial role of N terminus in function of cardiac L-type Ca2+ channel and its modulation by protein kinase C.

The role of the cytosolic N terminus of the main subunit (alpha1C) of cardiac L-type voltage-dependent Ca2+ channel was studied in Xenopus oocyte expression system. Deletion of the initial 46 or 139 amino acids (a.a.) of rabbit heart alpha1C caused a 5-10-fold increase in the whole cell Ca2+ channel current carried by Ba2+ (IBa), as reported previously (Wei, X., Neely, A., Olcese, R., Lang, W., Stefani, E., and Birnbaumer, L. (1996) Recept. Channels 4, 205-215). The plasma membrane content of alpha1C protein, measured immunochemically, was not altered by the 46-a.a. deletion. Patch clamp recordings in the presence of a dihydropyridine agonist showed that this deletion causes a approximately 10-fold increase in single channel open probability without changing channel density. Thus, the initial segment of the N terminus affects channel gating rather than expression. The increase in IBa caused by coexpression of the auxiliary beta2A subunit was substantially stronger in channels with full-length alpha1C than in 46- or 139-a.a. truncated mutants, suggesting an interaction between beta2A and N terminus. However, only the I-II domain linker of alpha1C, but not to N or C termini, bound beta2A in vitro. The well documented increase of IBa caused by activation of protein kinase C (PKC) was fully eliminated by the 46-a.a. deletion. Thus, the N terminus of alpha1C plays a crucial role in channel gating and PKC modulation. We propose that PKC and beta subunit enhance the activity of the channel in part by relieving an inhibitory control exerted by the N terminus. Since PKC up-regulation of L-type Ca2+ channels has been reported in many species, we predict that isoforms of alpha1C subunits containing the initial N-terminal 46 a.a. similar to those of the rabbit heart alpha1C are widespread in cardiac and smooth muscle cells.

Agonist-independent inactivation and agonist-induced desensitization of the G protein-activated K+ channel (GIRK) in Xenopus oocytes.

The G-protein-activated K+ channels of the GIRK (Kir 3) family are activated by Gbetagamma subunits of heterotrimeric Gi/Go proteins. Atrial GIRK currents evoked by acetylcholine (ACh)1 via muscarinic m2 receptors (m2R) display prominent desensitization. We studied desensitization of basal and ACh-evoked whole-cell GIRK currents in Xenopus oocytes. In the absence of receptor and/or agonist, the basal GIRK activity showed inactivation which was prominent when the preparation was bathed in a low-Na+, high-K+ extracellular solution (96 mM [K+]out and 2 mM [Na+]out) but did not occur in a normal physiological solution. Ion substitution experiments showed that this basal, agonist-independent inactivation was caused by the decrease in [Na+]out rather than by the increased [K+]out. We hypothesize that it reflects a depletion of intracellular Na+. ACh-evoked GIRK currents desensitized faster than the basal ones. The agonist-induced desensitization was present when the preparation was bathed in all solutions tested, independently of [Na+]out. A protein kinase C (PKC) activator inhibited the GIRK currents both in high and low [Na+]out, apparently mimicking agonist-induced desensitization; however, a potent serine/threonine protein kinase blocker, staurosporine, blocked only a minor part of desensitization. We conclude that basal inactivation and agonist-induced desensitization are separate processes, the former caused by changes in Na+ concentrations, and the latter by unknown factor(s) with only a minor contribution of PKC.

A C-terminal peptide of the GIRK1 subunit directly blocks the G protein-activated K+ channel (GIRK) expressed in Xenopus oocytes.

1. In order to find out the functional roles of cytosolic regions of a G protein-activated, inwardly rectifying potassium channel subunit we studied block of GIRK channels, expressed in Xenopus laevis oocytes, by synthetic peptides in isolated inside-out membrane patches. 2. A peptide (DS6) derived from the very end of the C-terminus of GIRK1 reversibly blocked GIRK activity with IC50 values of 7.9 +/- 2.0 or 3.5 +/- 0.5 micrograms ml-1 (corresponding to 3.7 +/- 0.9 or 1.7 +/- 0.2 mumol l-1) for GIRK1/GIRK5 or GIRK1/GIRK4 channels, respectively. 3. Dose dependency studies of GIRK activation by purified beta gamma subunits of the G protein (G beta gamma) showed that DS6 block of GIRK channels is not the result of competition of the peptide with functional GIRK channels for the available G beta gamma. 4. Burst duration of GIRK channels was reduced, whereas long closed times between bursts were markedly increased, accounting for the channel block observed. 5. Block by the DS6 peptide was slightly voltage dependent, being stronger at more negative potentials. 6. These data support the hypothesis that the distal part of the carboxy-terminus of GIRK1 is a part of the intrinsic gate that keeps GIRK channels closed in the absence of G beta gamma.

cAMP-dependent regulation of cardiac L-type Ca2+ channels requires membrane targeting of PKA and phosphorylation of channel subunits.

