Residues Tyr-110 through Gly-115 of serotonin transporter were replaced, one at a time, with cysteine. Of these mutants, only G113C retained full activity for transport, Q111C and N112C retained partial activity, but Y110C, G114C and G115C were inactive. Poor surface expression was at least partly responsible for the lack of transport by G114C and G115C. In membrane preparations, Y110C through G113C all bound a high affinity cocaine analog similarly to the wild type. Treatment with methanethiosulfonate reagents increased the transport activity of Q111C and N112C to essentially wild-type levels but had no measurable effect on the other mutants. The decreased activity of Q111C and N112C resulted from an increase in the K(M) for serotonin that was not accompanied by a decrease in serotonin binding affinity. Superfusion experiments indicated a defect in 5-HT exchange. Modification of the inserted cysteine residues reversed the increase in K(M) and the poor exchange, also with no effect on serotonin affinity. The results suggest that Gln-111 and Asn-112 are not required for substrate binding but participate in subsequent steps in the transport cycle.

中文翻译：

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血清素转运蛋白胞外环 1 参与血清素结合和转运。

血清素转运蛋白的残留 Tyr-110 到 Gly-115 被半胱氨酸一次一个替换。在这些突变体中，只有 G113C 保留了全部转运活性，Q111C 和 N112C 保留了部分活性，但 Y110C、G114C 和 G115C 没有活性。较差的表面表达至少是 G114C 和 G115C 缺乏转运的部分原因。在膜制剂中，Y110C 到 G113C 都与野生型类似，都结合了高亲和力的可卡因类似物。用甲硫基磺酸盐试剂处理将 Q111C 和 N112C 的转运活性增加到基本上野生型的水平，但对其他突变体没有可测量的影响。Q111C 和 N112C 的活性降低是由于血清素 K(M) 的增加而不伴随血清素结合亲和力的降低。灌注实验表明 5-HT 交换存在缺陷。插入的半胱氨酸残基的修改逆转了 K(M) 和不良交换的增加，也对血清素亲和力没有影响。结果表明 Gln-111 和 Asn-112 不是底物结合所必需的，而是参与运输循环的后续步骤。