Molecular mechanisms mediating inhibition of G protein-coupled inwardly-rectifying K+ channels

Neuronal G protein-coupled inwardly-rectifying potassium channels (GIRKs, Kir3.x) can be activated or inhibited by distinct classes of receptors (Galphai/o and Galphaq/11-coupled, respectively), providing dynamic regulation of neuronal excitability. In this mini-review, we highlight findings from our laboratory in which we used a mammalian heterologous expression system to address mechanisms of GIRK channel regulation by Galpha and Gbetagamma subunits. We found that, like beta1- and beta2-containing Gbetagamma dimers, GIRK channels are also activated by G protein betagamma dimers containing beta3 and beta4 subunits. By contrast, GIRK currents are inhibited by beta5-containing Gbetagamma dimers and/or by Galpha proteins of the Galphaq/11 family. The properties of Gbeta5-mediated inhibition suggest that beta5-containing Gbetagamma dimers act as competitive antagonists of other activating Gbetagamma pairs on GIRK channels. Inhibition of GIRK channels by Galpha subunits is specific to members of the Galphaq/11 family and appears to result, at least in part, from activation of phospholipase C (PLC) and the resultant decrease in membrane levels of phosphatidylinositol-4,5-bisphosphate (PIP2), an endogenous co-factor necessary for GIRK channel activity; this Galphaq/11 activated mechanism is largely responsible for receptor-mediated GIRK channel inhibition.     

Overexpression of beta 1 and beta 2 adrenergic receptors in rat atrial myocytes. Differential coupling to G protein-gated inward rectifier K(+) channels via G(s) and G(i)/o

G protein-activated inwardly rectifying K(+) (GIRK) channels, expressed in atrial myocytes, various neurons, and endocrine cells, represent the paradigmatic target of beta gamma subunits released from activated heterotrimeric G proteins. These channels contribute to physiological slowing of cardiac frequency and synaptic inhibition. They are activated by beta gamma dimers released upon stimulation of receptors coupled to pertussis toxin-sensitive G proteins (G(i/o)), whereas beta gamma released from G(s) do not converge on the channel subunits. This is in conflict with the finding that dimeric combinations of various beta and gamma subunits can activate GIRK channels with little specificity. In the present study, we have overexpressed the major subtypes of cardiac beta-adrenergic receptors (beta(1)-AR and beta(2)-AR) in atrial myocytes by transient transfection. Whereas in native cells beta-adrenergic stimulation with isoproterenol failed to induce measurable GIRK current, robust currents were recorded from myocytes overexpressing either beta(1)-AR or beta(2)-AR. Whereas the beta(2)-AR-induced current showed the same sensitivity to pertussis toxin as the current evoked by the endogenous G(i/o)-coupled muscarinic M(2) receptor, isoproterenol-activated currents were insensitive to pertussis toxin treatment in beta(1)-AR-overexpressing myocytes. In contrast to a recent publication (Leaney, J. L., Milligan, G., and Tinker, A. (2000) J. Biol. Chem. 275, 921-929), sizable GIRK currents could also be activated by isoproterenol when the signaling pathway was reconstituted by transient transfection in two different standard cell lines (Chinese hamster ovary and HEK293). These results demonstrate that specificity of receptor-G protein signaling can be disrupted by overexpression of receptors. Moreover, the alpha subunit of heterotrimeric G proteins does not confer specificity to G beta gamma-mediated signaling.     

G protein beta2 subunit-derived peptides for inhibition and induction of G protein pathways. Examination of voltage-gated Ca2+ and G protein inwardly rectifying K+ channels

