Corey S, Clapham D E
Neuroscience Program, Mayo Foundation, Rochester, Minnesota 55905, USA.
J Biol Chem. 2001 Apr 6;276(14):11409-13. doi: 10.1074/jbc.M100058200. Epub 2001 Jan 8.
G-protein-coupled inwardly rectifying K(+) (GIRK; Kir3.x) channels are the primary effectors of numerous G-protein-coupled receptors. GIRK channels decrease cellular excitability by hyperpolarizing the membrane potential in cardiac cells, neurons, and secretory cells. Although direct regulation of GIRKs by the heterotrimeric G-protein subunit Gbetagamma has been extensively studied, little is known about the number of Gbetagamma binding sites per channel. Here we demonstrate that purified GIRK (Kir 3.x) tetramers can be chemically cross-linked to exogenously purified Gbetagamma subunits. The observed laddering pattern of Gbetagamma attachment to GIRK4 homotetramers was consistent with the binding of one, two, three, or four Gbetagamma molecules per channel tetramer. The fraction of channels chemically cross-linked to four Gbetagamma molecules increased with increasing Gbetagamma concentrations and approached saturation. These results suggest that GIRK tetrameric channels have four Gbetagamma binding sites. Thus, GIRK (Kir 3.x) channels, like the distantly related cyclic nucleotide-gated channels, are tetramers and exhibit a 1:1 subunit/ligand binding stoichiometry.
G蛋白偶联内向整流钾离子（GIRK；Kir3.x）通道是众多G蛋白偶联受体的主要效应器。GIRK通道通过使心肌细胞、神经元和分泌细胞的膜电位超极化来降低细胞兴奋性。尽管异源三聚体G蛋白亚基Gβγ对GIRK的直接调节已得到广泛研究，但每个通道Gβγ结合位点的数量却知之甚少。在此，我们证明纯化的GIRK（Kir 3.x）四聚体可与外源纯化的Gβγ亚基发生化学交联。观察到的Gβγ与GIRK4同型四聚体结合的梯状模式与每个通道四聚体结合一个、两个、三个或四个Gβγ分子一致。与四个Gβγ分子发生化学交联的通道比例随Gβγ浓度增加而增加并趋于饱和。这些结果表明GIRK四聚体通道有四个Gβγ结合位点。因此，GIRK（Kir 3.x）通道与远亲的环核苷酸门控通道一样，是四聚体，且呈现1:1的亚基/配体结合化学计量比。
