Mim Carsten, Tao Zhen, Grewer Christof
Department of Physiology, University of Miami School of Medicine, 1600 NW 10th Avenue, Miami, Florida 33136, USA.
Biochemistry. 2007 Aug 7;46(31):9007-18. doi: 10.1021/bi7005465. Epub 2007 Jul 13.
Glutamate is transported across membranes by means of a carrier mechanism that is thought to require conformational changes of the transport protein. In this work, we have determined the thermodynamic parameters of glutamate and the Na+ binding steps to their extracellular binding sites along with the activation parameters of rapid, glutamate-induced processes in the transport cycle by analyzing the temperature dependence of glutamate transport at steady state and pre-steady state. Our results suggest that glutamate binding to the transporter is driven by a negative reaction enthalpy (DeltaH0 = -33 kJ/mol), whereas the tighter binding of the non-transportable inhibitor TBOA is caused by an additional increase in entropy. Processes linked to the binding of glutamate and Na+ to the transporter are associated with low activation barriers, indicative of diffusion-controlled reactions. The activation enthalpies of two processes in the glutamate translocation branch of the transport cycle were DeltaH++ = 95 kJ/mol and DeltaH++ = 120 kJ/mol, respectively. Such large values of DeltaH++ suggest that these processes are rate-limited by conformational changes of the transporter. We also found a large activation barrier for steady-state glutamate transport, which is rate-limited by the K+-dependent relocation of the empty transporter. Together, these results suggest that two conformational changes accompany glutamate translocation and at least one conformational change accompanies the relocation of the empty transporter. We interpret the data with an alternating access model that includes the closing and opening of an extracellular and an intracellular gate, respectively, in analogy to a hypothetical model proposed previously on the basis of the crystal structure of the bacterial glutamate transporter GltPh.
谷氨酸通过一种载体机制跨膜转运，这种机制被认为需要转运蛋白的构象变化。在这项工作中，我们通过分析稳态和预稳态下谷氨酸转运的温度依赖性，确定了谷氨酸和钠离子与它们细胞外结合位点结合步骤的热力学参数，以及转运循环中谷氨酸诱导的快速过程的活化参数。我们的结果表明，谷氨酸与转运体的结合由负反应焓驱动（ΔH0 = -33 kJ/mol），而非转运性抑制剂TBOA的更紧密结合是由熵的额外增加引起的。与谷氨酸和钠离子与转运体结合相关的过程具有低活化能垒，表明是扩散控制反应。转运循环中谷氨酸转运分支的两个过程的活化焓分别为ΔH++ = 95 kJ/mol和ΔH++ = 120 kJ/mol。如此大的ΔH++值表明这些过程受转运体构象变化的速率限制。我们还发现稳态谷氨酸转运存在一个大的活化能垒，它受空转运体的钾离子依赖性重新定位的速率限制。总之，这些结果表明谷氨酸转运伴随着两个构象变化，空转运体的重新定位至少伴随着一个构象变化。我们用交替访问模型来解释这些数据，该模型分别包括细胞外门和细胞内门的关闭和打开，类似于先前基于细菌谷氨酸转运体GltPh的晶体结构提出的假设模型。
