Expression and functional characterization of the cardiac L-type calcium channel carrying a skeletal muscle DHP-receptor mutation causing hypokalaemic periodic paralysis
Expression and functional characterization of the cardiac L-type calcium channel carrying a skeletal muscle DHP-receptor mutation causing hypokalaemic periodic paralysis
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DOI：
10.1007/s004240050021
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发表时间：
1996-01-01
影响因子：
4.5
通讯作者：
Melzer, W
中科院分区：
文献类型：
--
作者：
Lerche, H;Klugbauer, N;Melzer, W
据报道，人骨骼肌二氢吡啶(DHP)受体α(1)亚基II/S4中最外层的精氨酸被组氨酸取代，导致低血钾性周期性麻痹(HypoPP)。该突变使低PP患者肌管上L型钙电流失活的电压依赖性改变了-40 mV，而不影响激活。基于II/S4在心肌和骨骼肌α(1)中的高度同源性，我们将相应的突变引入兔心脏α(1)亚单位(R65OH)。用全细胞膜片钳技术将野生型(WT)和突变型(WT)载体与β和α(2)Delta亚基一起瞬时转染HEK细胞，并研究其钙电流和钙电流。与人肌管的实验结果相比，R650H的稳态激活和失活曲线均发生了较小的(-5 mV)但显著的漂移。当外界pH从7.4增加到8.4时，有利于H650去质子化，WT和突变通道之间唯一的区别是失活曲线的陡度略有降低。另外，只在骨骼肌中发现而在心肌中不存在的伽马亚基的额外共转染使WT和R650H的失活曲线移动了-20 mV。R650H对稳态失活的中点电压没有明显的选择性作用，因此R650对心肌L型钙通道的电压依赖性门控作用不同于人骨骼肌L型钙通道的相应残基。
A histidine substitution for the outermost arginine in II/S4 of the alpha(1) subunit of the human skeletal muscle dihydropyridine (DHP) receptor has been reported to cause hypokalaemic periodic paralysis (HypoPP). This mutation shifts the voltage dependence of L-type Ca current inactivation in myotubes from HypoPP patients by -40 mV without affecting activation. Based on the strong homology of II/S4 in cardiac and skeletal muscle alpha(1), we introduced the corresponding mutation into the rabbit cardiac alpha(1) subunit (R65OH). Wild type (WT) and mutant constructs were transiently transfected in HEK cells together with beta and alpha(2) delta subunits and Ca and Ba currents were studied using the whole-cell patch-clamp technique. In contrast to the results obtained from human myotubes, R650H produced a small (-5 mV) but significant shift of both the steady-state activation and inactivation curves. When external pH was increased from 7.4 to 8.4 in order to favour deprotonization of H650, the only difference between WT and mutant channels was a slightly reduced steepness of the inactivation curve. Additional cotransfection of the gamma subunit which is only found in skeletal but not in heart muscle, shifted the inactivation curves of both WT and R650H by -20 mV. We conclude that R650 plays a different role in voltage-dependent gating of the cardiac L-type Ca channel than the corresponding residue in the human skeletal muscle L-type channel, since a distinct and selective effect on the midpoint voltage of steady-state inactivation could not be found for R650H.