Voltage-gated Ca2+ channels of the N-, P/Q-, and R-type and G protein inwardly rectifying K+ channels (GIRK) are modulated via direct binding of G proteins. The modulation is mediated by G protein betagamma subunits. By using electrophysiological recordings and fluorescence resonance energy transfer, we characterized the modulatory domains of the G protein beta subunit on the recombinant P/Q-type channel and GIRK channel expressed in HEK293 cells and on native non-L-type Ca2+ currents of cultured hippocampal neurons. We found that Gbeta2 subunit-derived deletion constructs and synthesized peptides can either induce or inhibit G protein modulation of the examined ion channels. In particular, the 25-amino acid peptide derived from the Gbeta2 N terminus inhibits G protein modulation, whereas a 35-amino acid peptide derived from the Gbeta2 C terminus induced modulation of voltage-gated Ca2+ channels and GIRK channels. Fluorescence resonance energy transfer (FRET) analysis of the live action of these peptides revealed that the 25-amino acid peptide diminished the FRET signal between G protein beta2gamma3 subunits, indicating a reorientation between G protein beta2gamma3 subunits in the presence of the peptide. In contrast, the 35-amino acid peptide increased the FRET signal between GIRK1,2 channel subunits, similarly to the Gbetagamma-mediated FRET increase observed for this GIRK subunit combination. Circular dichroism spectra of the synthesized peptides suggest that the 25-amino acid peptide is structured. These results indicate that individual G protein beta subunit domains can act as independent, separate modulatory domains to either induce or inhibit G protein modulation for several effector proteins.     

G beta association and effector interaction selectivities of the divergent G gamma subunit G gamma(13).

G gamma(13) is a divergent member of the G gamma subunit family considered to be a component of the gustducin G-protein heterotrimer involved in bitter and sweet taste reception in taste bud cells. G gamma(13) contains a C-terminal asparagine-proline-tryptophan (NPW) tripeptide, a hallmark of RGS protein G gamma-like (GGL) domains which dimerize exclusively with G beta(5) subunits. In this study, we investigated the functional range of G gamma(13) assembly with G beta subunits using multiple assays of G beta association and G beta gamma effector modulation. G gamma(13) was observed to associate with all five G beta subunits (G beta(1-5)) upon co-translation in vitro, as well as function with all five G beta subunits in the modulation of Kir3.1/3.4 (GIRK1/4) potassium and N-type (alpha(1B)) calcium channels. Multiple G beta/G gamma(13) pairings were also functional in cellular assays of phospholipase C (PLC) beta 2 activation and inhibition of G alpha(q)-stimulated PLC beta 1 activity. However, upon cellular co-expression of G gamma(13) with different G beta subunits, only G beta(1)/G gamma(13), G beta(3)/G gamma(13), and G beta(4)/G gamma(13) pairings were found to form stable dimers detectable by co-immunoprecipitation under high-detergent cell lysis conditions. Collectively, these data indicate that G gamma(13) forms functional G beta gamma dimers with a range of G beta subunits. Coupled with our detection of G gamma(13) mRNA in mouse and human brain and retina, these results imply that this divergent G gamma subunit can act in signal transduction pathways other than that dedicated to taste reception in sensory lingual tissue.     

A G protein-gated K channel is activated via beta 2-adrenergic receptors and G beta gamma subunits in Xenopus oocytes

In many tissues, inwardly rectifying K channels are coupled to seven- helix receptors via the Gi/Go family of heterotrimeric G proteins. This activation proceeds at least partially via G beta gamma subunits. These experiments test the hypothesis that G beta gamma subunits activate the channel even if released from other classes of heterotrimeric G proteins. The G protein-gated K channel from rat atrium, KGA/GIRK1, was expressed in Xenopus oocytes with various receptors and G proteins. The beta 2-adrenergic receptor (beta 2AR), a Gs-linked receptor, activated large KGA currents when the alpha subunit, G alpha s, was also overexpressed. Although G alpha s augmented the coupling between beta 2AR and KGA, G alpha s also inhibited the basal, agonist-independent activity of KGA. KGA currents stimulated via beta 2AR activated, deactivated, and desensitized more slowly than currents stimulated via Gi/Go-linked receptors. There was partial occlusion between currents stimulated via beta 2AR and the m2 muscarinic receptor (a Gi/Go-linked receptor), indicating some convergence in the mechanism of activation by these two receptors. Although stimulation of beta 2AR also activates adenylyl cyclase and protein kinase A, activation of KGA via beta 2AR is not mediated by this second messenger pathway, because direct elevation of intracellular cAMP levels had no effect on KGA currents. Experiments with other coexpressed G protein alpha and beta gamma subunits showed that (a) a constitutively active G alpha s mutant did not suppress basal KGA currents and was only partially as effective as wild type G alpha s in coupling beta 2AR to KGA, and (b) beta gamma subunits increased basal KGA currents. These results reinforce present concepts that beta gamma subunits activate KGA, and also suggest that beta gamma subunits may provide a link between KGA and receptors not previously known to couple to inward rectifiers.     

