Mutational analysis of basic residues in the rat vesicular acetylcholine transporter - Identification of a transmembrane ion pair and evidence that histidine is not involved in proton translocation
Mutational analysis of basic residues in the rat vesicular acetylcholine transporter - Identification of a transmembrane ion pair and evidence that histidine is not involved in proton translocation
复制标题
DOI：
10.1074/jbc.275.9.6175
复制
发表时间：
2000-03-03
影响因子：
4.8
通讯作者：
Hersh, LB
中科院分区：
文献类型：
--
作者：
Kim, MH;Lu, M;Hersh, LB
本文研究了大鼠囊泡乙酰胆碱转运蛋白（rVAChT）中带正电荷残基的功能以及正、负电荷残基的相互作用。将跨膜结构域螺旋2（TM 2）中的Lys-131改变为Ala或Leu消除了转运活性，对vesamicol结合没有影响。然而，被His或Arg取代保留了运输活性，表明该位置的正电荷是至关重要的。TM 12中His-444或TM 10和TM 11之间胞质环中His-413的突变对ACh转运没有影响，但His-413突变体降低了vesamicol结合。将TM 8中的His-338改变为Ala或Lys并不影响ACh转运，然而用Cys或Arg替换则废除了活性。两个跨膜组氨酸或所有三个管腔环组氨酸的突变显示乙酰胆碱转运没有变化。跨膜结构域中带相反电荷的残基之间的突变体H338 A/D398 N显示不与vesamicol结合，然而电荷反转突变体H338 DID 398 H恢复结合，这表明His-338与Asp-398形成离子对。电荷中和突变体K131 A/D425 N或电荷交换突变体K131 D/D425 K不能恢复ACh转运。总之，这些结果提供了新的见解VAChT的三级结构。
The function of positively charged residues and the interaction of positively and negatively charged residues of the rat vesicular acetylcholine transporter (rVAChT) were studied. Changing Lys-131 in transmembrane domain helix 2 (TM2) to Ala or Leu eliminated transport activity, with no effect on vesamicol binding. However, replacement by His or Arg retained transport activity, suggesting a positive charge in this position is critical. Mutation of His-444 in TM12 or His-413 in the cytoplasmic loop between TM10 and TM11 was without effect on ACh transport, but vesamicol binding was reduced with His-413 mutants. Changing His-338 in TM8 to Ala or Lys did not effect ACh transport, however replacement with Cys or Arg abolished activity. Mutation of both of the transmembrane histidines or all three of the luminal loop histidines showed no change in acetylcholine transport. The mutant H338A/D398N between oppositely charged residues in transmembrane domains showed no vesamicol binding, however the charge reversal mutant H338DID398H restored binding, This suggests that His-338 forms an ion pair with Asp-398. The charge neutralizing mutant K131A/D425N or the charge exchanged mutant K131D/D425K did not restore ACh transport. Taken together these results provide new insights into the tertiary structure in VAChT.