Chen J G, Liu-Chen S, Rudnick G
Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06510, USA.
Biochemistry. 1997 Feb 11;36(6):1479-86. doi: 10.1021/bi962256g.
Hydropathy analysis predicts three cysteines (C109, C200, and C209) in extracellular loops of the rat serotonin transporter (SERT). We mutated these residues, singly and in combination, to either alanine or serine and expressed the mutant transporters in HeLa cells using the vaccinia-T7 transient expression system. Mutation of C109 to alanine had no effect on transport activity or surface expression of the transporter. In Na+-containing solutions, methanethiosulfonate (MTS) reagents had little effect on transport activity in the wild type or in the C109A mutant. When Na+ was replaced with Li+, inactivation of wild type by MTS reagents increased dramatically, but C109A was still resistant. The results suggest that C109 is exposed to the external medium in a manner dependent on cation binding. Replacing either C200 or C209 with serine resulted in either a partial (C200S) or almost total (C209S) loss of transport activity. MTS reagents rapidly inactivated transport activity in mutant C200S, suggesting increased accessibility of a previously unreactive cysteine residue. The double mutants C200S-C109A and C200S-C209S each retained partial activity. C200S-C109A was very sensitive to MTS reagents, but the C200S-C209S mutant was much less sensitive, similar to the wild type transporter. Replacement of C200 or C209 with serine dramatically decreased surface expression of the fully glycosylated transporter. Expression was normal, however, in the C200S-C209S double mutant. The Na+ dependence of transport and ligand binding was abnormal in both C200S and C200S-C209S mutants. Replacing C200 or C209 had similar effects on Na+ dependence and surface expression. Together with the increased MTS reactivity of C200S, these results support the possibility that C200 and C209 may be linked by a disulfide bond in the second external loop of SERT.
亲水性分析预测大鼠血清素转运体（SERT）的细胞外环中有三个半胱氨酸（C109、C200和C209）。我们将这些残基单独或组合突变为丙氨酸或丝氨酸，并使用痘苗-T7瞬时表达系统在HeLa细胞中表达突变型转运体。将C109突变为丙氨酸对转运体的转运活性或表面表达没有影响。在含Na+的溶液中，甲硫基磺酸盐（MTS）试剂对野生型或C109A突变体的转运活性影响很小。当用Li+取代Na+时，MTS试剂对野生型的失活作用显著增加，但C109A仍然具有抗性。结果表明，C109以依赖阳离子结合的方式暴露于外部介质中。用丝氨酸取代C200或C209会导致转运活性部分（C200S）或几乎完全（C209S）丧失。MTS试剂能迅速使突变体C200S的转运活性失活，这表明一个先前无反应性的半胱氨酸残基的可及性增加。双突变体C200S-C109A和C200S-C209S各自保留了部分活性。C200S-C109A对MTS试剂非常敏感，但C200S-C209S突变体的敏感性要低得多，类似于野生型转运体。用丝氨酸取代C200或C209会显著降低完全糖基化转运体的表面表达。然而，在C200S-C209S双突变体中表达正常。在C200S和C200S-C209S突变体中，转运和配体结合的Na+依赖性均异常。取代C200或C209对Na+依赖性和表面表达具有相似的影响。连同C200S增加的MTS反应性，这些结果支持了C200和C209可能在SERT的第二个外环中通过二硫键相连的可能性。