A dominant-negative strategy for studying roles of G proteins in vivo

G proteins play a critical role in transducing a large variety of signals into intracellular responses. Increasingly, there is evidence that G proteins may play other roles as well. Dominant-negative constructs of the alpha subunit of G proteins would be useful in studying the roles of G proteins in a variety of processes, but the currently available dominant-negative constructs, which target Mg2+-binding sites, are rather leaky. A variety of studies have implicated the carboxyl terminus of G protein alpha subunits in both mediating receptor-G protein interaction and in receptor selectivity. Thus we have made minigene plasmid constructs that encode oligonucleotide sequences corresponding to the carboxyl-terminal undecapeptide of Galphai, Galphaq, or Galphas. To determine whether overexpression of the carboxyl-terminal peptide would block cellular responses, we used as a test system the activation of the M2 muscarinic receptor activated K+ channels in HEK 293 cells. The minigenes were transiently transfected along with G protein-regulated inwardly rectifying K+ channels (GIRK) into HEK 293 cells that stably express the M2 muscarinic receptor. The presence of the Galphai carboxyl-terminal peptide results in specific inhibition of GIRK activity in response to agonist stimulation of the M2 muscarinic receptor. The Galphai minigene construct completely blocks agonist-mediated M2 mAChR K+ channel response whereas the control minigene constructs (empty vector, pcDNA3.1, and the Galpha carboxyl peptide in random order, pcDNA-GalphaiR) had no effect on agonist-mediated M2 muscarinic receptor GIRK response. The inhibitory effects of the Galphai minigene construct were specific because overexpression of peptides corresponding to the carboxyl terminus of Galphaq or Galphas had no effect on M2 muscarinic receptor stimulation of the K+ channel.     

G(alpha)(i) controls the gating of the G protein-activated K(+) channel, GIRK.

GIRK (Kir3) channels are activated by neurotransmitters coupled to G proteins, via a direct binding of G(beta)(gamma). The role of G(alpha) subunits in GIRK gating is elusive. Here we demonstrate that G(alpha)(i) is not only a donor of G(beta)(gamma) but also regulates GIRK gating. When overexpressed in Xenopus oocytes, GIRK channels show excessive basal activity and poor activation by agonist or G(beta)(gamma). Coexpression of G(alpha)(i3) or G(alpha)(i1) restores the correct gating parameters. G(alpha)(i) acts neither as a pure G(beta)(gamma) scavenger nor as an allosteric cofactor for G(beta)(gamma). It inhibits only the basal activity without interfering with G(beta)(gamma)-induced response. Thus, GIRK is regulated, in distinct ways, by both arms of the G protein. G(alpha)(i) probably acts in its GDP bound form, alone or as a part of G(alpha)(beta)(gamma) heterotrimer.     

Direct activation of trpl cation channels by G alpha11 subunits.

G proteins of the Gq/11 subfamily functionally couple cell surface receptors to phospholipase C beta (PLC beta) isoforms. Stimulation of PLC beta induces Ca2+ elevation by inositol 1,4,5-trisphosphate (InsP3)-mediated Ca2+ release and store-dependent 'capacitative' Ca2+ entry through Ca(2+)-permeable channels. The Drosophila trp gene, as well as some human trp homologs, code for such store-operated channels. The related trp-like (trpl) gene product also forms a Ca(2+)-permeable cation channel, but is not activated by store depletion. Co-expression of the constitutively active Gq subfamily member G alpha 11 (G alpha 11) with trpl enhanced trpl currents 33-fold in comparison with co-expression of trpl with other G alpha isoforms or G beta gamma complexes. This activation could not be attributed to signals downstream of PLC beta. In particular, InsP3 infusion, modulation of protein kinase C activity or elevation of intracellular calcium concentration failed to induce trpl currents. In contrast, purified G alpha 11 (but not other G protein subunits) activated trpl channels in inside-out patches. We conclude that trpl is regulated by G11 proteins in a membrane-confined manner not involving cytosolic factors. Thus, G proteins of the Gq subfamily may induce Ca2+ entry not only indirectly via store-operated mechanisms but also by directly stimulating cation channels.     

