Yamagishi Toshio, Xiong Wei, Kondratiev Andre, Vélez Patricio, Méndez-Fitzwilliam Ailsa, Balser Jeffrey R, Marbán Eduardo, Tomaselli Gordon F
Division of Cardiology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
Mol Pharmacol. 2009 Oct;76(4):861-71. doi: 10.1124/mol.109.055863. Epub 2009 Jul 20.
The pore of the Na+ channel is lined by asymmetric loops formed by the linkers between the fifth and sixth transmembrane segments (S5-S6). We investigated the role of the N-terminal portion (SS1) of the S5-S6 linkers in channel gating and local anesthetic (LA) block using site-directed cysteine mutagenesis of the rat skeletal muscle (Na(V)1.4) channel. The mutants examined have variable effects on voltage dependence and kinetics of fast inactivation. Of the cysteine mutants immediately N-terminal to the putative DEKA selectivity filter in four domains, only Q399C in domain I and F1236C in domain III exhibit reduced use-dependent block. These two mutations also markedly accelerated the recovery from use-dependent block. Moreover, F1236C and Q399C significantly decreased the affinity of QX-314 for binding to its channel receptor by 8.5- and 3.3-fold, respectively. Oddly enough, F1236C enhanced stabilization of slow inactivation by both hastening entry into and delaying recovery from slow inactivation states. It is noteworthy that symmetric applications of QX-314 on both external and internal sides of F1236C mutant channels reduced recovery from use-dependent block, indicating an allosteric effect of external QX-314 binding on the recovery of availability of F1236C. These observations suggest that cysteine mutation in the SS1 region, particularly immediate adjacent to the DEKA ring, may lead to a structural rearrangement that alters binding of permanently charged QX-314 to its receptor. The results lend further support for a role for the selectivity filter region as a structural determinant for local anesthetic block.
Na⁺通道的孔道由第五和第六跨膜片段（S5 - S6）之间的连接子形成的不对称环所环绕。我们利用大鼠骨骼肌（Na(V)1.4）通道的定点半胱氨酸诱变技术，研究了S5 - S6连接子的N端部分（SS1）在通道门控和局部麻醉药（LA）阻断中的作用。所检测的突变体对电压依赖性和快速失活动力学具有不同影响。在四个结构域中，位于假定的DEKA选择性过滤器紧邻N端的半胱氨酸突变体中，只有结构域I中的Q399C和结构域III中的F1236C表现出使用依赖性阻断的降低。这两个突变还显著加速了使用依赖性阻断后的恢复。此外，F1236C和Q399C分别使QX - 314与其通道受体结合的亲和力显著降低了8.5倍和3.3倍。奇怪的是，F1236C通过加速进入慢失活状态和延迟从慢失活状态恢复，增强了慢失活的稳定性。值得注意的是，在F1236C突变体通道的外侧和内侧对称应用QX - 314会降低使用依赖性阻断后的恢复，这表明外侧QX - 314结合对F1236C可用性恢复具有变构效应。这些观察结果表明，SS1区域的半胱氨酸突变，特别是紧邻DEKA环的突变，可能导致结构重排，从而改变带永久电荷的QX - 314与其受体的结合。这些结果进一步支持了选择性过滤器区域作为局部麻醉药阻断的结构决定因素的作用。
