Raab-Graham K F, Vandenberg C A
Department of Molecular, Cellular, and Developmental Biology, and Neuroscience Research Institute, University of California, Santa Barbara, California 93106, USA.
J Biol Chem. 1998 Jul 31;273(31):19699-707. doi: 10.1074/jbc.273.31.19699.
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.
Kir 2亚家族的强内向整流钾通道（IRK1、IRK2和IRK3）参与大脑和心脏细胞兴奋性的维持与调节。对在异源系统中表达的通道进行的电生理研究表明，孔传导途径包含四个亚基。然而，电生理研究得出的推论尚未在天然通道上得到验证，也未涉及非传导性辅助亚基的可能性。在此，我们研究了来自大鼠大脑的内源性内向整流钾通道Kir 2.2（IRK2）的亚基化学计量。通过化学交联、免疫沉淀和速度沉降，我们报告了证明天然通道四聚体结构的物理证据。Kir 2.2被依次交联，在SDS聚丙烯酰胺凝胶电泳上产生与单体、二聚体、三聚体以及三种四聚体形式相对应大小的条带。完全交联的通道以单一四聚体大小的条带形式存在。对生物素化膜进行免疫沉淀显示出一条对应于Kir 2.2的单一条带，表明该通道由单一类型的亚基组成。用3-[(3-胆酰胺丙基)二甲基铵]-1-丙烷磺酸溶解的通道的流体动力学性质用于计算通道的分子量。在H2O或D2O中进行速度沉降产生了一个沉降系数为17.3 S的尖锐峰。凝胶过滤得到的斯托克斯半径为5.92 nm。这些数据表明这是一种分子量为193 kDa的多亚基蛋白质，经计算含有3.98个亚基。总之，这些结果表明Kir 2.2通道是由Kir 2.2亚基的同型四聚体缔合形成的，并且不包含紧密结合的辅助亚基。这些研究表明，Kir 2.2通道在结构上不同于相关的异源八聚体ATP敏感性钾通道和异源四聚体G蛋白调节内向整流钾通道。