Novel inhibition of gbetagamma-activated potassium currents induced by M(2) muscarinic receptors via a pertussis toxin-insensitive pathway

G(i) protein-coupled receptors such as the M(2) muscarinic acetylcholine receptor (mAChR) and A(1) adenosine receptor have been shown to activate G protein-activated inwardly rectifying K(+) channels (GIRKs) via pertussis toxin-sensitive G proteins in atrial myocytes and in many neuronal cells. Here we show that muscarinic M(2) receptors not only activate but also reversibly inhibit these K(+) currents when stimulated with agonist for up to 2 min. The M(2) mAChR-mediated inhibition of the channel was also observed when the channels were first activated by inclusion of guanosine 5'-O-(thiotriphosphate) in the pipette. Under these conditions the M(2) mAChR-induced inhibition was quasi-irreversible, suggesting a role for G proteins in the inhibitory process. In contrast, when GIRK currents were maximally activated by co-expressing exogenous Gbetagamma, the extent of acetylcholine (ACh)-induced inhibition was significantly reduced, suggesting competition between the receptor-mediated inhibition and the large pool of available Gbetagamma subunits. The signaling pathway that led to the ACh-induced inhibition of GIRK channels was unaffected by pertussis toxin pretreatment. Furthermore, the internalization and agonist-induced phosphorylation of M(2) mAChR was not required because a phosphorylation- and internalization-deficient mutant of the M(2) mAChR was as potent as the wild-type counterpart. Pharmacological agents modulating various protein kinases or phosphatidylinositol 3-kinase did not affect the inhibition of GIRK currents. Furthermore, the signaling pathway that mediates GIRK current inhibition was found to be membrane-delimited because bath application of ACh did not inhibit GIRK channel activity in cell-attached patches. Other G protein-coupled receptors including M(4) mAChR and alpha(1A) adrenergic receptors also caused the inhibition, whereas other G protein-coupled receptors including A(1) and A(3) adenosine receptors and alpha(2A) and alpha(2C) adrenergic receptors could not induce the inhibition. The presented results suggest the existence of a novel signaling pathway that can be activated selectively by M(2) and M(4) mAChR but not by adenosine receptors and that involves non-pertussis toxin-sensitive G proteins leading to an inhibition of Gbetagamma-activated GIRK currents in a membrane-delimited fashion.     

Identification of critical residues controlling G protein-gated inwardly rectifying K(+) channel activity through interactions with the beta gamma subunits of G proteins.

G protein-sensitive inwardly rectifying potassium (GIRK) channels are activated through direct interactions of their cytoplasmic N- and C-terminal domains with the beta gamma subunits of G proteins. By using a combination of biochemical and electrophysiological approaches, we identified minimal N- and C-terminal G beta gamma -binding domains responsible for stimulation of GIRK4 channel activity. Within these domains one N-terminal residue, His-64, and one C-terminal residue, Leu-268, proved critical for G beta gamma-mediated GIRK4 activity. Moreover, mutations at these GIRK4 sites reduced significantly binding of the channel domains to G beta gamma . The corresponding residues in GIRK1 also showed a critical involvement in G beta gamma sensitivity. In GIRK4/GIRK1 heteromers the GIRK4 His-64 and Leu-268 residues showed greater contributions to G beta zeta sensitivity than did the corresponding GIRK1 His-57 and Leu-262 residues. These results identify functionally important channel interaction sites with the beta gamma subunits of G proteins, critical for channel activity.     

Identification of a potassium channel site that interacts with G protein betagamma subunits to mediate agonist-induced signaling

Activation of heterotrimeric GTP-binding (G) proteins by their coupled receptors, causes dissociation of the G protein alpha and betagamma subunits. Gbetagamma subunits interact directly with G protein-gated inwardly rectifying K+ (GIRK) channels to stimulate their activity. In addition, free Gbetagamma subunits, resulting from agonist-independent dissociation of G protein subunits, can account for a major component of the basal channel activity. Using a series of chimeric constructs between GIRK4 and a Gbetagamma-insensitive K+ channel, IRK1, we have identified a critical site of interaction of GIRK with Gbetagamma. Mutation of Leu339 to Glu within this site impaired agonist-induced sensitivity and decreased binding to Gbetagamma, without removing the Gbetagamma contribution to basal currents. Mutation of the corresponding residue in GIRK1 (Leu333) resulted in a similar phenotype. Both the GIRK1 and GIRK4 subunits contributed equally to the agonist-induced sensitivity of the heteromultimeric channel. Thus, we have identified a channel site that interacts specifically with Gbetagamma subunits released through receptor stimulation.     

