Mutational analysis of aspartate residues in the transmembrane regions and cytoplasmic loops of rat vesicular acetylcholine transporter
Mutational analysis of aspartate residues in the transmembrane regions and cytoplasmic loops of rat vesicular acetylcholine transporter
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DOI：
10.1074/jbc.274.2.673
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发表时间：
1999-01-08
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
囊泡乙酰胆碱转运蛋白（VAChT）是通过电化学梯度将神经递质乙酰胆碱（ACh）转运到突触囊泡中的载体。大鼠VAChT的跨膜结构域（TM）和胞浆环中含有大量天冬氨酸残基，这些残基在乙酰胆碱的转运过程中可能发挥重要的结构或功能作用。为了鉴定功能性带电残基，进行rVAChT的定点诱变。当胞质环中的5个天冬氨酸残基中的任何一个转化为天冬酰胺时，均未观察到对ACh转运的影响。同样，改变TM 1中的Asp-46（D46 N）或TM 6中的Asp-255（D255 N）对ACh转运或vesamicol无影响。然而，用Asn替换TM 10中的Asp-398完全消除了ACh转运和vesamicol结合。保守突变体D398 E保留了转运活性，但没有vesamicol结合，表明该残基对转运至关重要。突变的Asp-193在TM 4不影响乙酰胆碱转运活性，但是，vesamicol结合显着减少。与TM 11的突变体D425 N的ACh运输活性被完全阻断，没有对vesamicol结合的影响。在保守突变体D425 E中活性没有恢复，表明Asp-425的侧链以及负电荷对于底物结合是重要的。这些突变体，以及突变体D193 N，显然解离乙酰胆碱结合和运输从vesamicol结合。这些数据表明，TM 10中的Asp-398和TM 11中的Asp-425对于ACh结合和转运是重要的，而TM 4和TM 10中的Asp-193和Asp-398分别参与vesamicol结合。
The vesicular acetylcholine transporter (VAChT) is responsible for the transport of the neuotransmitter acetylcholine (ACh) into synaptic vesicles using an electrochemical gradient to drive transport, Rat VAChT has a number of aspartate residues within its predicted transmembrane domains (TM) and cytoplasmic loops, which may play important structural or functional roles in acetylcholine transport. In order to identify functional charged residues, site-directed mutagenesis of rVAChT was undertaken. No effect on ACh transport was observed when any of the five aspartate residues in the cytoplasmic loop were converted to asparagine, Similarly, changing Asp-46 (D46N) in TM1 or Asp-255 (D255N) in TM6 had no effect on ACh transport or vesamicol. binding, However, replacement of Asp-398 in TM10 with Asn completely eliminated both ACh transport and vesamicol binding. The conservative mutant D398E retained transport activity, but not vesamicol binding, suggesting this residue is critical for transport. Mutation of Asp-193 in TM4 did not affect ACh transport activity; however, vesamicol binding was dramatically reduced. With mutant D425N of TM11 transport activity for ACh was completely blocked, without an effect on vesamicol binding. Activity was not restored in the conservative mutant D425E, suggesting the side chain as well as the negative charge of Asp-425 is important for substrate binding. These mutants, as well as mutant D193N, clearly dissociated ACh binding and transport from vesamicol binding. These data suggest that Asp-398 in TM10 and Asp-425 in TM11 are important for ACh binding and transport, while Asp-193 and Asp-398 in TM4 and TM10, respectively, me involved in vesamicol binding.