Beurg M, Sukhareva M, Ahern C A, Conklin M W, Perez-Reyes E, Powers P A, Gregg R G, Coronado R
Department of Physiology, University of Wisconsin School of Medicine, Madison, Wisconsin 53706, USA.
Biophys J. 1999 Apr;76(4):1744-56. doi: 10.1016/S0006-3495(99)77336-4.
The dihydropyridine receptor (DHPR) of skeletal muscle functions as a Ca2+ channel and is required for excitation-contraction (EC) coupling. Here we show that the DHPR beta subunit is involved in the regulation of these two functions. Experiments were performed in skeletal mouse myotubes selectively lacking a functional DHPR beta subunit. These beta-null cells have a low-density L-type current, a low density of charge movements, and lack EC coupling. Transfection of beta-null cells with cDNAs encoding for either the homologous beta1a subunit or the cardiac- and brain-specific beta2a subunit fully restored the L-type Ca2+ current (161 +/- 17 pS/pF and 139 +/- 9 pS/pF, respectively, in 10 mM Ca2+). We compared the Boltzmann parameters of the Ca2+ conductance restored by beta1a and beta2a, the kinetics of activation of the Ca2+ current, and the single channel parameters estimated by ensemble variance analysis and found them to be indistinguishable. In contrast, the maximum density of charge movements in cells expressing beta2a was significantly lower than in cells expressing beta1a (2.7 +/- 0.2 nC/microF and 6.7 +/- 0. 4 nC/microF, respectively). Furthermore, the amplitude of Ca2+ transient measured by confocal line-scans of fluo-3 fluorescence in voltage-clamped cells were 3- to 5-fold lower in myotubes expressing beta2a. In summary, DHPR complexes that included beta2a or beta1a restored L-type Ca2+ channels. However, a DHPR complex with beta1a was required for complete restoration of charge movements and skeletal-type EC coupling. These results suggest that the beta1a subunit participates in key regulatory events required for the EC coupling function of the DHPR.
骨骼肌的二氢吡啶受体（DHPR）作为一种Ca2+通道，是兴奋-收缩（EC）偶联所必需的。在此我们表明，DHPRβ亚基参与这两种功能的调节。实验在选择性缺乏功能性DHPRβ亚基的小鼠骨骼肌管中进行。这些β亚基缺失的细胞具有低密度的L型电流、低密度的电荷移动，并且缺乏EC偶联。用编码同源β1a亚基或心脏和脑特异性β2a亚基的cDNA转染β亚基缺失的细胞，可完全恢复L型Ca2+电流（在10 mM Ca2+中，分别为161±17 pS/pF和139±9 pS/pF）。我们比较了由β1a和β2a恢复的Ca2+电导的玻尔兹曼参数、Ca2+电流的激活动力学以及通过总体方差分析估计的单通道参数，发现它们没有区别。相比之下，表达β2a的细胞中电荷移动的最大密度显著低于表达β1a的细胞（分别为2.7±0.2 nC/μF和6.7±0.4 nC/μF）。此外，在电压钳制的细胞中，通过共聚焦线扫描fluo-3荧光测量的Ca2+瞬变幅度在表达β2a的肌管中低3至5倍。总之，包含β2a或β1a的DHPR复合物恢复了L型Ca2+通道。然而，具有β1a的DHPR复合物是电荷移动和骨骼肌型EC偶联完全恢复所必需的。这些结果表明，β1a亚基参与了DHPR的EC偶联功能所需的关键调节事件。
