Bénitah J P, Chen Z, Balser J R, Tomaselli G F, Marbán E
Section of Molecular and Cellular Cardiology, Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
J Neurosci. 1999 Mar 1;19(5):1577-85. doi: 10.1523/JNEUROSCI.19-05-01577.1999.
Disulfide trapping studies have revealed that the pore-lining (P) segments of voltage-dependent sodium channels undergo sizable motions on a subsecond time scale. Such motions of the pore may be necessary for selective ion translocation. Although traditionally viewed as separable properties, gating and permeation are now known to interact extensively in various classes of channels. We have investigated the interaction of pore motions and voltage-dependent gating in micro1 sodium channels engineered to contain two cysteines within the P segments. Rates of catalyzed internal disulfide formation (kSS) were measured in K1237C+W1531C mutant channels expressed in oocytes. During repetitive voltage-clamp depolarizations, increasing the pulse duration had biphasic effects on the kSS, which first increased to a maximum at 200 msec and then decreased with longer depolarizations. This result suggested that occupancy of an intermediate inactivation state (IM) facilitates pore motions. Consistent with the known antagonism between alkali metals and a component of slow inactivation, kSS varied inversely with external [Na+]o. We examined the converse relationship, namely the effect of pore flexibility on gating, by measuring recovery from inactivation in Y401C+E758C (YC/EC) channels. Under oxidative conditions, recovery from inactivation was slower than in a reduced environment in which the spontaneous YC/EC cross-link is disrupted. The most prominent effects were slowing of a component with intermediate recovery kinetics, with diminution of its relative amplitude. We conclude that occupancy of an intermediate inactivation state facilitates motions of the P segments; conversely, flexibility of the P segments alters an intermediate component of inactivation.
二硫键捕获研究表明，电压依赖性钠通道的孔内衬（P）片段在亚秒时间尺度上会发生相当大的运动。孔的这种运动对于选择性离子转运可能是必要的。尽管传统上认为门控和通透是可分离的特性，但现在已知它们在各类通道中广泛相互作用。我们研究了在P片段中工程改造为含有两个半胱氨酸的微型1钠通道中孔运动与电压依赖性门控之间的相互作用。在卵母细胞中表达的K1237C + W1531C突变通道中测量了催化性内部二硫键形成的速率（kSS）。在重复的电压钳去极化过程中，增加脉冲持续时间对kSS有双相影响，kSS首先在200毫秒时增加到最大值，然后随着更长时间的去极化而降低。这一结果表明，占据中间失活状态（IM）有助于孔的运动。与碱金属和慢失活成分之间已知的拮抗作用一致，kSS与外部[Na +] o呈反比变化。我们通过测量Y401C + E758C（YC / EC）通道中失活的恢复情况来研究相反的关系，即孔的柔韧性对门控的影响。在氧化条件下，失活的恢复比在还原环境中慢，在还原环境中自发的YC / EC交联被破坏。最显著的影响是具有中间恢复动力学的成分的恢复减慢，其相对幅度减小。我们得出结论，占据中间失活状态有助于P片段的运动；相反，P片段的柔韧性改变了失活的一个中间成分。
