Studies on the transmembrane signalling mechanism of neurotransmitter receptors and ion channels in Xenopus oocytes injected with rat brain mRNA

为了阐明神经递质受体和离子通道的跨膜信号传导机制，我们利用注射大鼠脑mRNA的爪蟾卵母细胞，从生化和电生理上研究了几种药物对乙酰胆碱（Ach）或血清素（5-HT）诱导的膜电流和电压敏感钙通道（VSCC）活性的影响。我们在这里得到了以下结果。Ach/5-HT分别通过激活M_1或S_1受体和百日咳毒素敏感的gtp结合蛋白(G。/ G)。Ach/5-HT诱导的电流反应还可能涉及以下细胞内事件：1)磷脂酶C形成肌醇磷酸（IP_3）， 2)IP_3诱导储存细胞器释放Ca^<2+>, 3)Ca^<2+>。钙调素介导的膜C1通道的门控。此外，卵母细胞对Ach/5-HT的快速耐受性可以通过二酰基甘油和Ca^<2+>激活的蛋白激酶C磷酸化受体和/或G_i/G_o来解释。我们还观察了注射脑mRNA的卵母细胞中VSCC的表达，其特征如下：1) VSCC似乎是n型的，因为w- concontoxin GVTA有效地阻断了VSCC，而有机Ca拮抗剂没有；2)蛋白激酶A刺激VSCC，是因为环状amp诱导的VSCC电流的促进；3)蛋白激酶C以双相方式调节通道门控，因为phorbol酯（TPA）刺激低浓度影响VSCC电流，但在高浓度时具有抑制作用。因此，我们在此证明了脑mRNA微注射的爪蟾卵母细胞作为一个有用的模型来阐明从受体到离子通道的信号转导在中枢神经系统中的分子机制。

To clarify the transmembrane signalling mechanism in neurotransmitter receptors and ion channels, we biochemically and electrophysiologically investigated effects of several drugs on acetylcholine (Ach)- or serotonin (5-HT)-induced membrane current and on voltage-sensitive Ca channel (VSCC) activity using Xenopus oocytes injected with rat brain mRNA. We here obtained following results.Ach/5-HT elicited inward C1 current by activating M_1 or S_1 receptors respectively and pertussis toxinsensitive GTP-binding proteins(G./G ). The involvement of following intracellular events could also be suggested in the Ach/5-HT elicited current responses: 1)inositol phosphate (IP_3) formation by phospholipase C, 2)IP_3-induced release of Ca^<2+> from storage organella and 3)Ca^<2+>. calmodulin-mediated gating of membrane C1 channels. In addition, rapid refractoriness in oocytes to Ach/5-HT could be explained by phosphorylation of receptors and / or G_i/G_o by protein kinase C activated by diacylglycerol and Ca^<2+>.We also observed expression of VSCC in oocytes injected with brain mRNA and characterized as follows: 1) VSCC seems to be N-type, since w-conotoxin GVTA effectively blocked but organic Ca antagonists did not , 2) protei kinase A stimulates VSCC because of cyclic AMP-induced facilitation of VSCC current, and 3) protein kinase C modulates the channel gating in a biphasic manner, since a phorbol ester (TPA) stimulatorily affected VSCC current at a low concentration but inhibitorily at a high concentration.Thus we herewith demonstrated Xenopus oocytes microinjected with brain mRNA as a useful model in elucidating the molecular mechanism of signal transduction from receptors to ion channels in the CNS.