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G protein-gated inwardly rectifying potassium (GIRK) channels are essential regulators of cell excitability in the brain. While they are implicated in a variety of neurological diseases in both human and animal model studies, their therapeutic potential has been largely untapped. Here, we review recent advances in the development of small molecule compounds that specifically modulate GIRK channels and compare them with first-generation compounds that exhibit off-target activity. We describe the method of discovery of these small molecule modulators, their chemical features, and their effects in vivo. These studies provide a promising outlook on the future development of subunit-specific GIRK modulators to regulate neuronal excitability in a brain region-specific manner.

Addictive drugs usurp the brain's intrinsic mechanism for reward, leading to compulsive and destructive behaviors. In the ventral tegmental area (VTA), the center of the brain's reward circuit, GABAergic neurons control the excitability of dopamine (DA) projection neurons and are the site of initial psychostimulant-dependent changes in signaling. Previous work established that cocaine/methamphetamine exposure increases protein phosphatase 2A (PP2A) activity, which dephosphorylates the GABABR2 subunit, promotes internalization of the GABABreceptor (GABABR) and leads to smaller GABABR-activated G-protein-gated inwardly rectifying potassium (GIRK) currents in VTA GABA neurons. How the actions of PP2A become selective for a particular signaling pathway is poorly understood. Here, we demonstrate that PP2A can associate directly with a short peptide sequence in the C terminal domain of the GABABR1 subunit, and that GABABRs and PP2A are in close proximity in rodent neurons (mouse/rat; mixed sexes). We show that this PP2A-GABABR interaction can be regulated by intracellular Ca2+. Finally, a peptide that potentially reduces recruitment of PP2A to GABABRs and thereby limits receptor dephosphorylation increases the magnitude of baclofen-induced GIRK currents. Thus, limiting PP2A-dependent dephosphorylation of GABABRs may be a useful strategy to increase receptor signaling for treating diseases.SIGNIFICANCE STATEMENTDysregulation of GABABreceptors (GABABRs) underlies altered neurotransmission in many neurological disorders. Protein phosphatase 2A (PP2A) is involved in dephosphorylating and subsequent internalization of GABABRs in models of addiction and depression. Here, we provide new evidence that PP2A B55 regulatory subunit interacts directly with a small region of the C-terminal domain of the GABABR1 subunit, and that this interaction is sensitive to intracellular Ca2+. We demonstrate that a short peptide corresponding to the PP2A interaction site on GABABR1 competes for PP2A binding, enhances phosphorylation GABABR2 S783, and affects functional signaling through GIRK channels. Our study highlights how targeting PP2A dependent dephosphorylation of GABABRs may provide a specific strategy to modulate GABABR signaling in disease conditions.

Metabotropic GABA B receptors mediate a significant fraction of inhibitory neurotransmission in the brain. Native GABA B receptor complexes contain the principal subunits GABA B1 and GABA B2 , which form an obligate heterodimer, and auxiliary subunits, known as potassium channel tetramerization domain-containing proteins (KCTDs). KCTDs interact with GABA B receptors and modify the kinetics of GABA B receptor signaling. Little is known about the molecular mechanism governing the direct association and functional coupling of GABA B receptors with these auxiliary proteins. Here, we describe the high-resolution structure of the KCTD16 oligomerization domain in complex with part of the GABA B2 receptor. A single GABA B2 C-terminal peptide is bound to the interior of an open pentamer formed by the oligomerization domain of five KCTD16 subunits. Mutation of specific amino acids identified in the structure of the GABA B2 –KCTD16 interface disrupted both the biochemical association and functional modulation of GABA B receptors and G protein-activated inwardly rectifying K + channel (GIRK) channels. These interfacial residues are conserved among KCTDs, suggesting a common mode of KCTD interaction with GABA B receptors. Defining the binding interface of GABA B receptor and KCTD reveals a potential regulatory site for modulating GABA B -receptor function in the brain.

SUMMARY G-protein-gated inwardly rectifying potassium (GIRK) channels are important for determining neuronal excitability. In addition to G proteins, GIRK channels are potentiated by membrane cholesterol, which is elevated in the brains of people with neurodegenerative diseases such as Alzheimer’s dementia and Parkinson’s disease. The structural mechanism of cholesterol modulation of GIRK channels is not well understood. In this study, we present cryo-electron microscopy (cryoEM) structures of GIRK2 in the presence and absence of the cholesterol analog cholesteryl hemisuccinate (CHS) and phosphatidylinositol 4,5-bisphosphate (PIP2). The structures reveal that CHS binds near PIP2 in lipid-facing hydrophobic pockets of the transmembrane domain. Our structural analysis suggests that CHS stabilizes PIP2 interaction with the channel and promotes engagement of the cytoplasmic domain onto the transmembrane region. Mutagenesis of one of the CHS binding pockets eliminates cholesterol-dependent potentiation of GIRK2. Elucidating the structural mechanisms underlying cholesterol modulation of GIRK2 channels could facilitate the development of therapeutics for treating neurological diseases., Graphical Abstract, In brief Ion channels are important in determining neuronal excitability. Elevated cholesterol levels found in some neurodegenerative diseases can affect the function of ion channels. Mathiharan et al. take a structural and functional approach to identifying physical sites for cholesterol, and they provide details on how cholesterol potentiates ion channel activity.

G protein-gated inwardly rectifying potassium (GIRK) channels are important for determining neuronal excitability and have been implicated in a number of neurological diseases. In addition to G proteins, GIRK channels are also regulated by membrane cholesterol, which is elevated in the brain for neurodegenerative diseases like Alzheimer’s Dementia and Parkinson’s Disease. The structural mechanism of cholesterol modulation of GIRK channels is not well understood. Here, we present cryo-electron microscopy (cryoEM) structures of GIRK2 in the presence and absence of the cholesterol analog cholesteryl hemisuccinate (CHS) and PIP2. The structures reveal that CHS binds near PIP2 in lipid-facing hydrophobic pockets of the transmembrane domain. Our analysis suggests that CHS stabilizes PIP2 interaction with the channel and promotes the engagement of the cytoplasmic domain onto the transmembrane region. Mutagenesis of residues in one CHS binding pocket occludes cholesterol-dependent potentiation. Elucidating the cholesterol-binding site and characterizing the structural mechanisms underlying modulation of neuronal GIRK channels could facilitate the development of novel therapeutics for treating human diseases.

