Lei Q, Jones M B, Talley E M, Schrier A D, McIntire W E, Garrison J C, Bayliss D A
Department of Pharmacology, University of Virginia, Charlottesville 22908, USA.
Proc Natl Acad Sci U S A. 2000 Aug 15;97(17):9771-6. doi: 10.1073/pnas.97.17.9771.
G protein-coupled inwardly rectifying potassium (GIRK) channels can be activated or inhibited by different classes of receptors, suggesting a role for G proteins in determining signaling specificity. Because G protein betagamma subunits containing either beta1 or beta2 with multiple Ggamma subunits activate GIRK channels, we hypothesized that specificity might be imparted by beta3, beta4, or beta5 subunits. We used a transfection assay in cell lines expressing GIRK channels to examine effects of dimers containing these Gbeta subunits. Inwardly rectifying K(+) currents were increased in cells expressing beta3 or beta4, with either gamma2 or gamma11. Purified, recombinant beta3gamma2 and beta4gamma2 bound directly to glutathione-S-transferase fusion proteins containing N- or C-terminal cytoplasmic domains of GIRK1 and GIRK4, indicating that beta3 and beta4, like beta1, form dimers that bind to and activate GIRK channels. By contrast, beta5-containing dimers inhibited GIRK channel currents. This inhibitory effect was obtained with either beta5gamma2 or beta5gamma11, was observed with either GIRK1,4 or GIRK1,2 channels, and was evident in the context of either basal or agonist-induced currents, both of which were mediated by endogenous Gbetagamma subunits. In cotransfection assays, beta5gamma2 suppressed beta1gamma2-activated GIRK currents in a dose-dependent manner consistent with competitive inhibition. Moreover, we found that beta5gamma2 could bind to the same GIRK channel cytoplasmic domains as other, activating Gbetagamma subunits. Thus, beta5-containing dimers inhibit Gbetagamma-stimulated GIRK channels, perhaps by directly binding to the channels. This suggests that beta5-containing dimers could act as competitive antagonists of other Gbetagamma dimers on GIRK channels.
G蛋白偶联内向整流钾通道（GIRK）可被不同类型的受体激活或抑制，这表明G蛋白在决定信号特异性方面发挥作用。由于含有β1或β2以及多个Gγ亚基的G蛋白βγ亚基可激活GIRK通道，我们推测特异性可能由β3、β4或β5亚基赋予。我们在表达GIRK通道的细胞系中使用转染试验来检测含有这些Gβ亚基的二聚体的作用。在表达β3或β4以及γ2或γ11的细胞中，内向整流钾电流增加。纯化的重组β3γ2和β4γ2直接结合到含有GIRK1和GIRK4 N端或C端胞质结构域的谷胱甘肽-S-转移酶融合蛋白上，这表明β3和β4与β1一样，形成与GIRK通道结合并激活它的二聚体。相比之下，含β5的二聚体抑制GIRK通道电流。β5γ2或β5γ11均可产生这种抑制作用，在GIRK1,4或GIRK1,2通道中均观察到这种作用，并且在基础电流或激动剂诱导电流的情况下均很明显，这两种电流均由内源性Gβγ亚基介导。在共转染试验中，β5γ2以与竞争性抑制一致的剂量依赖性方式抑制β1γ2激活的GIRK电流。此外，我们发现β5γ2可与其他激活Gβγ亚基一样结合到相同的GIRK通道胞质结构域。因此，含β5的二聚体可能通过直接结合通道来抑制Gβγ刺激的GIRK通道。这表明含β5的二聚体可能作为GIRK通道上其他Gβγ二聚体的竞争性拮抗剂发挥作用。
