Regulation of ion channels by GTP-binding Proteins

GTP结合蛋白(G蛋白)由α、β、γ亚基组成，将信号从激动剂结合的受体传递到效应器，如酶或离子通道。由于霍乱或百日咳毒素将NAD^+的ADP-核糖基部分转移到G蛋白的α亚基，从而影响G蛋白的信号转导功能，毒素催化的ADP-核糖化被广泛地用作研究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蛋白亚基对内向整流性K~+通道和腺苷环化酶活性的调节机制。1)G蛋白的β亚基激活内向整流性K~+通道。豚鼠心肌细胞的内向整流性K~+通道可被M胆碱能或A^1-嘌呤能受体激活，这种激活对百日咳毒素敏感。在细胞…中由内向外的斑块膜越多，G蛋白的β亚基越能激活K~+通道。β-氨基丁酸诱导的K^+通道激活的特性与GTP依赖受体介导的K^+通道激活的特性相同。此外，受体介导的K~+通道激活可被转导蛋白G_t的GDP结合形式选择性地抑制。这些结果表明，内向整流性K~+通道偶联到膜结合受体的激活是通过从α-氨基丁酸中分解的β-氨基丁酸的作用来实现的。2)蛋白激酶C激活腺苷酸环化酶。在蛋白激酶C激活后，细胞内环状腺苷的形成会发生改变。但是，在环状腺苷形成途径中，被该酶修饰的位置(S)尚不清楚。因此，我们以纯化的蛋白为材料，研究了蛋白激酶C对腺苷环化酶活性的影响。蛋白激酶C可以直接磷酸化V型腺酰环化酶，使其催化活性提高20倍。这种激活作用远大于Forsklin刺激的激活(5倍)。当Forsklin和蛋白磷酸化结合时，5型环化酶活性比基础水平提高了100倍以上。因此，蛋白激酶C似乎直接并有效地激活腺苷环化酶。较少

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