Here, we describe a fourth case of a human with a de novo KCNJ6 (GIRK2) mutation, who presented with clinical findings of severe hyperkinetic movement disorder and developmental delay, similar to the Keppen–Lubinsky syndrome but without lipodystrophy. Whole-exome sequencing of the patient’s DNA revealed a heterozygous de novo variant in the KCNJ6 (c.512T>G, p.Leu171Arg). We conducted in vitro functional studies to determine if this Leu-to-Arg mutation alters the function of GIRK2 channels. Heterologous expression of the mutant GIRK2 channel alone produced an aberrant basal inward current that lacked G protein activation, lost K+ selectivity and gained Ca2+ permeability. Notably, the inward current was inhibited by the Na+ channel blocker QX-314, similar to the previously reported weaver mutation in murine GIRK2. Expression of a tandem dimer containing GIRK1 and GIRK2(p.Leu171Arg) did not lead to any currents, suggesting heterotetramers are not functional. In neurons expressing p.Leu171Arg GIRK2 channels, these changes in channel properties would be expected to generate a sustained depolarization, instead of the normal G protein-gated inhibitory response, which could be mitigated by expression of other GIRK subunits. The identification of the p.Leu171Arg GIRK2 mutation potentially expands the Keppen–Lubinsky syndrome phenotype to include severe dystonia and ballismus. Our study suggests screening for dominant KCNJ6 mutations in the evaluation of patients with severe movement disorders, which could provide evidence to support a causal role of KCNJ6 in neurological channelopathies.

Repeated exposure to psychostimulants induces locomotor sensitization and leads to persistent changes in the circuitry of the mesocorticolimbic dopamine (DA) system. G-protein-gated inwardly rectifying potassium (GIRK; also known as Kir3) channels mediate a slow IPSC and control the excitability of DA neurons. Repeated 5 d exposure to psychostimulants decreases the size of the GABABreceptor (GABABR)-activated GIRK currents (IBaclofen) in ventral tegmental area (VTA) DA neurons of mice, but the mechanism underlying this plasticity is poorly understood. Here, we show that methamphetamine-dependent attenuation of GABABR-GIRK currents in VTA DA neurons required activation of both D1R-like and D2R-like receptors. The methamphetamine-dependent decrease in GABABR-GIRK currents in VTA DA neurons did not depend on a mechanism of dephosphorylation of the GABABR2 subunit found previously for other neurons in the reward pathway. Rather, the presence of the GIRK3 subunit appeared critical for the methamphetamine-dependent decrease of GABABR-GIRK current in VTA DA neurons. Together, these results highlight different regulatory mechanisms in the learning-evoked changes that occur in the VTA with repeated exposure to psychostimulants.SIGNIFICANCE STATEMENTExposure to addictive drugs such as psychostimulants produces persistent adaptations in inhibitory circuits within the mesolimbic dopamine system, suggesting that addictive behaviors are encoded by changes in the reward neural circuitry. One form of neuroadaptation that occurs with repeated exposure to psychostimulants is a decrease in slow inhibition, mediated by a GABABreceptor and a potassium channel. Here, we examine the subcellular mechanism that links psychostimulant exposure with changes in slow inhibition and reveal that one type of potassium channel subunit is important for mediating the effect of repeated psychostimulant exposure. Dissecting out the components of drug-dependent plasticity and uncovering novel protein targets in the reward circuit may lead to the development of new therapeutics for treating addiction.

The coupling of GABA(B) receptors to G-protein-gated inwardly rectifying potassium (GIRK) channels constitutes an important inhibitory pathway in the brain. Here, we examined the mechanism underlying desensitization of agonist-evoked currents carried by homomeric GIRK2 channels expressed in HEK-293T cells. The canonical GABA(B) receptor agonist baclofen produced GIRK2 currents that decayed by 57.3+/-1.4% after 60 s of stimulation, and then deactivated rapidly (time constant of 3.90+/-0.21 s) upon removal of agonist. Surface labeling studies revealed that GABA(B) receptors, in contrast to micro opioid receptors (MOR), did not internalize with a sustained stimulation for 10 min, excluding receptor redistribution as the primary mechanism for desensitization. Furthermore, heterologous desensitization was observed between GABA(B) receptors and MOR, implicating downstream proteins, such G-proteins or the GIRK channel. To investigate the G-protein turnover cycle, the non-hydrolyzable GTP analogue (GTPgammaS) was included in the intracellular solution and found to attenuate desensitization to 38.3+/-2.0%. The extent of desensitization was also reduced (45.3+/-1.3%) by coexpressing a mutant form of the Galphaq G-protein subunit that has been designed to sequester endogenous RGS proteins. Finally, reconstitution of GABA(B) receptors with Galphao G-proteins rendered insensitive to RGS resulted in significantly less desensitization (28.5+/-3.2%). Taken together, our results demonstrate that endogenous levels of RGS proteins effectively enhance GABA(B) receptor-dependent desensitization of GIRK currents.

G-protein-gated inwardly rectifying K+(GIRK/Kir3) channel activation underlies key physiological effects of opioids, including analgesia and dependence. GIRK channel activation has also been implicated in the opioid-induced inhibition of midbrain GABA neurons and consequent disinhibition of dopamine (DA) neurons in the ventral tegmental area (VTA). Drug-induced disinhibition of VTA DA neurons has been linked to reward-related behaviors and underlies opioid-induced motor activation. Here, we demonstrate that mouse VTA GABA neurons express a GIRK channel formed by GIRK1 and GIRK2 subunits. Nevertheless, neither constitutive genetic ablation ofGirk1orGirk2, nor the selective ablation of GIRK channels in GABA neurons, diminished morphine-induced motor activity in mice. Moreover, direct activation of GIRK channels in midbrain GABA neurons did not enhance motor activity. In contrast, genetic manipulations that selectively enhanced or suppressed GIRK channel function in midbrain DA neurons correlated with decreased and increased sensitivity, respectively, to the motor-stimulatory effect of systemic morphine. Collectively, these data support the contention that the unique GIRK channel subtype in VTA DA neurons, the GIRK2/GIRK3 heteromer, regulates the sensitivity of the mouse mesolimbic DA system to drugs with addictive potential.

Activation of G protein-gated inwardly rectifying potassium (GIRK) channels leads to a hyperpolarization of the neuron’s membrane potential, providing an important component of inhibition in the brain. In addition to the canonical G protein-activation pathway, GIRK channels are activated by small molecules but less is known about the underlying gating mechanisms. One drawback to previous studies has been the inability to control intrinsic and extrinsic factors. Here we used a reconstitution strategy with highly purified mammalian GIRK2 channels incorporated into liposomes and demonstrate that cholesterol or intoxicating concentrations of ethanol, i.e., >20 mM, each activate GIRK2 channels directly, in the absence of G proteins. Notably, both activators require the membrane phospholipid PIP2 but appear to interact independently with different regions of the channel. Elucidating the mechanisms underlying G protein-independent pathways of activating GIRK channels provides a unique strategy for developing new types of neuronal excitability modulators.

