Gating of a G protein-sensitive Mammalian Kir3.1 Prokaryotic Kir Channel Chimera in Planar Lipid Bilayers

Edgar Leal-Pinto,Yacob Gómez-Llorente,Shobana Sundaram,Qiong-Yao Tang,Tatyana Ivanova-Nikolova,Rahul Mahajan,Lia Baki,Zhe Zhang,Jose Chavez,Iban Ubarretxena-Belandia,Diomedes E Logothetis

doi:10.1074/jbc.m110.151373

Edgar Leal-Pinto, Yacob Gómez-Llorente + Show 9 more

Open Access

https://doi.org/10.1074/jbc.m110.151373

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Abstract

Kir3 channels control heart rate and neuronal excitability through GTP-binding (G) protein and phosphoinositide signaling pathways. These channels were the first characterized effectors of the βγ subunits of G proteins. Because we currently lack structures of complexes between G proteins and Kir3 channels, their interactions leading to modulation of channel function are not well understood. The recent crystal structure of a chimera between the cytosolic domain of a mammalian Kir3.1 and the transmembrane region of a prokaryotic KirBac1.3 (Kir3.1 chimera) has provided invaluable structural insight. However, it was not known whether this chimera could form functional K(+) channels. Here, we achieved the functional reconstitution of purified Kir3.1 chimera in planar lipid bilayers. The chimera behaved like a bona fide Kir channel displaying an absolute requirement for PIP(2) and Mg(2+)-dependent inward rectification. The channel could also be blocked by external tertiapin Q. The three-dimensional reconstruction of the chimera by single particle electron microscopy revealed a structure consistent with the crystal structure. Channel activity could be stimulated by ethanol and activated G proteins. Remarkably, the presence of both activated Gα and Gβγ subunits was required for gating of the channel. These results confirm the Kir3.1 chimera as a valid structural and functional model of Kir3 channels.

Highlights

GTP-binding (G)6 protein-sensitive potassium (Kϩ) channels comprise the third subfamily of inwardly rectifying (Kir) channels, so called as they conduct more current in the inward than outward direction
Our study has succeeded in functionally reconstituting the Kir3.1 chimera in planar lipid bilayers and demonstrating that it behaves like a bona fide Kir3 channel: it displays sensitivity to PIP2 and its scavengers as well as Mg2ϩ-dependent inward rectification from the internal membrane side, and sensitivity to
Channel activity displayed an absolute requirement for PIP2, the lack of which potentially explains previous unsuccessful attempts to functionally reconstitute it [17]

Summary

Introduction

GTP-binding (G) protein-sensitive potassium (Kϩ) channels comprise the third subfamily of inwardly rectifying (Kir) channels, so called as they conduct more current in the inward than outward direction. Like all Kir family members, Kir channels depend on phosphoinositides to maintain their activity [1,2,3]. Kir3.1 channels do not form functional homomers and they localize poorly to the cell surface (e.g. Ref. 21). They potentiate the activity of other Kir channels upon assembly into heteromeric complexes (e.g. Refs. ). A model emerging from such studies proposes that opening of the cytosolic gates occurs as the cytosolic domains of Kir channels get tethered to the plasma membrane by virtue of electrostatic interactions between the acidic phosphoinositides and basic binding pockets on the channel surface near the inner leaflet of the lipid bilayer [26, 28]. Ethanol has been shown to activate Kir channels [6, 7]

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