Nguyen M L, Cox G D, Parsons S M
Department of Chemistry, Program in Biochemistry and Molecular Biology, University of California, Santa Barbara 93106, USA.
Biochemistry. 1998 Sep 22;37(38):13400-10. doi: 10.1021/bi9802263.
The vesicular acetylcholine transporter (VAChT) mediates ACh storage in synaptic vesicles by exchanging cytoplasmic ACh with vesicular protons. This study sought to determine the stoichiometry of exchange by analysis of ligand binding and transport kinetics. The effects of different pH values inside and outside, external ACh concentrations, and electrical potential gradients on ACh transport by vesicles isolated from the electric organ of Torpedo were determined using a pH-jump protocol. The equilibrium binding of a high-affinity analogue of ACh is inhibited by protonation with a pKa of 7.4 +/- 0.3. A two-proton model fits the transport data much better than a one-proton model does, and uptake increases at more positive internal electrical potential, as expected for the two-proton model. Thus, the results support the two-proton model. The transport cycle begins with binding of external ACh to outwardly oriented site 2 (KACho = 20 mM) and protonation of inwardly oriented site 1 (pKa1 = 4.73 +/- 0.05). Loaded VAChT reorients quickly (73 000 min-1) and releases ACh to the inside (KAChi = 44 000 mM) and the proton to the outside. Unloaded, internally oriented site 2 binds a proton (pKa2 = 7.0), after which VAChT reorients (150 +/- 20 min-1) in the rate-limiting step and releases the proton to the outside to complete the cycle. Rate constants for the reverse direction also were estimated. Two protons provide a thermodynamic driving force beyond that utilized in vivo, which suggests that vesicular filling is regulated. Other phenomena related to VAChT, namely the time required to fill synaptic vesicles, the fractional orientation of the ACh binding site toward cytoplasm, orientational lifetimes, and the rate of nonquantal release of ACh from cholinergic nerve terminals, were computer-simulated, and the results are compared with physiological observations.
囊泡乙酰胆碱转运体（VAChT）通过将胞质中的乙酰胆碱（ACh）与囊泡中的质子进行交换，介导ACh在突触囊泡中的储存。本研究旨在通过分析配体结合和转运动力学来确定交换的化学计量。使用pH跃变实验方案，测定了不同的内外pH值、外部ACh浓度以及电势梯度对从电鳐电器官分离的囊泡转运ACh的影响。ACh的一种高亲和力类似物的平衡结合受到质子化的抑制，其pKa为7.4±0.3。双质子模型比单质子模型能更好地拟合转运数据，并且如双质子模型所预期的那样，在内部电势更正时摄取增加。因此，结果支持双质子模型。转运循环始于外部ACh与向外定向的位点2结合（KACho = 20 mM）以及向内定向的位点1质子化（pKa1 = 4.73±0.05）。负载的VAChT迅速重新定向（73000 min-1），并将ACh释放到内部（KAChi = 44000 mM），同时将质子释放到外部。未负载时，向内定向的位点2结合一个质子（pKa2 = 7.0），之后VAChT在限速步骤中重新定向（150±20 min-1），并将质子释放到外部以完成循环。还估计了反向反应的速率常数。两个质子提供了超出体内利用的热力学驱动力，这表明囊泡填充是受调控的。对与VAChT相关的其他现象，即填充突触囊泡所需的时间、ACh结合位点朝向细胞质的分数取向、取向寿命以及ACh从胆碱能神经末梢的非量子释放速率进行了计算机模拟，并将结果与生理学观察结果进行了比较。