G protein-gated inwardly rectifying potassium (GIRK) channels are regulators of neuronal excitability in the brain. Knockout mice lacking GIRK channels display altered behavioral responses to multiple addictive drugs, implicating GIRK channels in addictive behaviors. Here, we review the effects of GIRK subunit deletions on the behavioral response to psychostimulants, such as cocaine and methamphetamine. Additionally, exposure of mice to psychostimulants produces alterations in the surface expression of GIRK channels in multiple types of neurons within the reward system of the brain. Thus, we compare the subcellular mechanisms by which drug exposure appears to alter GIRK expression in multiple cell types and provide an outlook on future studies examining the role of GIRK channels in addiction. A greater understanding of how GIRK channels are regulated by addictive drugs may enable the development of therapies to prevent or treat drug abuse.

G-protein-gated inwardly rectifying potassium (GIRK) channels, which help control neuronal excitability, are important for the response to drugs of abuse. Here, we describe a novel pathway for morphine-dependent enhancement of GIRK channel signaling in hippocampal neurons. Morphine treatment for ∼20 h increased the colocalization of GIRK2 with PSD95, a dendritic spine marker. Western blot analysis and quantitative immunoelectron microscopy revealed an increase in GIRK2 protein and targeting to dendritic spines.In vivoadministration of morphine also produced an upregulation of GIRK2 protein in the hippocampus. The mechanism engaged by morphine required elevated intracellular Ca2+and was insensitive to pertussis toxin, implicating opioid receptors that may couple to Gq G-proteins. Met-enkephalin, but not the μ-selective (DAMGO) and δ-selective (DPDPE) opioid receptor agonists, mimicked the effect of morphine, suggesting involvement of a heterodimeric opioid receptor complex. Peptide (KN-93) inhibition of CaMKII prevented the morphine-dependent change in GIRK localization, whereas expression of a constitutively activated form of CaMKII mimicked the effects of morphine. Coincident with an increase in GIRK2 surface expression, functional analyses revealed that morphine treatment increased the size of serotonin-activated GIRK currents and Ba2+-sensitive basal K+currents in neurons. These results demonstrate plasticity in neuronal GIRK signaling that may contribute to the abusive effects of morphine.

Many neurotransmitters and hormones signal by stimulating G protein-coupled neurotransmitter receptors (GPCRs), which activate G proteins and their downstream effectors. Whether these signalling proteins diffuse freely within the plasma membrane is not well understood. Recent studies have suggested that direct protein–protein interactions exist between GPCRs, G proteins and G protein-gated inwardly rectifying potassium (GIRK or Kir3) channels. Here, we used fluorescence resonance energy transfer (FRET) combined with total internal reflection fluorescence microscopy to investigate whether proteins within this signalling pathway move within 100 A of each other in the plasma membrane of living cells. GABAB R1 and R2 receptors, Kir3 channels, Gαo subunits and regulators of G protein signalling (RGS4) proteins were each fused to cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP) and first assessed for functional expression in HEK293 cells. The presence of the fluorophore did not significantly alter the signalling properties of these proteins. Possible FRET was then investigated for different protein pair combinations. As a positive control, FRET was measured between tagged GABAB R1 and R2 subunits (∼12% FRET), which are known to form heterodimers. We measured significant FRET between tagged RGS4 and GABAB R1 or R2 subunits (∼13% FRET), and between Gαo and GABAB R1 or R2 subunits (∼10% FRET). Surprisingly, FRET also occurred between tagged Kir3.2a/Kir3.4 channels and GABAB R1 or R2 subunits (∼10% FRET). FRET was not detected between Kir3.2a and RGS4 nor between Kir3.2a and Gαo. These data are discussed in terms of a model in which GABAB receptors, G proteins, RGS4 proteins and Kir3 channels are closely associated in a signalling complex.

G protein-gated inwardly rectifying potassium (GIRK) channels are critical regulators of neuronal excitability and can be directly activated by ethanol. Constitutive deletion of the GIRK3 subunit has minimal phenotypic consequences, except in response to drugs of abuse. Here we investigated how the GIRK3 subunit contributes to the cellular and behavioral effects of ethanol, as well as to voluntary ethanol consumption. We found that constitutive deletion of GIRK3 in knockout (KO) mice selectively increased ethanol binge-like drinking, without affecting ethanol metabolism, sensitivity to ethanol intoxication, or continuous-access drinking. Virally mediated expression of GIRK3 in the ventral tegmental area (VTA) reversed the phenotype of GIRK3 KO mice and further decreased the intake of their wild-type counterparts. In addition, GIRK3 KO mice showed a blunted response of the mesolimbic dopaminergic (DA) pathway to ethanol, as assessed by ethanol-induced excitation of VTA neurons and DA release in the nucleus accumbens. These findings support the notion that the subunit composition of VTA GIRK channels is a critical determinant of DA neuron sensitivity to drugs of abuse. Furthermore, our study reveals the behavioral impact of this cellular effect, whereby the level of GIRK3 expression in the VTA tunes ethanol intake under binge-type conditions: the more GIRK3, the less ethanol drinking.

G protein-gated inwardly rectifying potassium (GIRK) channels regulate cellular excitability in the heart and brain. Stimulation of G protein-coupled receptors that couple to Gi/o (pertussis toxin-sensitive) G proteins is the primary mechanism of activating GIRK channels. However, there are several small molecule components of GIRK channel activity that operate independently of the G protein-mediated pathway. The lipid environment surrounding GIRK channels in the plasma membrane is one important regulator of GIRK channel activity. Previous studies have established that the membrane phospholipid PIP2 is essential for GIRK channel gating. GIRK channel activators, such as Gβγ and alcohol (ethanol), appear to work by enhancing the interaction with PIP2. We studied the channel gating properties of the brain GIRK2 channel in a defined reconstituted system that allowed for precise control of small molecules and ions. We found that cholesterol, a major component of brain membranes, potentiates the neuronal GIRK2 channel activity in a PIP2 dependent manner. Cholesterol shifts the PIP2 dose-response curve to lower concentrations, demonstrating enhanced PIP2 binding to the channel. Ethanol activates GIRK channels independently of the agonist-mediated pathway via binding to an alcohol pocket in the cytoplasmic terminal domain. There is no apparent shift in alcohol sensitivity in the presence of cholesterol, suggesting alcohol and cholesterol interact structurally and mechanistically with different regions of the channel. Elucidating the molecular mechanism underlying the activation of GIRK channels by small compounds, such as ethanol and cholesterol, should aid in the design of new drugs for treating alcoholism and other neurological disorders.

