Regulation of ion channels by GTP-binding Proteins

GTP结合蛋白（G蛋白）由α、β、γ亚基组成，将激动剂结合受体的信号传递给效应物，如酶或离子通道。由于G蛋白作为信号转导分子的功能受到霍乱毒素或百日咳毒素的深刻影响，毒素将NAD^+的ADP-核糖基部分转移到G蛋白的α亚基上，因此毒素催化的ADP-核糖基化被广泛用作研究G蛋白在信号转导过程中的作用的工具。在本研究中，我们研究了G蛋白亚基调节内向整流钾通道和腺苷酸环化酶活性的机制：1）G蛋白β-γ亚基激活内向整流钾通道。豚鼠心房细胞内的内向整流钾离子通道可被毒蕈碱胆碱能或A^1-嘌呤能受体激活，激活方式与百日咳毒素敏感。在细胞的由内而外的小块中， ...更多信息 在细胞膜上，G蛋白的β-γ亚基激活了K^+通道。β-γ射线诱导的K^+通道激活的特性似乎与GTP依赖性受体介导的K^+通道激活中观察到的特性相同。此外，G_t（transducin）的GDP结合形式选择性地抑制受体介导的K^+通道激活。这些结果表明，与膜结合受体偶联的内向整流K^+通道的激活是通过从α-氨基甲烷中分离的β-氨基甲烷亚单位的作用介导的。2）蛋白激酶C激活腺苷酸环化酶。蛋白激酶C激活后，细胞内环腺苷酸的形成发生改变。然而，在环腺苷酸形成途径中，激酶修饰的位点仍不清楚。因此，用纯化的蛋白质研究了蛋白激酶C对腺苷酸环化酶活性的影响。蛋白激酶C可以直接磷酸化腺苷酸环化酶V型，导致其催化活性增加20倍。这种激活比毛喉素刺激的激活大得多（5倍）。当毛喉素和蛋白磷酸化相结合，5型环化酶活性增加超过100倍的基础。因此，似乎蛋白激酶C直接和有效地激活腺苷酸环化酶。少

GTP-binding protein (G proteins) consisting of alpha, beta, gamma subunits, carry signals from agonist-bound receptors to effectors such as enzymes or ion channels. Since the functions of G proteins as the signal transducers are profoundly affected by cholera or pertussis toxin which transfers the ADP-ribosyl moiety of NAD^+ to the alpha subunits of G proteins, the toxin-catalyzed ADP-ribosylation has widely been used as a tool to study the roles of G proteins in signal transduction processes. In the present studies, we investigated mechanisms by which the activities of an inwardly rectifying K^+ channel and adenylyl cyclase are regulated by the subunits of G proteins.1) Activation of inwardly rectifying K^+ channels by the betagamma subunits of G proteins. An inwardly rectifying K^+ channel in guinea pig cardiac atrial cells is activated by the stimulation of muscarinic cholinergic or A^1-purinergic receptors in a manner sensitive to pertussis toxin. In inside-out patches of the cell … More membranes, the betagamma subunits of G proteins activated the K^+ channels. Properties of the betagamma-induced activation of the K^+ channel appeared to be the same as those observed in GTP-dependent receptor-mediated activation of the K^+ channel. Moreover, the receptor-mediated activation of the K^+ channel was selectively inhibited by GDP-bound from of G_t (transducin). These results indicate that activation of inwardly rectifying K^+ channel coupling to membrane-bound receptors is mediated through the action] of betagammasubunits resolved from alphabetagammatrimer.2) Activation of adenylyl cyclase by protein kinase C.Cyclic AMP formation within cells is altered upon the activation of protein kinase C.However, the site (s) to be modified by the kinase in the cyclic AMP formation pathway remains unclear. Thus, effects of protein kinase C on the activity of adenylyl cyclase were investigated with purified proteins. Protein kinase C could phosphorylate adenylyl cyclase type V directly, leading to a 20-fold increase in its catalytic activity. This activation was much larger than that stimulated by forskolin (5 fold). When forskolin and the protein phosphorylation were combined, the type 5 cyclase activity was increased more than 100 fold over the basal. Thus, it appears that protein kinase C directly and potently activates adenylyl cyclase. Less