Sokolov S, Weiss R G, Timin E N, Hering S
Institut fur Biochemische Pharmakologie, Peter-Mayr-Strasse 1, A-6020 Innsbruck, Austria.
J Physiol. 2000 Sep 15;527 Pt 3(Pt 3):445-54. doi: 10.1111/j.1469-7793.2000.t01-1-00445.x.
beta-subunit modulation of slow inactivation of class A calcium (Ca2+) channels was studied with two-microlectrode voltage clamp after expression of the alpha1A- (BI-2) together with beta1a-, beta2a-, beta3- or beta4-subunits in Xenopus oocytes. On- and off-rates of slow inactivation were estimated from the kinetics of recovery from slow inactivation. Ca2+ channels with an alpha1A/beta-subunit composition inducing the slower rate of fast inactivation displayed the faster rate of slow inactivation. The corresponding order of slow inactivation time constants (tau[onset]) was: alpha1A/beta2a, 33 +/- 3 s; alpha1A/beta4, 42 +/- 4 s; alpha1A/beta1a, 59 +/- 4 s; alpha1A/beta3, 67 +/- 5 s (n >= 7). Recovery of class A Ca2+ channels from slow inactivation was voltage dependent and accelerated at hyperpolarized voltages. At a given holding potential recovery kinetics were not significantly modulated by different beta-subunits. Two mutations in segment IIIS6 (IF1612/1613AA) slowed fast inactivation and accelerated the onset of slow inactivation in the resulting mutant (alpha1A/IF-AA/beta3) in a similar manner as coexpression of the beta2a-subunit. Recovery from slow inactivation was slightly slowed in the double mutant. Our data suggest that class A Ca2+ channels enter the 'slow inactivated' state more willingly from the open than from the 'fast inactivated' state. The rate of slow inactivation is, therefore, indirectly modulated by different beta-subunits. Fast and slow inactivation in class A Ca2+ channels appears to represent structurally independent conformational changes. Fast inactivation is not a prerequisite for slow inactivation.
在非洲爪蟾卵母细胞中表达α1A-(BI-2)与β1a-、β2a-、β3-或β4-亚基后,利用双微电极电压钳研究了A类钙(Ca2+)通道缓慢失活的β亚基调节作用。根据从缓慢失活恢复的动力学来估计缓慢失活的开启和关闭速率。具有诱导更快快速失活速率的α1A/β亚基组成的Ca2+通道,其缓慢失活速率更快。相应的缓慢失活时间常数(tau[起始])顺序为:α1A/β2a,33±3秒;α1A/β4,42±4秒;α1A/β1a,59±4秒;α1A/β3,67±5秒(n≥7)。A类Ca2+通道从缓慢失活的恢复是电压依赖性的,并且在超极化电压下加速。在给定的钳制电位下,不同的β亚基对恢复动力学没有显著调节作用。III S6段的两个突变(IF1612/1613AA)减缓了快速失活,并以与β2a亚基共表达类似的方式加速了所得突变体(α1A/IF-AA/β3)中缓慢失活的起始。双突变体中从缓慢失活的恢复略有减慢。我们的数据表明,A类Ca2+通道从开放状态比从“快速失活”状态更易进入“缓慢失活”状态。因此,缓慢失活速率受到不同β亚基的间接调节。A类Ca2+通道中的快速和缓慢失活似乎代表结构上独立的构象变化。快速失活不是缓慢失活的先决条件。