Comparison of fura-2 imaging and electrophysiological analysis of murine calcium channel alpha 1 subunits coexpressed with novel beta 2 subunit isoforms.
Comparison of fura-2 imaging and electrophysiological analysis of murine calcium channel alpha 1 subunits coexpressed with novel beta 2 subunit isoforms.
复制标题
DOI：
--
复制
发表时间：
1995-04
影响因子：
3.6
通讯作者：
E. Massa;K. Kelly;D. Yule;R. Macdonald;M. Uhler
中科院分区：
文献类型：
--
作者：
E. Massa;K. Kelly;D. Yule;R. Macdonald;M. Uhler
聚合酶链反应产物用于分离编码电压依赖性Ca 2+通道β亚基亚型的小鼠脑cDNA克隆。所描述的两个小鼠脑β 2亚基cDNA克隆，β 2a和β 2b，通过编码区内的选择性剪接而不同，但具有在其他β 2亚基cDNA中尚未报道的独特氨基末端。北方印迹和RNA酶保护分析表明，这两种mRNA异构体可以检测到在心脏和大脑中的丰度最高，在肺，肾和睾丸中的水平较低。在β 2亚基功能的新测定中，用α 1和β 2亚基表达载体转染COS-1细胞，并通过使用Fura-2成像测定细胞内Ca 2+浓度的增加。用小鼠脑C-1 α 1亚基表达载体和β 2亚基表达载体共转染COS-1细胞，在用升高的K+和二氢吡啶激动剂Bay K 8644刺激后，导致细胞内Ca 2+浓度增加。单独转染α 1或β 2亚基表达载体不会导致细胞内Ca 2+浓度升高。电生理记录的人胚肾293细胞转染的表达载体的α 1亚基单独或与任一β 2亚基表现出表达的电压依赖性钙离子通道是二氢吡啶敏感。仅表达α 1亚基形成的电流很小，并且缓慢失活。相反，由α 1亚基与β 2亚基共表达形成的电流更大，失活更快。二氢吡啶结合研究表明，与单独表达α 1亚基相比，α 1亚基与β 2亚基的共表达增加了功能性受体的密度。这些实验表明，α 1和β 2亚基的共表达产生功能性的二氢吡啶敏感的Ca 2+通道，这两种β亚基亚型对这些通道具有调节作用。
A polymerase chain reaction product was used to isolate mouse brain cDNA clones coding for isoforms of the beta subunit of voltage-dependent Ca2+ channels. The two mouse brain beta 2 subunit cDNA clones described, beta 2a and beta 2b, differed by alternative splicing within the coding region but possessed a unique amino terminus not yet reported in other beta 2 subunit cDNAs. Northern blot and RNase protection analyses demonstrated that both mRNA isoforms could be detected in highest abundance in heart and brain and at lower levels in lung, kidney, and testis. In a novel assay for beta 2 subunit function, COS-1 cells were transfected with alpha 1 and beta 2 subunit expression vectors and assayed for increases in intracellular Ca2+ concentration by using fura-2 imaging. Co-transfection of COS-1 cells with the mouse brain class C-1 alpha 1 subunit expression vector and either of the beta 2 subunit expression vectors resulted in increases in intracellular Ca2+ concentration after stimulation with elevated K+ and the dihydropyridine agonist Bay K 8644. Transfection of either alpha 1 or beta 2 subunit expression vectors alone did not result in an elevation of intracellular Ca2+ concentration. Electrophysiological recording of human embryonic kidney 293 cells transfected with the expression vector for the alpha 1 subunit alone or with either beta 2 subunit demonstrated expression of voltage-dependent Ca2+ channels that were dihydropyridine sensitive. Currents formed by expression of only the alpha 1 subunit were small and slowly inactivated. In contrast, the currents formed by coexpression of alpha 1 subunits with either beta 2 subunit were larger and inactivated more rapidly. Dihydropyridine binding studies demonstrated that coexpression of alpha 1 subunits with beta 2 subunits increased the density of functional receptors, compared with expression of alpha 1 subunits alone. These experiments suggested that coexpression of the alpha 1 and beta 2 subunits produced functional dihydropyridine-sensitive Ca2+ channels and that both beta subunit isoforms had modulatory effects on these channels.