Inhibition of a Gi-activated potassium channel (GIRK1/4) by the Gq-coupled m1 muscarinic acetylcholine receptor

The G protein-coupled inwardly rectifying K+ channel, GIRK1/GIRK4, can be activated by receptors coupled to the Galpha(i) subunit. An opposing role for Galpha(q) receptor signaling in GIRK regulation has only recently begun to be established. We have studied the effects of m1 muscarinic acetylcholine receptor (mAChR) stimulation, which is known to mobilize calcium and activate protein kinase C (PKC) by a Galpha(q)-dependent mechanism, on whole cell GIRK1/4 currents in Xenopus oocytes. We found that stimulation of the m1 mAChR suppresses both basal and dopamine 2 receptor-activated GIRK 1/4 currents. Overexpression of Gbetagamma subunits attenuates this effect, suggesting that increased binding of Gbetagamma to the GIRK channel can effectively compete with the G(q)-mediated inhibitory signal. This G(q) signal requires the use of second messenger molecules; pharmacology implicates a role for PKC and Ca2+ responses as m1 mAChR-mediated inhibition of GIRK channels is mimicked by PMA and Ca2+ ionophore. We have analyzed a series of mutant and chimeric channels suggesting that the GIRK4 subunit is capable of responding to G(q) signals and that the resulting current inhibition does not occur via phosphorylation of a canonical PKC site on the channel itself.     

A switch mechanism for G beta gamma activation of I(KACh)

G protein-gated inwardly rectifying potassium (GIRK) channels are a family of K(+)-selective ion channels that slow the firing rate of neurons and cardiac myocytes. GIRK channels are directly bound and activated by the G protein G beta gamma subunit. As heterotetramers, they comprise the GIRK1 and the GIRK2, -3, or -4 subunits. Here we show that GIRK1 but not the GIRK4 subunit is phosphorylated when heterologously expressed. We found also that phosphatase PP2A dephosphorylation of a protein in the excised patch abrogates channel activation by G beta gamma. Experiments with the truncated molecule demonstrated that the GIRK1 C-terminal is critical for both channel phosphorylation and channel regulation by protein phosphorylation, but the critical phosphorylation sites were not located on the C terminus. These data provide evidence for a novel switch mechanism in which protein phosphorylation enables G beta gamma gating of the channel complex.     

Interaction sites of the G protein beta subunit with brain G protein-coupled inward rectifier K+ channel

G protein-coupled inward rectifier K(+) channels (GIRK channels) are activated directly by the G protein betagamma subunit. The crystal structure of the G protein betagamma subunits reveals that the beta subunit consists of an N-terminal alpha helix followed by a symmetrical seven-bladed propeller structure. Each blade is made up of four antiparallel beta strands. The top surface of the propeller structure interacts with the Galpha subunit. The outer surface of the betagamma torus is largely made from outer beta strands of the propeller. We analyzed the interaction between the beta subunit and brain GIRK channels by mutating the outer surface of the betagamma torus. Mutants of the outer surface of the beta(1) subunit were generated by replacing the sequences at the outer beta strands of each blade with corresponding sequences of the yeast beta subunit, STE4. The mutant beta(1)gamma(2) subunits were expressed in and purified from Sf9 cells. They were applied to inside-out patches of cultured locus coeruleus neurons. The wild type beta(1)gamma(2) induced robust GIRK channel activity with an EC(50) of about 4 nm. Among the eight outer surface mutants tested, blade 1 and blade 2 mutants (D1 and CD2) were far less active than the wild type in stimulating GIRK channels. However, the ability of D1 and CD2 to regulate type I and type II adenylyl cyclases was not very different from that of the wild type beta(1)gamma(2). As to the activities to stimulate phospholipase Cbeta(2), D1 was more potent and CD2 was less potent than the wild type beta(1)gamma(2). Additionally we tested four beta(1) mutants in which mutated residues are located in the top Galpha/beta interacting surface. Among them, mutant W332A showed far less ability than the wild type to activate GIRK channels. These results suggest that the outer surface of blade 1 and blade 2 of the beta subunit might specifically interact with GIRK and that the beta subunit interacts with GIRK both over the outer surface and over the top Galpha interacting surface.     

