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

我们在非洲爪哇卵母细胞表达系统中检测了β-肾上腺素能刺激对L钙通道的影响。L钙通道(α1C)与钙通道α2、β2a亚基和刺激性谷丙转氨酶结合蛋白(Gs)共表达。腺苷环化酶、β2-肾上腺素能受体和PKA结合蛋白(AKAP79)也在这些蛋白的不同组合中共表达。用传统的双微电极电压钳方法测量钙通道电流。我们已经克隆了这些蛋白的cDNA，并用体外转录的方法获得了每个蛋白的cRNA。微量注射不同CRNA的组合，观察异丙肾上腺素对膜电流幅度调制的影响。然而，该蛋白的结合不能重建β-肾上腺素能刺激的L型钙通道，这种钙通道在骨骼肌和肌细胞中都很常见。我们将钙通道的α亚基从AlphalC改变为AlphalA(P/Q型)。我们观察到PKA对通道的刺激，而Rp-cAMP和H89，PKA抑制剂，降低了膜电流。我们计划研究这两个字母亚单位的结构-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效果。这一结果作为支持的研究发表。

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.