Wang D W, George A L, Bennett P B
Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-6602, USA.
Biophys J. 1996 Jan;70(1):238-45. doi: 10.1016/S0006-3495(96)79566-8.
In this study we have expressed and characterized recombinant cardiac and skeletal muscle sodium channel alpha subunits in tsA-201 cells under identical experimental conditions. Unlike the Xenopus oocyte expression system, in tsA-201 cells (transformed human embryonic kidney) both channels seem to gate rapidly, as in native tissue. In general, hSkM1 gating seemed faster than hH1 both in terms of rate of inactivation and rate of recovery from inactivation as well as time to peak current. The midpoint of the steady-state inactivation curve was approximately 25 mV more negative for hH1 compared with hSkM1. In both isoforms, the steady-state channel availability relationships ("inactivation curves") shifted toward more negative membrane potentials with time. The cardiac isoform showed a minimal shift in the activation curve as a function of time after whole-cell dialysis, whereas hSkM1 showed a continued and marked negative shift in the activation voltage dependence of channel gating. This observation suggests that the mechanism underlying the shift in inactivation voltage dependence may be similar to the one that is causing the shift in the activation voltage dependence in hSkM1 but that this is uncoupled in the cardiac isoform. These results demonstrate the utility and limitations of measuring cardiac and skeletal muscle recombinant Na+ channels in tsA-201 cells. This baseline characterization will be useful for future investigations on channel mutants and pharmacology.
在本研究中,我们在相同实验条件下,于tsA - 201细胞中表达并表征了重组心肌和骨骼肌钠通道α亚基。与非洲爪蟾卵母细胞表达系统不同,在tsA - 201细胞(转化的人胚胎肾细胞)中,这两种通道似乎都能像在天然组织中一样快速开启。总体而言,就失活速率、从失活中恢复的速率以及达到峰值电流的时间而言,hSkM1的门控似乎比hH1更快。与hSkM1相比,hH1的稳态失活曲线中点负移约25 mV。在这两种同工型中,稳态通道可用性关系(“失活曲线”)都随时间向更负的膜电位移动。在全细胞透析后的一段时间内,心脏同工型的激活曲线变化最小,而hSkM1的通道门控激活电压依赖性则持续且显著地向负向移动。这一观察结果表明,失活电压依赖性变化的潜在机制可能与导致hSkM1激活电压依赖性变化的机制相似,但在心脏同工型中二者是解耦的。这些结果证明了在tsA - 201细胞中测量心肌和骨骼肌重组Na⁺通道的实用性和局限性。这一基线表征将有助于未来对通道突变体和药理学的研究。