The cardiac L-type Ca2+ channel is a textbook example of an ion channel regulated by protein phosphorylation; however, the molecular events that underlie its regulation remain unknown. Here, we report that in transiently transfected HEK293 cells expressing L-type channels, elevations in cAMP resulted in phosphorylation of the alpha1C and beta2a channel subunits and increases in channel activity. Channel phosphorylation and regulation were facilitated by submembrane targeting of protein kinase A (PKA), through association with an A-kinase anchoring protein called AKAP79. In transfected cells expressing a mutant AKAP79 that is unable to bind PKA, phosphorylation of the alpha1C subunit and regulation of channel activity were not observed. Furthermore, we have demonstrated that the association of an AKAP with PKA was required for beta-adrenergic receptor-mediated regulation of L-type channels in native cardiac myocytes, illustrating that the events observed in the heterologous expression system reflect those occurring in the native system. Mutation of Ser1928 to alanine in the C-terminus of the alpha1C subunit resulted in a complete loss of cAMP-mediated phosphorylation and a loss of channel regulation. Thus, the PKA-mediated regulation of L-type Ca2+ channels is critically dependent on a functional AKAP and phosphorylation of the alpha1C subunit at Ser1928.

Positive and negative coupling of the metabotropic glutamate receptors to a G protein-activated K+ channel, GIRK, in Xenopus oocytes.

Metabotropic glutamate receptors (mGluRs) control intracellular signaling cascades through activation of G proteins. The inwardly rectifying K+ channel, GIRK, is activated by the beta gamma subunits of G proteins and is widely expressed in the brain. We investigated whether an interaction between mGluRs and GIRK is possible, using Xenopus oocytes expressing mGluRs and a cardiac/brain subunit of GIRK, GIRK1, with or without another brain subunit, GIRK2. mGluRs known to inhibit adenylyl cyclase (types 2, 3, 4, 6, and 7) activated the GIRK channel. The strongest response was observed with mGluR2; it was inhibited by pertussis toxin (PTX). This is consistent with the activation of GIRK by Gi/Go-coupled receptors. In contrast, mGluR1a and mGluR5 receptors known to activate phospholipase C, presumably via G proteins of the Gq class, inhibited the channel's activity. The inhibition was preceded by an initial weak activation, which was more prominent at higher levels of mGluR1a expression. The inhibition of GIRK activity by mGluR1a was suppressed by a broad-specificity protein kinase inhibitor, staurosporine, and by a specific protein kinase C (PKC) inhibitor, bis-indolylmaleimide, but not by PTX, Ca(2-)chelation, or calphostin C. Thus, mGluR1a inhibits the GIRK channel primarily via a pathway involving activation of a PTX-insensitive G protein and, eventually, of a subtype of PKC, possibly PKC-mu. In contrast, the initial activation of GIRK1 caused by mGluR1a was suppressed by PTX but not by the protein kinase inhibitors. Thus, this activation probably results from a promiscuous coupling of mGluR1a to a Gi/Go protein. The observed modulations may be involved in the mGluRs effects on neuronal excitability in the brain. Inhibition of GIRK by phospholipase C-activating mGluRs bears upon the problem of specificity of G protein (GIRK interaction) helping to explain why receptors coupled to Gq are inefficient in activating GIRK.

Signalling via the G protein-activated K+ channels.

The inwardly rectifying K+ channels of the GIRK (Kir3) family, members of the superfamily of inwardly rectifying K+ channels (Kir), are important physiological tools to regulate excitability in heart and brain by neurotransmitters, and the only ion channels conclusively shown to be activated by a direct interaction with heterotrimeric G protein subunits. During the last decade, especially since their cloning in 1993, remarkable progress has been made in understanding the structure, mechanisms of gating, activation by G proteins, and modulation of these channels. However, much of the molecular details of structure and of gating by G protein subunits and other factors, mechanisms of modulation and desensitization, and determinants of specificity of coupling to G proteins, remain unknown. This review summarizes both the recent advances and the unresolved questions now on the agenda in GIRK studies.

Inhibition of an inwardly rectifying K+ channel by G-protein alpha-subunits.

Cholinergic muscarinic, serotonergic, opioid and several other G-protein-coupled neurotransmitter receptors activate inwardly rectifying K+ channels of the GIRK family, slowing the heartbeat and decreasing the excitability of neuronal cells. Inhibitory modulation of GIRKs by G-protein-coupled receptors may have important implications in cardiac and brain physiology. Previously G alpha and G beta gamma subunits of heterotrimeric G proteins have both been implicated in channel opening, but recent studies attribute this role primarily to the G beta gamma dimer that activates GIRKs in a membrane-delimited fashion, probably by direct binding to the channel protein. We report here that free GTP gamma S-activated G alpha i 1, but not G alpha i 2 or G alpha i 3, potently inhibits G beta 1 gamma 2-induced GIRK activity in excised membrane patches of Xenopus oocytes expressing GIRK1. High-affinity but partial inhibition is produced by G alpha s-GTP gamma S. G alpha i 1-GTP gamma S also inhibits G beta 1 gamma 2-activated GIRK in atrial myocytes. Antagonistic interactions between G alpha and G beta gamma may be among the mechanisms determining specificity of G protein coupling to GIRKs.

A potential site of functional modulation by protein kinase A in the cardiac Ca2+ channel alpha 1C subunit.

The well-characterized enhancement of the cardiac Ca2+ L-type current by protein kinase A (PKA) is not observed when the corresponding channel is expressed in Xenopus oocytes, possibly because it is fully phosphorylated in the basal state. However, the activity of the expressed channel is reduced by PKA inhibitors. Using this paradigm as an assay to search for PKA sites relevant to channel modulation, we have found that mutation of serine 1928 of the alpha 1C subunit to alanine abolishes the modulation of the expressed channel by PKA inhibitors. This effect was independent of the presence of the beta subunit. Phosphorylation of serine 1928 of alpha 1C may mediate the modulatory effect of PKA on the cardiac voltage-dependent ca2+ channel.