Calcium channel beta subunit promotes voltage-dependent modulation of alpha 1 B by G beta gamma

Voltage-dependent calcium channels (VDCCs) are heteromultimers composed of a pore-forming alpha1 subunit and auxiliary subunits, including the intracellular beta subunit, which has a strong influence on the channel properties. Voltage-dependent inhibitory modulation of neuronal VDCCs occurs primarily by activation of G-proteins and elevation of the free G beta gamma dimer concentration. Here we have examined the interaction between the regulation of N-type (alpha 1 B) channels by their beta subunits and by G beta gamma dimers, heterologously expressed in COS-7 cells. In contrast to previous studies suggesting antagonism of G protein inhibition by the VDCC beta subunit, we found a significantly larger G beta gamma-dependent inhibition of alpha 1 B channel activation when the VDCC alpha 1 B and beta subunits were coexpressed. In the absence of coexpressed VDCC beta subunit, the G beta gamma dimers, either expressed tonically or elevated via receptor activation, did not produce the expected features of voltage-dependent G protein modulation of N-type channels, including slowed activation and prepulse facilitation, while VDCC beta subunit coexpression restored all of the hallmarks of G beta gamma modulation. These results suggest that the VDCC beta subunit must be present for G beta gamma to induce voltage-dependent modulation of N-type calcium channels.     

Subunit composition of G(o) proteins functionally coupling galanin receptors to voltage-gated calcium channels.

The neuropeptide galanin is widely expressed in the central nervous system and other tissues and induces different cellular reactions, e.g. hormone release from pituitary and inhibition of insulin release from pancreatic B cells. By microinjection of antisense oligonucleotides we studied the question as to which G proteins mediate the galanin-induced inhibition of voltage-gated Ca2+ channels in the rat pancreatic B-cell line RINm5F and in the rat pituitary cell line GH3. Injection of antisense oligonucleotides directed against alpha 01, beta 2, beta 3, gamma 2 and gamma 4 G protein subunits reduced the inhibition of Ca2+ channel current which was induced by galanin, whereas no change was seen after injection of cells with antisense oligonucleotides directed against alpha i, alpha q, alpha 11, alpha 14, alpha 15, beta 1, beta 4, gamma 1, gamma 3, gamma 5, or gamma 7 G protein subunits or with sense control oligonucleotides. In view of these data and of previous results, we conclude that the galanin receptors in GH3 and in RINm5F cells couple mainly to the G(0) protein consisting of alpha 01 beta 2 gamma 2 to inhibit Ca2+ channels and use alpha 01beta 3 gamma 4 less efficiently. The latter G protein composition was previously shown to be used by muscarinic M4 receptors to inhibit Ca2+ channels.     

Beta gamma dimers of G proteins inhibit atrial muscarinic K+ channels

It has been proposed that beta gamma dimers of signal-transducing G proteins mediate muscarinic activation of atrial K+ channels. We examined this hypothesis by testing the effects of beta gamma dimers from four sources (human erythrocytes, human placenta, bovine brain, and bovine retina) on single channel muscarinic K+ (K+[acetylcholine (ACh)]) currents in inside-out membrane patches of adult guinea pig atria. None of the four beta gamma dimer preparations stimulated K+[ACh] currents; on the contrary, each inhibited the currents whether the currents were activated with GTP alone (agonist-independent activity) or with GTP plus a muscarinic agonist (agonist-dependent activity). Detergents at concentrations used to suspend erythrocyte, brain, and placental beta gamma dimers had no effect by themselves, and detergents were not used with the retinal beta gamma dimers. We conclude that beta gamma dimers do not mediate stimulatory effects of the endogenous G protein that regulates the K+ channels. In fact beta gamma dimers appear to inhibit activation by the endogenous G alpha subunits. Further insight into the role of beta gamma dimers came from the observation that agonist-independent GTP-activated K+[ACh] currents were inhibited by beta gamma dimers at about one-tenth the concentration required to inhibit agonist-dependent activation. One possibility is that dimeric beta gamma may have a higher affinity for free alpha subunits than for alpha subunits associated with agonist-occupied receptors. Thus, in addition to the known requirement of beta gamma dimers for the interaction of alpha subunits with receptors, beta gamma dimers may also improve the signal-to-noise ratio for agonists by reducing agonist-independent background activities.     

