GAT1 (GABA:Na+:Cl-) cotransport function. Database reconstruction with an alternating access model.
GAT1 (GABA:Na+:Cl-) cotransport function. Database reconstruction with an alternating access model.
复制标题
DOI：
10.1085/jgp.114.3.459
复制
发表时间：
1999-09
期刊：
影响因子：
--
通讯作者：
Lu CC
中科院分区：
文献类型：
--
作者：
Hilgemann DW;Lu CC
我们建立了一个交替通道转运模型，该模型很好地解释了非洲爪哇卵母细胞膜上GAT1(GABA：Na+：CL−)的共转运功能。为此，将许多替代模型与GAT1函数数据库进行了匹配，并对差异进行了分析。该模型假设GAT1主要以两种状态存在，即E In和E Out。在E-In状态下，一个氯离子和两个钠离子可以从细胞质一侧依次结合。在EOUT状态下，一个钠离子被封闭在转运体内，一个氯化物、一个钠和一个γ-氨基丁酸(GABA)分子可以从胞外结合。当E-In位点为空时，向E-Out状态的转变打开了与外部的结合位点，并封闭了一个细胞外钠离子。这种构象变化是主要的电生GAT1反应，它限制了0 mV的前向转运(即GABA摄取)。从E_OUT状态，一个GABA可以与一个钠离子一起转移到细胞质一侧，从而形成*E_IN状态。此后，细胞外的氯离子可以被转移，被封闭的钠离子被释放到细胞质，这使转运蛋白返回到E的状态。GABA-GABA交换可以在没有细胞外氯离子的情况下发生，但必须运输氯离子才能完成前向运输循环。在反向转运循环中，一个细胞质氯离子首先与E结合，然后是两个钠离子。一个氯离子和一个钠离子被封闭在一起，然后第二个钠离子和GABA被封闭和移位。这些反应的弱电压依赖性决定了外向电流-电压关系的斜率。精确模拟的实验结果包括(A)所有的电流-电压关系，(B)到目前为止描述的所有底物相关性，(C)顺-顺和顺-反底物的相互作用，(D)在没有传输电流的情况下的电荷运动，(E)电荷运动动力学对底物浓度的依赖，(F)在底物存在时的稳态前的电流瞬变，(G)底物诱导的电容变化，(H)GABA-GABA交换，以及(I)在名义上没有细胞外氯的情况下内向传输电流和GABA-GABA交换的存在。
We have developed an alternating access transport model that accounts well for GAT1 (GABA:Na+:Cl−) cotransport function in Xenopus oocyte membranes. To do so, many alternative models were fitted to a database on GAT1 function, and discrepancies were analyzed. The model assumes that GAT1 exists predominantly in two states, E in and E out. In the E in state, one chloride and two sodium ions can bind sequentially from the cytoplasmic side. In the E out state, one sodium ion is occluded within the transporter, and one chloride, one sodium, and one γ-aminobutyric acid (GABA) molecule can bind from the extracellular side. When E in sites are empty, a transition to the E out state opens binding sites to the outside and occludes one extracellular sodium ion. This conformational change is the major electrogenic GAT1 reaction, and it rate-limits forward transport (i.e., GABA uptake) at 0 mV. From the E out state, one GABA can be translocated with one sodium ion to the cytoplasmic side, thereby forming the *E in state. Thereafter, an extracellular chloride ion can be translocated and the occluded sodium ion released to the cytoplasm, which returns the transporter to the E in state. GABA–GABA exchange can occur in the absence of extracellular chloride, but a chloride ion must be transported to complete a forward transport cycle. In the reverse transport cycle, one cytoplasmic chloride ion binds first to the E in state, followed by two sodium ions. One chloride ion and one sodium ion are occluded together, and thereafter the second sodium ion and GABA are occluded and translocated. The weak voltage dependence of these reactions determines the slopes of outward current–voltage relations. Experimental results that are simulated accurately include (a) all current–voltage relations, (b) all substrate dependencies described to date, (c) cis–cis and cis–trans substrate interactions, (d) charge movements in the absence of transport current, (e) dependencies of charge movement kinetics on substrate concentrations, (f) pre–steady state current transients in the presence of substrates, (g) substrate-induced capacitance changes, (h) GABA–GABA exchange, and (i) the existence of inward transport current and GABA–GABA exchange in the nominal absence of extracellular chloride.