Lorenz E, Alekseev A E, Krapivinsky G B, Carrasco A J, Clapham D E, Terzic A
Department of Medicine, Mayo Clinic, Mayo Foundation, Rochester, Minnesota 55905, USA.
Mol Cell Biol. 1998 Mar;18(3):1652-9. doi: 10.1128/MCB.18.3.1652.
Structurally unique among ion channels, ATP-sensitive K+ (KATP) channels are essential in coupling cellular metabolism with membrane excitability, and their activity can be reconstituted by coexpression of an inwardly rectifying K+ channel, Kir6.2, with an ATP-binding cassette protein, SUR1. To determine if constitutive channel subunits form a physical complex, we developed antibodies to specifically label and immunoprecipitate Kir6.2. From a mixture of Kir6.2 and SUR1 in vitro-translated proteins, and from COS cells transfected with both channel subunits, the Kir6.2-specific antibody coimmunoprecipitated 38- and 140-kDa proteins corresponding to Kir6.2 and SUR1, respectively. Since previous reports suggest that the carboxy-truncated Kir6.2 can form a channel independent of SUR, we deleted 114 nucleotides from the carboxy terminus of the Kir6.2 open reading frame (Kir6.2deltaC37). Kir6.2deltaC37 still coimmunoprecipitated with SUR1, suggesting that the distal carboxy terminus of Kir6.2 is unnecessary for subunit association. Confocal microscopic images of COS cells transfected with Kir6.2 or Kir6.2deltaC37 and labeled with fluorescent antibodies revealed unique honeycomb patterns unlike the diffuse immunostaining observed when cells were cotransfected with Kir6.2-SUR1 or Kir6.2deltaC37-SUR1. Membrane patches excised from COS cells cotransfected with Kir6.2-SUR1 or Kir6.2deltaC37-SUR1 exhibited single-channel activity characteristic of pancreatic KATP channels. Kir6.2deltaC37 alone formed functional channels with single-channel conductance and intraburst kinetic properties similar to those of Kir6.2-SUR1 or Kir6.2deltaC37-SUR1 but with reduced burst duration. This study provides direct evidence that an inwardly rectifying K+ channel and an ATP-binding cassette protein physically associate, which affects the cellular distribution and kinetic behavior of a KATP channel.
ATP敏感性钾离子（KATP）通道在离子通道中结构独特，在将细胞代谢与膜兴奋性耦合方面至关重要，其活性可通过内向整流钾离子通道Kir6.2与ATP结合盒蛋白SUR1共表达来重建。为了确定组成型通道亚基是否形成物理复合物，我们制备了特异性标记和免疫沉淀Kir6.2的抗体。在体外翻译的Kir6.2和SUR1蛋白混合物中，以及在用两种通道亚基转染的COS细胞中，Kir6.2特异性抗体共免疫沉淀了分别对应于Kir6.2和SUR1的38 kDa和140 kDa蛋白。由于先前的报道表明羧基末端截短的Kir6.2可以形成独立于SUR的通道，我们从Kir6.2开放阅读框的羧基末端删除了114个核苷酸（Kir6.2deltaC37）。Kir6.2deltaC37仍与SUR1共免疫沉淀，这表明Kir6.2的远端羧基末端对于亚基缔合并非必需。用Kir6.2或Kir6.2deltaC37转染并用荧光抗体标记的COS细胞的共聚焦显微镜图像显示出独特的蜂窝状模式，这与用Kir6.2 - SUR1或Kir6.2deltaC37 - SUR1共转染细胞时观察到的弥漫性免疫染色不同。从用Kir6.2 - SUR1或Kir6.2deltaC37 - SUR1共转染的COS细胞中切除的膜片表现出胰腺KATP通道的单通道活性特征。单独的Kir6.2deltaC37形成了具有与Kir6.2 - SUR1或Kir6.2deltaC37 - SUR1相似的单通道电导和爆发内动力学特性的功能性通道，但爆发持续时间缩短。这项研究提供了直接证据，即内向整流钾离子通道和ATP结合盒蛋白发生物理缔合，这会影响KATP通道的细胞分布和动力学行为。