The activity of G protein-activated inwardly rectifying K+ channels (GIRK or Kir3) is important for regulating membrane excitability in neuronal, cardiac and endocrine cells. Although Gβγ subunits are known to bind the N- and C-termini of GIRK channels, the mechanism underlying Gβγ activation of GIRK is not well understood. Here, we used chimeras and point mutants constructed from GIRK2 and IRK1, a G protein-insensitive inward rectifier, to determine the region within GIRK2 important for Gβγ binding and activation. An analysis of mutant channels expressed in Xenopus oocytes revealed two amino acid substitutions in the C-terminal domain of GIRK2, GIRK2L344E and GIRK2G347H, that exhibited decreased carbachol-activated currents but significantly enhanced basal currents with coexpression of Gβγ subunits. Combining the two mutations (GIRK2EH) led to a more severe reduction in carbachol-activated and Gβγ-stimulated currents. Ethanol-activated currents were normal, however, suggesting that G protein-independent gating was unaffected by the mutations. Both GIRK2L344E and GIRK2EH also showed reduced carbachol activation and normal ethanol activation when expressed in HEK-293T cells. Using epitope-tagged channels expressed in HEK-293T cells, immunocytochemistry showed that Gβγ-impaired mutants were expressed on the plasma membrane, although to varying extents, and could not account completely for the reduced Gβγ activation. In vitro Gβγ binding assays revealed an ∼60% decrease in Gβγ binding to the C-terminal domain of GIRK2L344E but no statistical change with GIRK2EH or GIRK2G347H, though both mutants exhibited Gβγ-impaired activation. Together, these results suggest that L344, and to a lesser extent, G347 play an important functional role in Gβγ activation of GIRK2 channels. Based on the 1.8 A structure of GIRK1 cytoplasmic domains, L344 and G347 are positioned in the βL–βM loop, which is situated away from the pore and near the N-terminal domain. The results are discussed in terms of a model for activation in which Gβγ alters the interaction between the βL–βM loop and the N-terminal domain.

The rewarding effect of drugs of abuse is mediated by activation of the mesolimbic dopamine system, which is inhibited by putative anti-craving compounds. Interestingly, different GABA(B) receptor agonists can exert similarly opposing effects on the reward pathway, but the cellular mechanisms involved are unknown. Here we found that the coupling efficacy (EC(50)) of G-protein-gated inwardly rectifying potassium (GIRK, Kir3) channels to GABA(B) receptor was much lower in dopamine neurons than in GABA neurons of the ventral tegmental area (VTA), depending on the differential expression of GIRK subunits. Consequently, in rodent VTA slices, a low concentration of the canonical agonist baclofen caused increased activity, whereas higher doses eventually inhibited dopamine neurons. At behaviorally relevant dosages, baclofen activated GIRK channels in both cell types, but the drug of abuse gamma-hydroxy-butyric acid (GHB) activated GIRK channels only in GABAergic neurons. Thus GABA(B) receptor agonists exert parallel cellular and behavioral effects due to the cell-specific expression of GIRK subunits.

G protein-gated inwardly rectifying potassium (GIRK; Kir3) channels, which are members of the large family of inwardly rectifying potassium channels (Kir1-Kir7), regulate excitability in the heart and brain. GIRK channels are activated following stimulation of G protein-coupled receptors that couple to the G(i/o) (pertussis toxin-sensitive) G proteins. GIRK channels, like all other Kir channels, possess an extrinsic mechanism of inward rectification involving intracellular Mg(2+) and polyamines that occlude the conduction pathway at membrane potentials positive to E(K). In the past 17 years, more than 20 high-resolution atomic structures containing GIRK channel cytoplasmic domains and transmembrane domains have been solved. These structures have provided valuable insights into the structural determinants of many of the properties common to all inward rectifiers, such as permeation and rectification, as well as revealing the structural bases for GIRK channel gating. In this chapter, we describe advances in our understanding of GIRK channel function based on recent high-resolution atomic structures of inwardly rectifying K(+) channels discussed in the context of classical structure-function experiments.

Alcohol (ethanol)-induced behaviors may arise from direct interaction of alcohol with discrete protein cavities within brain proteins. Recent structural and biochemical studies have provided new insights into the mechanism of alcohol-dependent activation of G protein-gated inwardly rectifying potassium (GIRK) channels, which regulate neuronal responses in the brain reward circuit. GIRK channels contain an alcohol binding pocket formed at the interface of two adjacent channel subunits. Here, we discuss the physiochemical properties of the alcohol pocket and the roles of G protein βγ subunits and membrane phospholipid PIP2 in regulating the alcohol response of GIRK channels. Some of the features of alcohol modulation of GIRK channels may be common to other alcohol-sensitive brain proteins. We discuss the possibility of alcohol-selective therapeutics that block alcohol access to the pocket. Understanding alcohol recognition and modulation of brain proteins is essential for development of therapeutics for alcohol abuse and addiction.

Alcohol (ethanol) produces a wide range of pharmacological effects on the nervous system through its actions on ion channels. The molecular mechanism underlying ethanol modulation of ion channels is poorly understood. Here we used a unique method of alcohol-tagging to demonstrate that alcohol activation of a G-protein–gated inwardly rectifying potassium (GIRK or Kir3) channel is mediated by a defined alcohol pocket through changes in affinity for the membrane phospholipid signaling molecule phosphatidylinositol 4,5-bisphosphate. Surprisingly, hydrophobicity and size, but not the canonical hydroxyl, were important determinants of alcohol-dependent activation. Altering levels of G protein Gβγ subunits, conversely, did not affect alcohol-dependent activation, suggesting a fundamental distinction between receptor and alcohol gating of GIRK channels. The chemical properties of the alcohol pocket revealed here might extend to other alcohol-sensitive proteins, revealing a unique protein microdomain for targeting alcohol-selective therapeutics in the treatment of alcoholism and addiction.

Acetylcholine released during parasympathetic stimulation of the vagal nerve slows the heart rate through the activation of muscarinic receptors and subsequent opening of an inwardly rectifying potassium channel. The activation of these muscarinic potassium channels is mediated by a pertussis toxin-sensitive heterotrimeric GTP-binding protein (G protein). It has not been resolved whether exogenously applied G alpha or G beta gamma, or both, activate the channel. Using a heterologous expression system, we have tested the ability of different G protein subunits to activate the cloned muscarinic potassium channel, GIRK1. We report here that coexpression of GIRK1 with G beta gamma but not G alpha beta gamma in Xenopus oocytes results in channel activity that persists in the absence of cytoplasmic GTP. This activity is reduced by fusion proteins of the beta-adrenergic receptor kinase and of recombinant G alpha i-GDP, both of which are known to interact with G beta gamma. Moreover, application of recombinant G beta gamma, but not G alpha i-GTP-gamma S, activates GIRK1 channels. Thus G beta gamma appears to be sufficient for the activation of GIRK1 muscarinic potassium channels.

