Identification of a Potassium Channel Site That Interacts with G Protein βγ Subunits to Mediate Agonist-induced Signaling

He, Cheng; Zhang, Hailin; Mirshahi, Tooraj; Logothetis, Diomedes E.

doi:10.1074/jbc.274.18.12517

Cited by 111 publications

(154 citation statements)

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“…Previous reports suggested a difference in alcohol and receptor activation mechanisms (4,5). In support of this finding, the rate of MTS-HE-dependent inhibition of basal currents in L344C, part of the Gβγ binding site in the βL-βM loop (28)(29)(30), was dependent on Gβγ levels, whereas MTS-HE activation of L257C channels was unaffected by varying Gβγ levels. However, alcohol-dependent (this study), Gβγ-dependent (19), and Na +z -dependent (25,(40)(41)(42) activation all involve changes in PIP 2 -GIRK affinity, suggesting a convergence of Fig.…”

Section: Discussionsupporting

confidence: 75%

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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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Section: Discussionsupporting

confidence: 75%

“…Receptor-dependent Gβγ activation, however, relies on direct protein-protein interaction between GIRKs and G proteins (21,(28)(29)(30). Previous reports suggested a difference in alcohol and receptor activation mechanisms (4,5).…”

Section: Discussionmentioning

confidence: 99%

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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“…Tel./Fax: 81-3-3815-6540; E-mail: shimada@ iw-nmr.f.u-tokyo.ac.jp. 3 The abbreviations used are: GIRK, G protein-activated inwardly rectifying potassium channel; G␤␥, heterotrimeric G protein ␤␥ subunits; GIRK CP , cytoplasmic pore domain of GIRK; Kir, inwardly rectifying potassium channel; TM, transmembrane; CP, cytoplasmic pore; ITC, isothermal titration calorimetry; TROSY, transverse relaxation-optimized spectroscopy; TCS, transferred cross-saturation; CSP, chemical shift perturbation; PDB, Protein Data Bank.…”

Section: Expression and Purification Of The Cytoplasmic Regions Ofmentioning

confidence: 99%

“…G protein-activated inwardly rectifying potassium channel (GIRK) 3 is a member of the inwardly rectifying potassium channel (Kir) family, which regulates heart rate and neuronal excitability (1,2). The Kir proteins function as tetramers, consisting of a transmembrane (TM) region and a cytoplasmic pore (CP) region.…”

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NMR Analyses of the Gβγ Binding and Conformational Rearrangements of the Cytoplasmic Pore of G Protein-activated Inwardly Rectifying Potassium Channel 1 (GIRK1)

Yokogawa

Osawa

Takeuchi

et al. 2011

Journal of Biological Chemistry

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G protein-activated inwardly rectifying potassium channel (GIRK) plays crucial roles in regulating heart rate and neuronal excitability in eukaryotic cells. GIRK is activated by the direct binding of heterotrimeric G protein ␤␥ subunits (G␤␥) upon stimulation of G protein-coupled receptors, such as M2 acetylcholine receptor. The binding of G␤␥ to the cytoplasmic pore (CP) region of GIRK causes structural rearrangements, which are assumed to open the transmembrane ion gate. However, the crucial residues involved in the G␤␥ binding and the structural mechanism of GIRK gating have not been fully elucidated. Here, we have characterized the interaction between the CP region of GIRK and G␤␥, by ITC and NMR. The ITC analyses indicated that four G␤␥ molecules bind to a tetramer of the CP region of GIRK with a dissociation constant of 250 M. The NMR analyses revealed that the G␤␥ binding site spans two neighboring subunits of the GIRK tetramer, which causes conformational rearrangements between subunits. A possible binding mode and mechanism of GIRK gating are proposed.G protein-activated inwardly rectifying potassium channel (GIRK) 3 is a member of the inwardly rectifying potassium channel (Kir) family, which regulates heart rate and neuronal excitability (1, 2). The Kir proteins function as tetramers, consisting of a transmembrane (TM) region and a cytoplasmic pore (CP) region. The helix bundle at the cytoplasmic side of the TM region is assumed to be a K ϩ -ion gate. The opening and closing in the gate (gating) of Kirs are regulated by a variety of cytoplasmic factors. The gating of GIRK is triggered by the binding of its CP region with the heterotrimeric G-protein ␤␥ subunits (G␤␥), which are released from the pertussis toxin-sensitive G protein ␣ subunit (G␣ i/o ), subsequent to the stimulation of a G protein-coupled receptor, such as M2 acetylcholine receptor.Extensive mutational analyses to identify the GIRK residues that are critical for the G␤␥-induced activation revealed several critical residues such as His 57 , Leu 262 , Leu 333, and Gly 336 of GIRK1 (3-5). However, when these residues were mapped on the recently reported crystal structures of Kirs, they did not form a cluster on the protein surface (6 -9). Therefore, no clear consensus has been obtained regarding the region of GIRK that is essential for G␤␥ binding and/or GIRK activation. One of the reasons might be a structural alteration introduced by the mutagenesis (10), which could change the gating property of the channel. Although the various crystal structures have provided little information about the conformational change involved in the gating of the channel, FRET analyses have clearly demonstrated the conformational rearrangements in the CP region of GIRK upon G␤␥ binding (11). However, the resolution of the structural information obtained by the FRET analyses is low, primarily due to the large size of the fluorescent probe proteins.To reveal the structural mechanism by which G␤␥ binding activates GIRK, we have investigated the direct interacti...

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K ⁺ Channels: Function‐Structural Overview

González

Báez-Nieto

Valencia

et al. 2012

Comprehensive Physiology

210

139

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Potassium channels are particularly important in determining the shape and duration of the action potential, controlling the membrane potential, modulating hormone secretion, epithelial function and, in the case of those K + channels activated by Ca 2+ , damping excitatory signals. The multiplicity of roles played by K + channels is only possible to their mammoth diversity that includes at present 70 K + channels encoding genes in mammals. Today, thanks to the use of cloning, mutagenesis, and the more recent structural studies using x‐ray crystallography, we are in a unique position to understand the origins of the enormous diversity of this superfamily of ion channels, the roles they play in different cell types, and the relations that exist between structure and function. With the exception of two‐pore K + channels that are dimers, voltage‐dependent K + channels are tetrameric assemblies and share an extremely well conserved pore region, in which the ion‐selectivity filter resides. In the present overview, we discuss in the function, localization, and the relations between function and structure of the five different subfamilies of K + channels: (a) inward rectifiers, Kir; (b) four transmembrane segments‐2 pores, K 2P ; (c) voltage‐gated, Kv; (d) the Slo family; and (e) Ca 2+ ‐activated SK family, SKCa. © 2012 American Physiological Society. Compr Physiol 2:2087‐2149, 2012.

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Identification of a Potassium Channel Site That Interacts with G Protein βγ Subunits to Mediate Agonist-induced Signaling

Cited by 111 publications

References 31 publications

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

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

NMR Analyses of the Gβγ Binding and Conformational Rearrangements of the Cytoplasmic Pore of G Protein-activated Inwardly Rectifying Potassium Channel 1 (GIRK1)

K ⁺ Channels: Function‐Structural Overview

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Identification of a Potassium Channel Site That Interacts with G Protein βγ Subunits to Mediate Agonist-induced Signaling

Cited by 111 publications

References 31 publications

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

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

NMR Analyses of the Gβγ Binding and Conformational Rearrangements of the Cytoplasmic Pore of G Protein-activated Inwardly Rectifying Potassium Channel 1 (GIRK1)

K + Channels: Function‐Structural Overview

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K ⁺ Channels: Function‐Structural Overview