X-ray structure of the mammalian GIRK2–βγ G-protein complex

Whorton, Matthew R.; MacKinnon, Roderick

doi:10.1038/nature12241

Cited by 272 publications

(376 citation statements)

References 62 publications

(78 reference statements)

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“…We postulated that Na ϩ binds to a site defined, in part, by acidic residues, consistent with Na ϩ -bound protein structures (43,44) and the predominance of oxygen atoms coordinating Na ϩ in model small molecules (45). We also reasoned that the preference of the channel for Na ϩ over other cations as an inhibitory effector originates from the binding site(s) rather than from subsequent transduction steps.…”

Section: Discussionmentioning

confidence: 68%

Na+ Inhibits the Epithelial Na+ Channel by Binding to a Site in an Extracellular Acidic Cleft

Kashlan

Blobner

Zuzek

et al. 2015

Journal of Biological Chemistry

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Section: Discussionmentioning

confidence: 68%

Na+ Inhibits the Epithelial Na+ Channel by Binding to a Site in an Extracellular Acidic Cleft

Kashlan

Blobner

Zuzek

et al. 2015

Journal of Biological Chemistry

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“…With PIP 2 present, GIRK channels are "primed" for activation. Indeed, Gβγ binding (in the presence of PIP 2 ) leads to opening of the inner-helical gate and the G-loop gate, which is formed by the inner face of the cytosolic domains; Gβγ in the absence of PIP 2 can only open the G-loop gate (27,30). Our data suggest that ML297, like other channel agonists, ultimately activates GIRK channels by opening inner-helical and G-loop gates.…”

Section: Discussionmentioning

confidence: 74%

“…Clear resolution of the channel-Gβγ interaction was obtained with the cocrystallization of Gβγ and a GIRK2 homomer (27). Gβγ binds to an outward-facing surface created by two adjacent 1% DMSO), baclofen (100 μM), and ML297 (10 μM) on holding currents in neurons from wild-type (Upper) and Girk1 −/− (Lower) mice.…”

Section: Discussionmentioning

confidence: 99%

Mechanisms underlying the activation of G-protein–gated inwardly rectifying K⁺(GIRK) channels by the novel anxiolytic drug, ML297

Wydeven¹,

Velasco²,

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

136

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ML297 was recently identified as a potent and selective small molecule agonist of G-protein-gated inwardly rectifying K + (GIRK/Kir3) channels. Here, we show ML297 selectively activates recombinant neuronal GIRK channels containing the GIRK1 subunit in a manner that requires phosphatidylinositol-4,5-bisphosphate (PIP 2 ), but is otherwise distinct from receptor-induced, G-protein-dependent channel activation. Two amino acids unique to the pore helix (F137) and second membrane-spanning (D173) domain of GIRK1 were identified as necessary and sufficient for the selective activation of GIRK channels by ML297. Further investigation into the behavioral effects of ML297 revealed that in addition to its known antiseizure efficacy, ML297 decreases anxiety-related behavior without sedative or addictive liabilities. Importantly, the anxiolytic effect of ML297 was lost in mice lacking GIRK1. Thus, activation of GIRK1-containing channels by ML297 or derivatives may represent a new approach to the treatment of seizure and/or anxiety disorders.electrophysiology | structure-activity relationship S ignal transduction involving inhibitory (G i/o ) G proteins titrates the excitability of neurons, cardiac myocytes, and endocrine cells, influencing behavior, cardiac output, and energy homeostasis (1). G-protein-gated inwardly rectifying potassium (K + ) (GIRK/Kir3) channels are a common effector for G i/odependent signaling pathways in the heart and nervous system (2, 3). Polymorphisms and mutations in human GIRK channels have been linked to arrhythmias, hyperaldosteronism (and associated hypertension), schizophrenia, sensitivity to analgesics, and alcohol dependence (1).GIRK channels are activated by binding of the G protein Gβγ subunit (1-3). Gβγ binding strengthens channel affinity for phosphatidylinositol-4,5-bisphosphate (PIP 2 ), a necessary cofactor for channel gating (4, 5). GIRK channels are also activated in a G-protein-independent manner by ethanol (6, 7), volatile anesthetics (8, 9), and naringin (10). Many psychoactive and clinically relevant compounds with other primary molecular targets inhibit GIRK channels, albeit at relatively high doses (1, 11). The lack of selective GIRK channel modulators, and in particular, drugs that discriminate among GIRK channel subtypes, has hampered investigation into their physiological relevance and therapeutic potential.GIRK channels are homo-and heterotetramers formed by GIRK1, GIRK2, GIRK3, and GIRK4 subunits (2, 3). GIRK subunits exhibit overlapping but distinct cellular expression patterns, potentially yielding multiple channel subtypes (1). Although it cannot form functional homomers (12), GIRK1 is an integral subunit of the cardiac GIRK channel and most neuronal GIRK channels (13,14). GIRK1 confers robust basal and receptordependent activity to GIRK heteromers, attributable in part to unique residues in the pore and second transmembrane domain (15-17). The intracellular C-terminal domain also contributes to the potentiating influence of GIRK1 on channel activity, likely due to th...