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

G protein-gated inwardly rectifying potassium (GIRK) channels hyperpolarize neurons in response to the activation of many G-protein coupled receptors and thus control the excitability of neurons through GIRK-mediated self-inhibition, slow synaptic potentials and volume transmission. GIRK channel function and trafficking are highly dependent on their subunit composition. Pharmacological investigations of GIRK channels and studies in animal models suggest that GIRK activity has an important role in physiological responses, including pain perception and memory modulation. Moreover, abnormal GIRK function has been implicated in altering neuronal excitability and cell death that may be important in the pathophysiology of human diseases such as epilepsy, Down’s syndrome, Parkinson’s disease and drug addiction. GIRK channels may therefore prove to be a valuable new therapeutic target for treating these health problems.

GABA(B) receptors have been found to play a key role in regulating membrane excitability and synaptic transmission in the brain. The GABA(B) receptor is a G-protein coupled receptor (GPCR) that associates with a subset of G-proteins (pertussis toxin sensitive Gi/o family), that in turn regulate specific ion channels and trigger cAMP cascades. In this review, we describe the relationships between the GABA(B) receptor, its effectors and associated proteins that mediate GABA(B) receptor function within the brain. We discuss a unique feature of the GABA(B) receptor, the requirement for heterodimerization to produce functional receptors, as well as an increasing body of evidence that suggests GABA(B) receptors comprise a macromolecular signaling heterocomplex, critical for efficient targeting and function of the receptors. Within this complex, GABA(B) receptors associate specifically with Gi/o G-proteins that regulate voltage-gated Ca(2+) (Ca(V)) channels, G-protein activated inwardly rectifying K(+) (GIRK) channels, and adenylyl cyclase. Numerous studies have revealed that lipid rafts, scaffold proteins, targeting motifs in the receptor, and regulators of G-protein signaling (RGS) proteins also contribute to the function of GABA(B) receptors and affect cellular processes such as receptor trafficking and activity-dependent desensitization. This complex regulation of GABA(B) receptors in the brain may provide opportunities for new ways to regulate GABA-dependent inhibition in normal and diseased states of the nervous system.

Downregulation of G-protein-coupled receptors (GPCRs) provides an important mechanism for reducing neurotransmitter signaling during sustained stimulation. Chronic stimulation of M2muscarinic receptors (M2Rs) causes internalization of M2R and G-protein-activated inwardly rectifying potassium (GIRK) channels in neuronal PC12 cells, resulting in loss of function. Here, we show that coexpression of GABABR2 receptors (GBR2s) rescues both surface expression and function of M2R, including M2R-induced activation of GIRKs and inhibition of cAMP production. GBR2 showed significant association with M2R at the plasma membrane but not other GPCRs (M1R, μ-opioid receptor), as detected by fluorescence resonance energy transfer measured with total internal reflection fluorescence microscopy. Unique regions of the proximal C-terminal domains of GBR2 and M2R mediate specific binding between M2R and GBR2. In the brain, GBR2, but not GBR1, biochemically coprecipitates with M2R and overlaps with M2R expression in cortical neurons. This novel heteromeric association between M2R and GBR2 provides a possible mechanism for altering muscarinic signaling in the brain and represents a previously unrecognized role for GBR2.

Ethanol modifies neural activity in the brain by modulating ion channels. Ethanol activates G protein-gated inwardly rectifying K(+) channels, but the molecular mechanism is not well understood. Here, we used a crystal structure of a mouse inward rectifier containing a bound alcohol and structure-based mutagenesis to probe a putative alcohol-binding pocket located in the cytoplasmic domains of GIRK channels. Substitutions with bulkier side-chains in the alcohol-binding pocket reduced or eliminated activation by alcohols. By contrast, alcohols inhibited constitutively open channels, such as IRK1 or GIRK2 engineered to strongly bind PIP(2). Mutations in the hydrophobic alcohol-binding pocket of these channels had no effect on alcohol-dependent inhibition, suggesting an alternate site is involved in inhibition. Comparison of high-resolution structures of inwardly rectifying K(+) channels suggests a model for activation of GIRK channels using this hydrophobic alcohol-binding pocket. These results provide a tool for developing therapeutic compounds that could mitigate the effects of alcohol.

Regulator of G-protein signaling (RGS) proteins are strong modulators of G-protein-mediated pathways in the nervous system. One function of RGS proteins is to accelerate the activation-deactivation kinetics of G-protein-coupled inwardly rectifying potassium (GIRK) channels. The opening of GIRK channels reduces the firing rates of neurons. Recent studies indicate that RGS proteins also modulate the coupling efficiency between gamma-aminobutyric acid type B (GABA(B)) receptors and GIRK channels in dopamine neurons of the ventral tegmental area (VTA), the initial target for addictive drugs in the brain reward pathway. Chronic drug exposure can dynamically regulate the expression levels of RGS. Functional and behavioral studies now reveal that levels of RGS2 protein, through selective association with GIRK3, critically determine whether GABA(B) agonists are excitatory or inhibitory in the VTA. The regulation of RGS protein in the reward pathway might underlie adaptation to different types of addictive drugs.

Agonists of GABA(B) receptors exert a bi-directional effect on the activity of dopamine (DA) neurons of the ventral tegmental area, which can be explained by the fact that coupling between GABA(B) receptors and G protein-gated inwardly rectifying potassium (GIRK) channels is significantly weaker in DA neurons than in GABA neurons. Thus, low concentrations of agonists preferentially inhibit GABA neurons and thereby disinhibit DA neurons. This disinhibition might confer reinforcing properties on addictive GABA(B) receptor agonists such as gamma-hydroxybutyrate (GHB) and its derivatives. Here we show that, in DA neurons of mice, the low coupling efficiency reflects the selective expression of heteromeric GIRK2/3 channels and is dynamically modulated by a member of the regulator of G protein signaling (RGS) protein family. Moreover, repetitive exposure to GHB increases the GABA(B) receptor-GIRK channel coupling efficiency through downregulation of RGS2. Finally, oral self-administration of GHB at a concentration that is normally rewarding becomes aversive after chronic exposure. On the basis of these results, we propose a mechanism that might underlie tolerance to GHB.

Neuronal G-protein-gated inwardly rectifying potassium (Kir3; GIRK) channels are activated by G-protein-coupled receptors that selectively interact with PTX-sensitive (Galphai/o) G proteins. Although the Gbetagamma dimer is known to activate GIRK channels, the role of the Galphai/o subunit remains unclear. Here, we established that Galphao subunits co-immunoprecipitate with neuronal GIRK channels. In vitro binding studies led to the identification of six amino acids in the GIRK2 C-terminal domain essential for Galphao binding. Further studies suggested that the Galphai/obetagamma heterotrimer binds to the GIRK2 C-terminal domain via Galpha and not Gbetagamma. Galphai/o binding-impaired GIRK2 channels exhibited reduced receptor-activated currents, but retained normal ethanol- and Gbetagamma-activated currents. Finally, PTX-insensitive Galphaq or Galphas subunits did not bind to the GIRK2 C-terminus. Together, these results suggest that the interaction of PTX-sensitive Galphai/o subunit with the GIRK2 C-terminal domain regulates G-protein receptor coupling, and may be important for establishing specific Galphai/o signaling pathways.

