Zhen Xiao-Guang, Xie Cheng, Yamada Yoichi, Zhang Yun, Doyle Christina, Yang Jian
Department of Biological Sciences, 917 Fairchild Center, MC2462, Columbia University, New York, NY 10027, USA.
FEBS Lett. 2006 Oct 16;580(24):5733-8. doi: 10.1016/j.febslet.2006.09.027. Epub 2006 Sep 22.
The activity of voltage-gated calcium channels (VGCCs) decreases with time in whole-cell and inside-out patch-clamp recordings. In this study we found that substituting a single amino acid (I1520) at the intracellular end of IIIS6 in the alpha(1) subunit of P/Q-type Ca(2+) channels with histidine or aspartate greatly attenuated channel rundown in inside-out patch-clamp recordings. The homologous mutations also slowed rundown of N- and L-type Ca(2+) channels, albeit to a lesser degree. In P/Q-type channels, the attenuation of rundown is accompanied by an increased apparent affinity for phosphatidylinositol-4,5-bisphosphate, which has been shown to be critical for maintaining Ca(2+) channel activity [L. Wu, C.S. Bauer, X.-G. Zhen, C. Xie, J. Yang, Dual regulation of voltage-gated calcium channels by PtdIns(4,5)P2. Nature 419 (2002) 947-952]. Furthermore, the histidine mutation significantly stabilized the open state, making the channels easier to open, slower to close, harder to inactivate and faster to recover from inactivation. Our finding that mutation of a single amino acid can greatly attenuate rundown provides an easy and efficient way to slow the rundown of VGCCs, facilitating functional studies that require direct access to the cytoplasmic side of the channel.
在全细胞和内面向外膜片钳记录中，电压门控钙通道（VGCCs）的活性会随时间降低。在本研究中，我们发现在P/Q型Ca(2+)通道α(1)亚基IIIS6胞内端将单个氨基酸（I1520）替换为组氨酸或天冬氨酸，在内面向外膜片钳记录中可极大减弱通道电流衰减。同源突变也减缓了N型和L型Ca(2+)通道的电流衰减，尽管程度较小。在P/Q型通道中，电流衰减的减弱伴随着对磷脂酰肌醇-4,5-二磷酸的表观亲和力增加，而磷脂酰肌醇-4,5-二磷酸已被证明对维持Ca(2+)通道活性至关重要[L. Wu, C.S. Bauer, X.-G. Zhen, C. Xie, J. Yang, PtdIns(4,5)P2对电压门控钙通道的双重调节。《自然》419 (2002) 947 - 952]。此外，组氨酸突变显著稳定了开放状态，使通道更易开放、关闭更慢、更难失活且从失活中恢复更快。我们发现单个氨基酸突变可极大减弱电流衰减，这为减缓VGCCs电流衰减提供了一种简便有效的方法，便于进行需要直接接触通道胞质侧的功能研究。
