Jones L P, Wei S K, Yue D T
Program in Molecular and Cellular Systems Physiology, Departments of Biomedical Engineering and Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
J Gen Physiol. 1998 Aug;112(2):125-43. doi: 10.1085/jgp.112.2.125.
Voltage-gated calcium channels are composed of a main pore-forming alpha1 moiety, and one or more auxiliary subunits (beta, alpha2 delta) that modulate channel properties. Because modulatory properties may vary greatly with different channels, expression systems, and protocols, it is advantageous to study subunit regulation with a uniform experimental strategy. Here, in HEK 293 cells, we examine the expression and activation gating of alpha1E calcium channels in combination with a beta (beta1-beta4) and/or the alpha2 delta subunit, exploiting both ionic- and gating-current measurements. Furthermore, to explore whether more than one auxiliary subunit can concomitantly specify gating properties, we investigate the effects of cotransfecting alpha2delta with beta subunits, of transfecting two different beta subunits simultaneously, and of COOH-terminal truncation of alpha1E to remove a second beta binding site. The main results are as follows. (a) The alpha2delta and beta subunits modulate alpha1E in fundamentally different ways. The sole effect of alpha2 delta is to increase current density by elevating channel density. By contrast, though beta subunits also increase functional channel number, they also enhance maximum open probability (Gmax/Qmax) and hyperpolarize the voltage dependence of ionic-current activation and gating-charge movement, all without discernible effect on activation kinetics. Different beta isoforms produce nearly indistinguishable effects on activation. However, beta subunits produced clear, isoform-specific effects on inactivation properties. (b) All the beta subunit effects can be explained by a gating model in which subunits act only on weakly voltage-dependent steps near the open state. (c) We find no clear evidence for simultaneous modulation by two different beta subunits. (d) The modulatory features found here for alpha1E do not generalize uniformly to other alpha1 channel types, as alpha1C activation gating shows marked beta isoform dependence that is absent for alpha1E. Together, these results help to establish a more comprehensive picture of auxiliary-subunit regulation of alpha1E calcium channels.
电压门控钙通道由一个主要的成孔α1部分和一个或多个调节通道特性的辅助亚基（β、α2δ）组成。由于调节特性可能因不同的通道、表达系统和实验方案而有很大差异，因此采用统一的实验策略来研究亚基调节是有利的。在这里，在人胚肾293细胞中，我们利用离子电流和门控电流测量，研究了α1E钙通道与β（β1-β4）和/或α2δ亚基组合时的表达和激活门控。此外，为了探究是否有多个辅助亚基能同时决定门控特性，我们研究了共转染α2δ和β亚基、同时转染两种不同β亚基以及α1E的羧基末端截短以去除第二个β结合位点的影响。主要结果如下。（a）α2δ和β亚基以根本不同的方式调节α1E。α2δ的唯一作用是通过提高通道密度来增加电流密度。相比之下，虽然β亚基也增加功能性通道数量，但它们还增强最大开放概率（Gmax/Qmax），并使离子电流激活和门控电荷移动的电压依赖性超极化，所有这些对激活动力学均无明显影响。不同的β亚型对激活产生几乎难以区分的影响。然而，β亚基对失活特性产生了明显的、亚型特异性的影响。（b）所有β亚基的作用都可以用一个门控模型来解释，即亚基仅作用于开放状态附近弱电压依赖性步骤。（c）我们没有发现两种不同β亚基同时调节的明确证据。（d）这里发现的α1E的调节特征并不能统一推广到其他α1通道类型，因为α1C激活门控显示出明显的β亚型依赖性，而α1E则没有。总之，这些结果有助于建立α1E钙通道辅助亚基调节的更全面图景。