Alcohol is widely used and often abused. Yet, the molecular understanding of its action on brain function is poorly understood. Alcohol directly modulates the activity of several ion channels, including the G protein-gated inwardly-rectifying potassium (GIRK) channel. GIRK channels are directly activated by alcohol, independent of their typical G-protein mediated pathway. Currently, however, the molecular mechanism underlying alcohol activation of GIRK is poorly understood. We recently demonstrated that introduction of a Cysteine into the alcohol pocket of GIRK2(L257C) created a channel that could be chemically activated with alcohol-like cysteine-reacting reagents. Here, we studied the channel gating properties of purified GIRK2-L257C channels in a defined reconstituted system that allowed precise control of the lipids, G proteins and ions. We expressed a truncated cys-less GIRK2-L257C(GIRK2Δ∗-L257C) in Pichia pastoris, reconstituted purified protein into liposomes and studied the function of purified GIRK2Δ∗ L257C using a high throughput potassium flux assay. Reconstitution of GIRK2Δ∗-L257C into POPE:POPG:PIP2 containing liposomes exhibited a basal, barium-sensitive flux that was potently enhanced by pre-incubation with MTS-hydroxyethyl(MTS-HE) as well as the Gβγ G-protein subunits. Propanol treatment also enhanced the K+ flux. Thus, in the absence of any other proteins or cytoplasmic regulators, these experiments demonstrate the direct activation of GIRK2 channels by three distinct ligands, Gβγ G-proteins, alcohol and MTS-HE. Interestingly, MTS-HE-activated GIRK2Δ∗-L257C channels were inhibited by propanol in a dose-dependent manner. Inclusion of TCEP (tris(2-carboxyethyl)phosphine), which reduces disulfides, decreased MTS-HE activation but converted the propanol response from inhibition to activation. These experiments reveal that GIRK2 channels can also be inhibited by alcohol, perhaps through a different site, depending on the level of basal channel activation. Elucidating the details underlying alcohol's effects on channel proteins is paramount to developing selective pharmacological tools that could be used in the treatment of alcohol abuse and addiction.

Local anesthetics, commonly used for treating cardiac arrhythmias, pain, and seizures, are best known for their inhibitory effects on voltage-gated Na + channels. Cardiovascular and central nervous system toxicity are unwanted side-effects from local anesthetics that cannot be attributed to the inhibition of only Na + channels. Here, we report that extracellular application of the membrane-permeant local anesthetic bupivacaine selectively inhibited G protein-gated inwardly rectifying K + channels (GIRK:Kir3) but not other families of inwardly rectifying K + channels (ROMK:Kir1 and IRK:Kir2). Bupivacaine inhibited GIRK channels within seconds of application, regardless of whether channels were activated through the muscarinic receptor or directly via coexpressed G protein G β γ subunits. Bupivacaine also inhibited alcohol-induced GIRK currents in the absence of functional pertussis toxin-sensitive G proteins. The mutated GIRK1 and GIRK2 (GIRK1/2) channels containing the high-affinity phosphatidylinositol 4,5-bisphosphate (PIP 2 ) domain from IRK1, on the other hand, showed dramatically less inhibition with bupivacaine. Surprisingly, GIRK1/2 channels with high affinity for PIP 2 were inhibited by ethanol, like IRK1 channels. We propose that membrane-permeant local anesthetics inhibit GIRK channels by antagonizing the interaction of PIP 2 with the channel, which is essential for G β γ and ethanol activation of GIRK channels.

Stimulation of inhibitory neurotransmitter receptors, such as γ-aminobutyric acid type B (GABA B ) receptors, activates G protein-gated inwardly rectifying K + channels (GIRK) which, in turn, influence membrane excitability. Seizure activity has been reported in a Girk2 null mutant mouse lacking GIRK2 channels but showing normal cerebellar development as well as in the weaver mouse, which has mutated GIRK2 channels and shows abnormal development. To understand how the function of GIRK2 channels differs in these two mutant mice, we compared the G protein-activated inwardly rectifying K + currents in cerebellar granule cells isolated from Girk2 null mutant and weaver mutant mice with those from wild-type mice. Activation of GABA B receptors in wild-type granule cells induced an inwardly rectifying K + current, which was sensitive to pertussis toxin and inhibited by external Ba 2+ ions. The amplitude of the GABA B receptor-activated current was severely attenuated in granule cells isolated from both weaver and Girk2 null mutant mice. By contrast, the G protein-gated inwardly rectifying current and possibly the agonist-independent basal current appeared to be less selective for K + ions in weaver but not Girk2 null mutant granule cells. Our results support the hypothesis that a nonselective current leads to the weaver phenotype. The loss of GABA B receptor-activated GIRK current appears coincident with the absence of GIRK2 channel protein and the reduction of GIRK1 channel protein in the Girk2 null mutant mouse, suggesting that GABA B receptors couple to heteromultimers composed of GIRK1 and GIRK2 channel subunits.

G-protein gated inwardly rectifying potassium (GIRK) channels are implicated in alcohol abuse. Ethanol directly activates GIRKs through interaction with a discrete alcohol-binding pocket that we identified in the cytoplasmic domain. The structural mechanism underlying ethanol-dependent activation, however, is not well understood. Here, we hypothesized that chemically modifying the alcohol pocket with alcohol-like reagents would activate the channel. To examine this, we engineered GIRK2 containing a single cytoplasmic cysteine substitution in the βD-βE domain of the alcohol pocket; S246 is located near the edge of the pocket, and L257 is located within the pocket. All Cys substitutions were introduced into GIRK2c lacking four native Cys residues (GIRK2c(cys-)) and transiently expressed in HEK293T cells. We studied the effect of 2-Hydroxyethyl MTS (MTS-HE, alcohol-like) and two hydrophobic MTS reagents- Benzyl-MTS (MTS-Bn), and 4-hydroxy benzyl-MTS (MTS-Y), on basal (Ba2+-sensitive) and alcohol-activated GIRK currents. MTS-HE (100 μM) increased basal GIRK currents for S246C (34% ± 12%, n=7) and L257C (120% ± 24%, n=7). Similarly, MTS-Bn (10 μM) modification increased basal currents by 74% ± 18% (n=7) for S246C, and by 333% ± 31% (n=4) for L257C. MTS-Y (100 μM) increased the basal currents by 102% ± 25% (n= 5) for L257C but did not affect S246C. In addition, MTS modification decreased MPD-activated current. For L257C, we confirmed that intracellular application of MTS-HE produced a concentration-dependent increase in the time course of channel activation, having a rate constant of 90.6±17.2 M−1s−1 (n=11). Taken together, these data demonstrate that MTS modification of a single amino acid in the alcohol pocket can engage channel activation, and limit the access of bulkier diols (e.g., MPD). These results reveal novel mechanisms for chemical activation of GIRKs and underscore the importance of hydrophobic interactions in alcohol-mediated activation of GIRKs.

