Kontis K J, Goldin A L
Department of Microbiology and Molecular Genetics, University of California, Irvine 92717.
Mol Pharmacol. 1993 Apr;43(4):635-44.
We have used site-directed mutagenesis to examine the functional role of each of the eight acidic amino acid residues in the region between proposed transmembrane segments 5 and 6 (S5-S6) of domain II of the rat brain IIA sodium channel alpha subunit. The mutant sodium channels were expressed in Xenopus oocytes and analyzed by two-microelectrode voltage clamping with respect to voltage-dependent activation, inactivation, ion selectivity, and sensitivity to the pore-blocking neurotoxins tetrodotoxin (TTX) and saxitoxin (STX). None of the mutations had significant effects on voltage-dependent gating, ion selectivity, or block by protons or calcium. Three of the mutations had significant effects on the sensitivity of the channel to block by TTX and STX. Neutralization of negative charges at positions 942 and 945 greatly reduced the block by TTX and STX, suggesting that these two residues interact directly with the toxins. Substitution of a nearby negative charge at position 949 resulted in a smaller decrease in TTX and STX block, although analysis of TTX block of this mutant at low ionic strength suggests that the interaction is not simply by an electrostatic through-space mechanism. None of the other five mutations had any effects on block by either TTX or STX. The two acidic residues that had dramatic effects on toxin binding had significantly smaller effects at a depolarized membrane potential. The sodium channel interacts with TTX and STX with higher affinity at depolarized potentials, so these two residues must make a greater contribution to toxin binding in the low affinity state. These results define a small segment of the sodium channel alpha subunit domain II S5-S6 region that interacts with TTX and STX and therefore must lie near the mouth of the channel pore.
我们利用定点诱变技术研究了大鼠脑IIA钠通道α亚基结构域II中假定的跨膜片段5和6（S5-S6）之间区域的八个酸性氨基酸残基各自的功能作用。将突变的钠通道在非洲爪蟾卵母细胞中表达，并通过双微电极电压钳技术分析其电压依赖性激活、失活、离子选择性以及对孔道阻断性神经毒素河豚毒素（TTX）和石房蛤毒素（STX）的敏感性。这些突变均未对电压依赖性门控、离子选择性或质子或钙的阻断产生显著影响。其中三个突变对通道对TTX和STX阻断的敏感性有显著影响。中和942位和945位的负电荷极大地降低了TTX和STX的阻断作用，表明这两个残基直接与毒素相互作用。在949位取代附近的一个负电荷导致TTX和STX阻断作用的降低幅度较小，尽管在低离子强度下对该突变体的TTX阻断分析表明这种相互作用并非简单的静电空间机制。其他五个突变对TTX或STX的阻断均无任何影响。对毒素结合有显著影响的两个酸性残基在去极化膜电位下的影响明显较小。钠通道在去极化电位下与TTX和STX的亲和力更高，因此这两个残基在低亲和力状态下对毒素结合的贡献必定更大。这些结果确定了钠通道α亚基结构域II S5-S6区域中与TTX和STX相互作用且必定位于通道孔口附近的一小段区域。
