Tetrameric subunit structure of the native brain inwardly rectifying potassium channel Kir 2.2.
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.
Department of Molecular, Cellular, and Developmental Biology, and Neuroscience Research Institute, University of California, Santa Barbara, California 93106, USA.
K F
KF
Raab-Graham
C A
CA
Vandenberg
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism*; D002462:Cell Membrane / Q000737:chemistry; D002499:Centrifugation, Density Gradient; D003432:Cross-Linking Reagents / Q000378:metabolism; D003902:Detergents / Q000378:metabolism; D005976:Glutaral / Q000378:metabolism; D015151:Immunoblotting; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000276:immunology; D015221:Potassium Channels / Q000737:chemistry*; D024661:Potassium Channels, Inwardly Rectifying; D011487:Protein Conformation; D051381:Rats
D003432:Cross-Linking Reagents; D003902:Detergents; D010446:Peptide Fragments; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; C089327:inward rectifier potassium channel 2; D005976:Glutaral