Tetrameric Subunit Structure of the Native Brain Inwardly Rectifying Potassium Channel Kir 2.2*
- From the Department of Molecular, Cellular, and Developmental Biology, and Neuroscience Research Institute, University of California, Santa Barbara, California 93106
Abstract
Strongly inwardly rectifying potassium channels of the Kir 2 subfamily (IRK1, IRK2, and IRK3) are involved in maintenance and modulation of cell excitability in brain and heart. Electrophysiological studies of channels expressed in heterologous systems have suggested that the pore-conducting pathway contains four subunits. However, inferences from electrophysiological studies have not been tested on native channels and do not address the possibility of nonconducting auxiliary subunits. Here, we investigate the subunit stoichiometry of endogenous inwardly rectifying potassium channel Kir 2.2 (IRK2) from rat brain. Using chemical cross-linking, immunoprecipitiation, and velocity sedimentation, we report physical evidence demonstrating the tetrameric organization of the native channel. Kir 2.2 was sequentially cross-linked to produce bands on SDS-polyacrylamide gel electrophoresis corresponding in size to monomer, dimer, trimer, and three forms of tetramer. Fully cross-linked channel was present as a single band of tetrameric size. Immunoprecipitation of biotinylated membranes revealed a single band corresponding to Kir 2.2, suggesting that the channel is composed of a single type of subunit. Hydrodynamic properties of 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonic acid-solubilized channel were used to calculate the molecular mass of the channel. Velocity sedimentation in H2O or D2O gave a sharp peak with a sedimentation coefficient of 17.3 S. Gel filtration yielded a Stokes radius of 5.92 nm. These data indicate a multisubunit protein with a molecular mass of 193 kDa, calculated to contain 3.98 subunits. Together, these results demonstrate that Kir 2.2 channels are formed by the homotetrameric association of Kir 2.2 subunits and do not contain tightly associated auxiliary subunits. These studies suggest that Kir 2.2 channels differ in structure from related heterooctomeric ATP-sensitive K channels and heterotetrameric G-protein-regulated inward rectifier K channels.
Footnotes
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↵* This work was supported by National Institutes of Health Grant HL41656, California Tobacco-related Disease Research Program Grant 4RT-0289, and the American Heart Association, California Affiliate, Predoctoral Fellowship (to K. R.-G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‡ To whom correspondence should be addressed. Tel.: 805-893-8505; Fax: 805-893-2005; E-mail: vandenbe{at}lifesci.lscf.ucsb.edu.
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↵1 The abbreviations used are: IR channel, inwardly rectifying potassium channel; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonic acid; Kir 2.2, IRK2 inwardly rectifying potassium channel; TBS, Tris-buffered saline; PAGE, polyacrylamide gel electrophoresis.
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↵2 K. Raab-Graham, unpublished observations.
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- Received April 23, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