G protein {beta}{gamma} gating confers volatile anesthetic inhibition to Kir3 channels

G protein-activated inwardly rectifying potassium (GIRK or Kir3) channels are directly gated by the βγ subunits of G proteins and contribute to inhibitory neurotransmitter signaling pathways. Paradoxically, volatile anesthetics such as halothane inhibit these channels. We find that neuronal Kir3 currents are highly sensitive to inhibition by halothane. Given that Kir3 currents result from increased Gβγ available to the channels, we asked whether reducing available Gβγ to the channel would adversely affect halothane inhibition. Remarkably, scavenging Gβγ using the C-terminal domain of β-adrenergic receptor kinase (cβARK) resulted in channel activation by halothane. Consistent with this effect, channel mutants that impair Gβγ activation were also activated by halothane. A single residue, phenylalanine 192, occupies the putative Gβγ gate of neuronal Kir3.2 channels. Mutation of Phe-192 at the gate to other residues rendered the channel non-responsive, either activated or inhibited by halothane. These data indicated that halothane predominantly interferes with Gβγ-mediated Kir3 currents, such as those functioning during inhibitory synaptic activity. Our report identifies the molecular correlate for anesthetic inhibition of Kir3 channels and highlights the significance of these effects in modulating neurotransmitter-mediated inhibitory signaling.     

G alpha(q/11) coupled to mammalian phospholipase C beta 3-like enzyme mediates the ginsenoside effect on Ca(2+)-activated Cl(-) current in the Xenopus oocyte.

Recently we demonstrated that ginsenosides, the active ingredients of Panax ginseng, enhanced Ca(2+)-activated Cl(-) current in the Xenopus oocyte through a signal transduction mechanism involving the activation of pertussis toxin-insensitive G protein and phospholipase C (PLC). However, it has not yet been determined precisely which G protein subunit(s) and which PLC isoform(s) participate in the ginsenoside signaling. To provide answers to these questions, we investigated the changes in ginsenoside effect on the Cl(-) current after intraoocyte injections of the cRNAs coding various G protein subunits, a regulator of G protein signaling (RGS2), and G beta gamma-binding proteins. In addition, we examined which of mammalian PLC beta 1-3 antibodies injected into the oocyte inhibited the action of ginsenosides on the Cl(-) current. Injection of G alpha(q) or G alpha(11) cRNA increased the basal Cl(-) current recorded 48 h after, and it further prevented ginsenosides from enhancing the Cl(-) current, whereas G alpha(i2) and G alpha(oA) cRNA injection had no significant effect. The changes following G alpha(q) cRNA injection were prevented when G beta(1)gamma(2) and G alpha(q) subunits were co-expressed by simultaneous injection of the cRNAs coding these subunits. Injection of cRNA coding G alpha(q)Q209L, a constitutively active mutant that does not bind to G beta gamma, produced effects similar to those of G alpha(q) cRNA injection. The effects of G alpha(q)Q209L cRNA injection, however, were not prevented by co-injection of G beta(1)gamma(2) cRNA. Injection of the cRNA coding RGS2, which interacts most selectively with G alpha(q/11) among various identified RGS isoforms and stimulates the hydrolysis of GTP to GDP in active GTP-bound G alpha subunit, resulted in a severe attenuation of ginsenoside effect on the Cl(-) current. Finally, antibodies against PLC beta 3, but not -beta 1 and -beta 2, markedly attenuated the ginsenoside effect examined at 3-h postinjection. These results suggest that G alpha(q/11) coupled to mammalian PLC beta 3-like enzyme mediates ginsenoside effect on Ca(2+)-activated Cl(-) current in the Xenopus oocyte.     

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.