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“…After submission of our study, Whorton and MacKinnon described the high-resolution structure of Gβγ in complex with GIRK2 (51). The overlap between the Gβγ sites of interaction and the alcohol pocket is striking (Fig.…”

Section: Discussionmentioning

confidence: 93%

Molecular mechanism underlying ethanol activation of G-protein–gated inwardly rectifying potassium channels

Bodhinathan

Slesinger

2013

Proc. Natl. Acad. Sci. U.S.A.

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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 alcoholsensitive proteins, revealing a unique protein microdomain for targeting alcohol-selective therapeutics in the treatment of alcoholism and addiction.A lcohol (ethanol) produces a wide range of pharmacological effects on the nervous system, ranging from anxiolytic effects to intoxication and alcohol addiction in certain individuals. Although the neural circuits underlying such addictive disorders are becoming better understood (1, 2), little is known about the molecular mechanisms underlying ethanol's interaction with specific target proteins, such as ion channels. G-proteingated inwardly rectifying K + (GIRK or Kir3) channels are activated by concentrations of ethanol relevant to human consumption (18 mM ethanol or 0.08% blood alcohol level) (3-5) and have been found to play a key role in alcohol-related disorders (6-9). For example, mice lacking GIRK2 (or Kir3.2) channels self-administer more ethanol and fail to develop conditioned place preference for ethanol, compared with wild-type (WT) littermates (6, 10). These results support a model in which ethanol may have lost its target in GIRK knockout mice, thus failing to elicit behaviors associated with ethanol consumption. Receptor activation of GIRK channels generates an outward, slow inhibitory postsynaptic current, which reduces neuronal activity (11). In addition to directly activating GIRKs, ethanol potentiates the slow inhibitory postsynaptic potential in midbrain dopamine neurons of the ventral tegmental area (8), which is produced by GABA B receptor activation of GIRK channels (7,12,13). Together these observations implicate GIRK channels in the etiology of alcohol dependence and addiction; however, the molecular details underlying ethanol activation of GIRK channels remain unknown.A major challenge is to understand how ethanol, with its simple chemistry of only two carbons and a hydroxyl, can produce behavioral changes with rapidity and reproducibility. Ethanol has little volume or distinguishing stereochemistry. Although it was once thought to interact nonspecifically with membrane lipids, a preponderance of evidence sugg...

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X-ray structure of the mammalian GIRK2–βγ G-protein complex

Cited by 272 publications

References 62 publications

Na+ Inhibits the Epithelial Na+ Channel by Binding to a Site in an Extracellular Acidic Cleft

Na+ Inhibits the Epithelial Na+ Channel by Binding to a Site in an Extracellular Acidic Cleft

Mechanisms underlying the activation of G-protein–gated inwardly rectifying K⁺(GIRK) channels by the novel anxiolytic drug, ML297

Molecular mechanism underlying ethanol activation of G-protein–gated inwardly rectifying potassium channels

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X-ray structure of the mammalian GIRK2–βγ G-protein complex

Cited by 272 publications

References 62 publications

Na+ Inhibits the Epithelial Na+ Channel by Binding to a Site in an Extracellular Acidic Cleft

Na+ Inhibits the Epithelial Na+ Channel by Binding to a Site in an Extracellular Acidic Cleft

Mechanisms underlying the activation of G-protein–gated inwardly rectifying K+(GIRK) channels by the novel anxiolytic drug, ML297

Molecular mechanism underlying ethanol activation of G-protein–gated inwardly rectifying potassium channels

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Mechanisms underlying the activation of G-protein–gated inwardly rectifying K⁺(GIRK) channels by the novel anxiolytic drug, ML297