Parasympathetic nerve stimulation causes slowing of the heart rate by activation of muscarinic receptors and the subsequent opening of muscarinic K+ channels in the sinoatrial node and atrium. This inwardly rectifying K+ channel is coupled directly with G protein. Based on sequence homology with cloned inwardly rectifying K+ channels, ROMK1 (ref. 11) and IRK1 (ref. 12), we have isolated a complementary DNA for a G-protein-coupled inwardly rectifying K+ channel (GIRK1) from rat heart. The GIRK1 channel probably corresponds to the muscarinic K+ channel because (1) its functional properties resemble those of the atrial muscarinic K+ channel and (2) its messenger RNA is much more abundant in the atrium than in the ventricle. In addition, GIRK1 mRNA is expressed not only in the heart but also in the brain.

In addition to G proteins, ethanol can activate G protein-gated inwardly rectifying K (GIRK) channels. The mechanism underlying GIRK channel activation by alcohol is not well understood. Based on a crystal structure of a related IRK1 channel which contains the alcohol (2-methyl,2-4-pentanediol- MPD) bound to a cytoplasmic hydrophobic pocket, we used structure-based mutagenesis and patch-clamp electrophysiology to investigate the role of the homologous alcohol pocket in GIRK2 channels. In HEK293T cells transfected with GIRK2 cDNA, both ethanol and MPD activated GIRK2 channels. Replacing a conserved Leucine (L257) in this pocket with a bulkier Tyrosine or Tryptophan led to significant attenuation or loss of alcohol-dependent activation of GIRK2 channels, suggesting these larger hydrophobic side-chains filled the pocket. Based on structure and functional evidence, we conclude that this hydrophobic pocket is the site for alcohol activation of GIRK channels. We hypothesized that tethering a hydrophobic group near the pocket might mimic alcohol mediated activation of the channel. To test this idea, we introduced a S246C mutation in a Cysteine-less GIRK2 channel and examined the effect of bath applied MTS-Benzene. Application of 10 micromolar MTS-Benzene dramatically increased the size of basal GIRK currents by 336+66% n=5. This rapid activation was reversed by application of reducing agent DTT (10 mM), indicating a disulfide bond had formed. In addition to the change in basal current, MTS modification of S246C channel altered the rank order for alcohol activation -with significantly less activation by the larger alcohol MPD. These results suggest that attachment of a bulky hydrophobic amino acid near the hydrophobic alcohol-binding pocket can produce sustained activation of the channel by associating with the activation site. These experiments provide a launching point to study molecular events at this hydrophobic pocket that lead to activation of GIRK channels.

SummaryThe subcellular pathways that regulate G protein-gated inwardly rectifying potassium (GIRK or Kir3) channels are important for controlling the excitability of neurons. Sorting nexin 27 (SNX27) is a PDZ-containing protein known to bind GIRK2c/GIRK3 channels, but its function in vivo is poorly understood. Here, we investigated the role of SNX27 in regulating GIRK currents in dopamine (DA) neurons of the ventral tegmental area (VTA). Mice lacking SNX27 in DA neurons exhibited reduced GABABR-activated GIRK currents but had normal Ih currents and DA D2R-activated GIRK currents. Expression of GIRK2a, an SNX27-insensitive splice variant, restored GABABR-activated GIRK currents in SNX27-deficient DA neurons. Remarkably, mice with significantly reduced GABABR-activated GIRK currents in only DA neurons were hypersensitive to cocaine and could be restored to a normal locomotor response with GIRK2a expression. These results identify a pathway for regulating excitability of VTA DA neurons, highlighting SNX27 as a promising target for treating addiction.

Psychostimulants induce neuroadaptations in excitatory and fast inhibitory transmission in the ventral tegmental area (VTA). Mechanisms underlying drug-evoked synaptic plasticity of slow inhibitory transmission mediated by GABA(B) receptors and G protein-gated inwardly rectifying potassium (GIRK/Kir(3)) channels, however, are poorly understood. Here, we show that 1 day after methamphetamine (METH) or cocaine exposure both synaptically evoked and baclofen-activated GABA(B)R-GIRK currents were significantly depressed in VTA GABA neurons and remained depressed for 7 days. Presynaptic inhibition mediated by GABA(B)Rs on GABA terminals was also weakened. Quantitative immunoelectron microscopy revealed internalization of GABA(B1) and GIRK2, which occurred coincident with dephosphorylation of serine 783 (S783) in GABA(B2), a site implicated in regulating GABA(B)R surface expression. Inhibition of protein phosphatases recovered GABA(B)R-GIRK currents in VTA GABA neurons of METH-injected mice. This psychostimulant-evoked impairment in GABA(B)R signaling removes an intrinsic brake on GABA neuron spiking, which may augment GABA transmission in the mesocorticolimbic system.

The weaver mutation (G156S) in G-protein-gated inwardly rectifying K + (GIRK) channels alters ion selectivity and reveals sensitivity to inhibition by a charged local anesthetic, QX-314, applied extracellularly. In this paper, disrupting the ion selectivity in another GIRK channel, chimera I1G1(M), generates a GIRK channel that is also inhibited by extracellular local anesthetics. I1G1(M) is a chimera of IRK1 (G-protein-insensitive) and GIRK1 and contains the hydrophobic domains (M1–pore-loop–M2) of GIRK1 (G1(M)) with the N- and C-terminal domains of IRK1 (I1). The local anesthetic binding site in I1G1(M) is indistinguishable from that in GIRK2 wv channels. Whereas chimera I1G1(M) loses K + selectivity, although there are no mutations in the pore-loop complex, chimera I1G2(M), which contains the hydrophobic domain from GIRK2, exhibits normal K + selectivity. Mutation of two amino acids that are unique in the pore-loop complex of GIRK1 (F137S and A143T) restores K + selectivity and eliminates the inhibition by extracellular local anesthetics, suggesting that the pore-loop complex prevents QX-314 from reaching the intrapore site. Alanine mutations in the extracellular half of the M2 transmembrane domain alter QX-314 inhibition, indicating the M2 forms part of the intrapore binding site. Finally, the inhibition of G-protein-activated currents by intracellular QX-314 appears to be different from that observed in nonselective GIRK channels. The results suggest that inward rectifiers contain an intrapore-binding site for local anesthetic that is normally inaccessible from extracellular charged local anesthetics.

