External pore residue mediates slow inactivation in mu 1 rat skeletal muscle sodium channels
External pore residue mediates slow inactivation in mu 1 rat skeletal muscle sodium channels
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DOI：
10.1113/jphysiol.1996.sp021503
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发表时间：
1996-07-15
影响因子：
5.5
通讯作者：
Tomaselli, GF
中科院分区：
文献类型：
--
作者：
Balser, JR;Nuss, HB;Tomaselli, GF
1.在去极化时，电压门控钠通道呈现非传导失活状态，其可根据恢复所需的复极化期的长度表征为“快”或“慢”。非洲爪蟾卵母细胞中表达的骨骼肌Na+通道a亚基显示异常门控行为，在短暂去极化后实质性缓慢失活。我们利用这种动力学行为来研究缓慢失活的结构基础。虽然Na+通道中的快速失活是由III-IV接头残基对孔的细胞质闭塞介导的，但缓慢失活的结构特征尚不清楚。由于外部孔衬残基调节钾通道中的C型失活，我们在大鼠骨骼肌Na+通道（mu 1）的渗透环（P-loop）中进行了系列半胱氨酸诱变，以确定类似位置的残基是否参与Na+通道的缓慢失活.野生型和突变体α-亚基异源表达非洲爪蟾卵母细胞，Na+电流记录使用双电极电压钳。短暂去极化后的缓慢失活被结构域I中的W 402 C突变消除。结构域II、III和IV中同源色氨酸残基的半胱氨酸取代并不改变慢失活。与W 402 C突变类似，野生型α亚基与大鼠脑Na+通道β（1）亚基的共表达减弱了慢失活。然而，W 402 C突变使快速失活的恢复延迟，而β（1）亚基共表达没有。我们认为W 402 C突变和β（1）亚基通过不同的机制调节门控。去除野生型α亚基中的快速失活与III-IV接头突变I1303 Q; F1304 Q; M1305 Q显着减缓了缓慢失活的发展。我们建议，在Na+通道的缓慢失活涉及的外部孔的构象变化。影响快失活和慢失活的突变似乎相互作用，尽管它们在通道中的位置很远。
1. Upon depolarization, voltage-gated sodium channels assume non-conducting inactivated states which may be characterized as 'fast' or 'slow' depending on the length of the repolarization period needed for recovery. Skeletal muscle Na+ channel a-subunits expressed in Xenopus laevis oocytes display anomalous gating behaviour, with substantial slow inactivation after brief depolarizations. We exploited this kinetic behaviour to examine the structural basis for slow inactivation.2. While fast inactivation in Na+ channels is mediated by cytoplasmic occlusion of the pore by III-IV linker residues, the structural features of slow inactivation are unknown. Since external pore-lining residues modulate C-type inactivation in potassium channels, we performed serial cysteine mutagenesis in the permeation loop (P-loop) of the rat skeletal muscle Na+ channel (mu 1) to determine whether similarly placed residues are involved in Na+ channel slow inactivation.3. Wild-type and mutant alpha-subunits were heterologously expressed in Xenopus oocytes, and Na+ currents were recorded using a two-electrode voltage clamp. Slow inactivation after brief depolarizations was eliminated by the W402C mutation in domain I. Cysteine substitution of the homologous tryptophan residues in domains II, III and IV did not alter slow inactivation.4. Analogous to the W402C mutation, coexpression of the wild-type alpha-subunit with rat brain Na+ channel beta(1)-subunit attenuated slow inactivation. However, the W402C mutation imposed a delay on recovery from fast inactivation, while beta(1)-subunit coexpression did not. We propose that the W402C mutation and the beta(1)-subunit modulate gating through distinct mechanisms.5. Removal of fast inactivation in wild-type alpha-subunits with the III-IV linker mutation I1303Q; F1304Q; M1305Q markedly slowed the development of slow inactivation. We propose that slow inactivation in Na+ channels involves conformational changes in the external pore. Mutations that affect fast and slow inactivation appear to inter act despite their remote positions in the channel.