Xiong Wei, Li Ronald A, Tian Yanli, Tomaselli Gordon F
Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
J Gen Physiol. 2003 Sep;122(3):323-32. doi: 10.1085/jgp.200308881. Epub 2003 Aug 11.
In contrast to fast inactivation, the molecular basis of sodium (Na) channel slow inactivation is poorly understood. It has been suggested that structural rearrangements in the outer pore mediate slow inactivation of Na channels similar to C-type inactivation in potassium (K) channels. We probed the role of the outer ring of charge in inactivation gating by paired cysteine mutagenesis in the rat skeletal muscle Na channel (rNav1.4). The outer charged ring residues were substituted with cysteine, paired with cysteine mutants at other positions in the external pore, and coexpressed with rat brain beta1 in Xenopus oocytes. Dithiolthreitol (DTT) markedly increased the current in E403C+E758C double mutant, indicating the spontaneous formation of a disulfide bond and proximity of the alpha carbons of these residues of no more than 7 A. The redox catalyst Cu(II) (1,10-phenanthroline)3 (Cu(phe)3) reduced the peak current of double mutants (E403C+E758C, E403C+D1241C, E403C+D1532C, and D1241C+D1532C) at a rate proportional to the stimulation frequency. Voltage protocols that favored occupancy of slow inactivation states completely prevented Cu(phe)3 modification of outer charged ring paired mutants E403C+E758C, E403C+D1241C, and E403C+D1532C. In contrast, voltage protocols that favored slow inactivation did not prevent Cu(phe)3 modification of other double mutants such as E403C+W756C, E403C+W1239C, and E403C+W1531C. Our data suggest that slow inactivation of the Na channel is associated with a structural rearrangement of the outer ring of charge.
与快速失活相反，钠（Na）通道缓慢失活的分子基础目前了解甚少。有人提出，外孔的结构重排介导了Na通道的缓慢失活，这类似于钾（K）通道中的C型失活。我们通过对大鼠骨骼肌Na通道（rNav1.4）进行成对半胱氨酸诱变，探究了电荷外环在失活门控中的作用。将带电荷的外环残基替换为半胱氨酸，与外孔中其他位置的半胱氨酸突变体配对，并与大鼠脑β1在非洲爪蟾卵母细胞中共表达。二硫苏糖醇（DTT）显著增加了E403C + E758C双突变体中的电流，表明自发形成了二硫键，且这些残基的α碳原子之间的距离不超过7埃。氧化还原催化剂Cu(II)(1,10 - 菲咯啉)3（Cu(phe)3）以与刺激频率成比例的速率降低双突变体（E403C + E758C、E403C + D1241C、E403C + D1532C和D1241C + D1532C）的峰值电流。有利于占据缓慢失活状态的电压方案完全阻止了Cu(phe)3对外环电荷配对突变体E403C + E758C、E403C + D1241C和E403C + D1532C的修饰。相反，有利于缓慢失活的电压方案并未阻止Cu(phe)3对其他双突变体（如E403C + W756C、E403C + W1239C和E403C + W1531C）的修饰。我们的数据表明，Na通道的缓慢失活与电荷外环的结构重排有关。
