大鼠脑mRNA在爪蟾卵母细胞内表达的电压门控钙通道的电压箝研究

A VOLTAGE--CLAMP STUDY ON VOLTAGE--GATED CALCIUM CHANNELS TRANSLATED IN XENOPUS OOCYTES BY RAT BRAIN mRNA

本工作用电压箝记录方法研究了爪蟾卵母细胞经注射大鼠脑mRNA后表达的电压门控钙通道。钙通道的特性由通过钙通道的钡离子流(I_(Ba))来描述。本研究采用的卵母细胞均取自被鉴定的爪蟾。这些爪蟾卵母细胞内源性I_(Ba)大多为零,或小于15nA。将从出生后10d的大鼠全脑中提取的mRNA微量注入这些卵母细胞。在注射mRNA后的5d内,I_(Ba)逐渐增大。在mRNA注射后第三天,由大鼠脑mRNA表达的电压依赖性I_(Ba)最大值一般超过100nA。作为对比,在注射从胚胎大鼠脑提取的mRNA的卵母细胞,几乎测不到电压依赖性I_(Ba)的表达。我们研究了由大鼠脑mRNA表达的I_(Ba)的电压依赖性激活及失活特性和I_(Ba)的药理。发现镧系金属离子(La^(+3),Nd^(+3),Sm^(+3),Eu^(+3),Gd^(+3),Dy^(+3),Er^(+3)在微摩尔浓度数量级即能有效抑制I_(Ba)。L-型钙通道配体nifedipine和Bay K 8644在浓度100μmmol/L时,抑制I_(Ba),而另一dihydropyridine类配体(±)-nimodipine在相同浓度却增加I_(Ba)。

Voltage--gated calcium channels, expressed in Xenopus oocytes after injection ofrat brain mRNA, were studied by using voltage--clamp technique. The properties ofthe calcium channels were characterized by barium current (I_(Ba)) passed through thechannels. All oocytes used in this study were taken from five identified donors. En-dogenous voltage--activated barium current measured in most oocytes from thesedonors were not detectable, or smalleer than 15 nA, mRNA was extracted from thewhole brains of 10 day postnatal rats and microinjected into the oocytes. I_(Ba) increasedgradually during five days after mRNA injection. The maximum amplitude of the ex-pressed voltage--activated barium current was usually larger than one hundred of nAon the third day after mRNA injection. In comparison, the expresion of voltage--acti-vated barium current was hardly detectable in oocytes injected by mRNA extractedfrom brains of embryonic rats. The voltaged--dependence of activation and inactiva-tion pharmacology of I_(Ba) were studied. It was found that I_(Ba) was inhibited potently bylanthanide cations (La^(+3), Nd^(+3), Sm^(+3), Eu^(+3), Gd^(+3), Dy^(+3), Er^(+3) at μmol/L concentra--tion level. L--type calcium channel ligands, nifedipineand Bay K 8644 inhibited I_(Ba) at100 μmol/L, while another dihydropyridine ligand (±) nimodipine enhanced I_(Ba) atthe same concentration.