G protein-gated inwardly rectifying potassium (GIRK) channels play an important role in regulating neuronal excitability. Sorting nexin 27b (SNX27b), which reduces surface expression of GIRK channels through a PDZ domain interaction, contains a putative Ras-association (RA) domain with unknown function. Deleting the RA domain in SNX27b (SNX27b-ΔRA) prevents the down-regulation of GIRK2c/GIRK3 channels. Similarly, a point mutation (K305A) in the RA domain disrupts regulation of GIRK2c/GIRK3 channels and reduces H-Ras binding in vitro. Finally, the dominant-negative H-Ras (S17N) occludes the SNX27b-dependent decrease in surface expression of GIRK2c/GIRK3 channels. Thus, the presence of a functional RA domain and the interaction with Ras-like G proteins comprise a novel mechanism for modulating SNX27b control of GIRK channel surface expression and cellular excitability.

G-protein-gated potassium inwardly rectifying (GIRK) channels play an important role in regulating neuronal excitability in the brain. We have previously shown that trafficking and surface expression of GIRK channels are regulated by sorting nexin 27 (SNX27). SXN27 contains three domains; a phosphoinositide-binding Phox (PX) domain that targets SNX27 preferentially to early endosomes, a PDZ domain that associates directly with carboxyl-terminal tailof GIRK3 and GIRK2c subunits, and a Ras-associated RA domain that could potentially bind to small monomeric G proteins. The functional consequence of this interaction with the RA domain was unknown. Here we show for the first time that expression of a SNX27 mutant devoid of the RA domain (SNX27ΔRA) in HEK293T cells abrogates the down-regulation of baclofen-induced GIRK2c/3 currents that is typically observed with wild-type SNX27 (12.4±3.6 pA/pF (SNX27 WT group) vs. 55.8±8.2 pA/pF (SNX27ΔRA) vs. 47.1±6.7pA/pF (control group without SNX27); mean current density±SEM). Using bimolecular fluorescence complementation (BiFC) technique to follow the intracellular localization of GIRK2c/3 heterotetramers, we observed that SNX27 but not SNX27ΔRA increases the number of GIRK2c/3 heterotetramers in early endosomes. Moreover, a single point mutation within the RA domain (K305A) was sufficient to disrupt SNX27-dependent clustering of GIRK2c/3 heterotetramers, mimicking the functional effects of the RA deletion. Recently, it was shown that the RA domain of SNX27 interacts with H-Ras in vitro. We postulate that K305A disrupts RA interaction with H-Ras. In conclusion, we provide the first functional evidence that intact SNX27 RA domain and interaction with small monomeric G proteins is necessary for the regulation of GIRK channels. These new findings suggest that H-Ras signaling directly affects the magnitude of GIRK currents through regulation of SNX27.

Alcohols can activate G protein-gated inwardly rectifying K (GIRK) channels but the molecular mechanism is not well understood. To investigate the possibility of a physical alcohol pocket located in the cytoplasmic domain of GIRK channels, we used a crystal structure of related IRK1 channel containing a bound alcohol (2-methyl, 2-4-pentanediol - MPD) and structure-based mutagenesis of GIRK2 and GIRK4 channels combined with patch-clamp electrophysiology. In transiently transfected HEK293 cells, both wild-type GIRK2 and GIRK4 channels were activated by 100 mM ethanol, MPD and 1-Propanol. Replacing a conserved Leucine (GIRK2-L257 / GIRK4-L252) in the betaD-betaE region of the cytoplasmic domain with bulkier Tyrosine or Tryptophan led to significant attenuation or loss of alcohol-dependent activation for both GIRK2 and GIRK4 channels. Constitutively open channels, such as IRK1 and GIRK2-PIP2 (engineered to bind PIP2 with high affinity), on the other hand, were inhibited by ethanol, 1-propanol, 1-butanol and MPD. Mutating the homologous Leucine in IRK1 (L257) or in GIRK2-PIP2 channels did not alter the sensitivity to inhibition by these alcohols, suggesting a second site in the channel is involved in inhibition by alcohols. Consistent with this, mutagenesis of the extracellular pore-helix of GIRK4 and GIRK2-PIP2 channels reduced the sensitivity to alcohol-mediated inhibition. Interestingly, mutation of the conserved Leucine (L257/L252) in the betaD-betaE domain also disrupted G protein-dependent activation, suggesting a common mechanism of activation by alcohols and G-proteins. Using our data and an analysis of high-resolution structures of inwardly rectifying K channels, we propose a novel model for alcohol activation of GIRK channels that is mediated by the cytoplasmic hydrophobic alcohol pocket.

G protein-gated inwardly rectifying potassium (GIRK) channels are implicated in alcohol abuse and addiction. We discovered a discrete alcohol-binding pocket in the channel mediating ethanol-dependent activation. Here, we investigated the role of G proteins and PIP2 in ethanol-dependent gating. We engineered GIRK2 with single, modifiable cysteine at L257 in alcohol pocket and found that alcohol-like methanthiosulfonate (MTS) reagents activate GIRK2-L257C, similar to ethanol-dependent activation. We assessed role of G proteins in alcohol activation by either increasing levels of Gβγ (+Gβ1γ2) or decreasing Gβγ through chelation with membrane bound Phosducin (+mPhos). Neither Gβ1γ2 nor mPhos altered the rate of MTS-HE-mediated GIRK2-L257C activation. For comparison, we examined GIRK2-L344C, a key site for Gβγ activation that is inhibited by MTS modification. In contrast to L257C, rate of MTS modification showed a clear dependence on Gβγ levels. These results suggest that alcohol activation of GIRK channel is independent of G proteins.To investigate the role of PIP2, we used voltage-sensitive phosphatase DR-VSP to transiently deplete PIP2 in the membrane. Activation of DR-VSP completely reversed MTS-HE activated current of GIRK2-L257C. Furthermore, MTS-HE treatment significantly slowed the rate of GIRK2-L257C current inhibition following DR-VSP activation, suggesting an increase in apparent affinity for PIP2 and GIRK2-L257C channels modified by MTS-HE. Lastly, we examined the role of PIP2 on alcohol-dependent activation of wild-type GIRK2. Addition of propanol significantly slowed the rate of wild-type GIRK2 current inhibition following PIP2 depletion. Taken together, these data demonstrate that alcohol-dependent activation of GIRK involves an increase in apparent affinity for PIP2, with little influence of Gβγ subunits. The fundamental dichotomy between alcohol and Gβγ arises from distinct gating mechanisms converging on PIP2-dependent opening, revealing novel pathways for antagonizing alcohol's effects on ion channels.