Mutational and pH analysis of ionic residues in transmembrane domains of vesicular acetylcholine transporter
Mutational and pH analysis of ionic residues in transmembrane domains of vesicular acetylcholine transporter
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DOI：
10.1021/bi047442y
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发表时间：
2005-06-07
期刊：
影响因子：
2.9
通讯作者：
Parsons, SM
中科院分区：
文献类型：
--
作者：
Bravo, DT;Kolmakova, NG;Parsons, SM
本研究探讨了7个保守离子残基在囊泡型乙酰胆碱转运体（VAChT） 12个推测的跨膜结构域（TMDs）中的作用。野生型和突变型大鼠VAChT在PC12（A123.7）细胞中表达。在不同的pH值下，用过滤法对转运和配体结合进行了表征。ACh结合位点显示出高或低亲和力（K-d值分别接近10和200 mM）。TMDs II和IV中赖氨酸和天冬氨酸残基的突变可降低高亲和力位点的比例。在相关转运体的三维结构中，这些TMDs彼此相邻，远离TMDs VIII和X，它们可能包含ACh和变构抑制剂vesamicol的结合位点。重要的是，TMD XI中天冬氨酸的突变可以产生ACh （K-d约为4 mM）和vesamicol （K-d约为2 nM，而不是约为20 nM）的超高亲和力。不同外部pH值对转运的影响表明，必须质子化的位点（表观pK(a)接近7.6）可能是TMD XI中的天冬氨酸。这些观察结果表明，TMD 11中的赖氨酸和TMD XI中的天冬氨酸之间的已知离子对控制了TMD XI的构象或相对位置，从而控制了VAChT c端一半中的其他TMDs。pH效应还表明，运输过程中必须未质子化的位点（表观pK(a)约为6.4）和维萨霉素结合位点（表观pK(a)约为6.3）仍未确定。
This research investigated the roles of 7 conserved ionic residues in the 12 putative transmenibrane domains (TMDs) of vesicular acetylcholine transporter (VAChT). Rat VAChT in wildtype and mutant forms was expressed in PC12(A123.7) cells. Transport and ligand binding were characterized at different pH values using filter assays. The ACh binding site is shown to exhibit high or low affinity (K-d values are approximate to 10 and 200 mM, respectively). Mutation of the lysine and aspartate residues in TMDs II and IV, respectively, can decrease the fraction of sites having high affinity. In three-dimensional structures of related transporters, these TMDs lie next to each other and distantly from TMDs VIII and X, which probably contain the binding sites for ACh and the allosteric inhibitor vesamicol. Importantly, mutation of the aspartate in TMD XI can create extra-high affinities for ACh (K-d approximate to 4 mM) and vesamicol (K-d approximate to 2 nM compared to approximate to 20 nM). Effects of different external pH values on transport indicate a site that must be protonated (apparent pK(a) approximate to 7.6) likely is the aspartate in TMD XI. The observations suggest a model in which the known ion pair between lysine in TMD 11 and aspartate in TMD XI controls the conformation or relative position of TMD XI, which in turn controls additional TMDs in the C-terminal half of VAChT. The pH effects also indicate that sites that must be unprotonated for transport (apparent pK(a) approximate to 6.4) and vesamicol binding (apparent pK(a) approximate to 6.3) remain unidentified.