A Research for L-type Calcium Channel Modulation by b2-adrenergic Stimulation

我们在非洲爪蟾卵母细胞表达系统中检测了β-肾上腺素能刺激对L-型钙通道的影响。L型钙通道（α 1 C）与钙通道α 2、β 2a亚基和刺激性GPT结合蛋白（Gs）共表达。腺苷酸环化酶、β 2-肾上腺素能受体和PKA结合蛋白（AKAP 79）也以这些蛋白的各种组合共表达。采用双微电极电压钳法测定细胞膜电流。这些蛋白质的cDNA已经被克隆，并且我们使用体外转录方法产生每种蛋白质的cRNA。进行各种cRNA的组合的显微注射，并测量异丙肾上腺素的β刺激对膜电流幅度的调节的影响。然而，没有蛋白质的组合重建β-肾上腺素能刺激的L-型钙通道，这是常见的骨骼肌和心肌细胞。我们观察到PKA对通道的刺激，PKA抑制剂Rp-cAMPS和H89降低了膜电流。我们计划研究这两个亚基的结构-功能关系，在研究过程中，我们使用相同的表达系统测量了许多钙通道拮抗剂的作用。研究结果作为支持研究发表。

We measured the effects of beta-adrenergic stimulations on L-type calcium channel in Xenopus oocyte expression system. L-type calcium channel (alpha 1 C) was co-expressed with calcium channel alpha2, beta2a subunits and stimulatory GPT binding protein (Gs). Adenylate cyclase, beta2-adrenergic receptor and PKA binding protein (AKAP79) were also co-expressed in various combinations of these proteins. Membrane current through expressed calcium channel was measured by conventional two-microelectrodes voltage clamp methods. cDNA for these proteins were already cloned and we produced cRNA for each proteins using in-vitro-transcription method. Microinjections of the combination of various cRNA were performed, and measured the effect of beta-stimulation by isoproterenol on the modulation of membrane current amplitude. However, no combination of the protein re-constructs the beta-adrenergic stimulation of L-type calcium channel, which is common in both skeletal muscles and myocytes.We changed the expressing alphal subunit of calcium channel from alphalC to alphalA (P/Q-type). We observed PKA stimulation of the channel, and Rp-cAMPS and H89, PKA inhibitory agents, decreased the membrane current. We are planning to investigate the structure-function relationships of the two alphal subunits.During the investigation we measured the effect of numerous calcium channel antagonists using the same expression system. The results were published as the supported research.