Conformational changes in CIC chloride channels

这是该项目的第三年，使用生化方法研究ClC型氯离子通道蛋白的构象变化。ClC家族的氯离子传导离子通道参与了一系列的生物过程，这些通道维持骨骼肌的静息膜电位，调节中枢神经元的兴奋性，并参与多种细胞内隔室的pH动态平衡。尽管它们具有重要的生理意义，但人们对这些通道的作用机制知之甚少。我们试图通过研究几个家族成员的功能来了解这些蛋白的功能特性，包括真核和原核同源物。在这个项目中，我们使用原核生物的CLC来生化研究运输过程激活时的构象变化。为了实现这一目标，我们已经在运输蛋白的关键区域构建了一系列半胱氨酸突变体，并可以将荧光探针共价连接到这些位置。然后，我们研究了这些蛋白质重组成脂质体后的荧光性质；我们发现，在低pH下激活运输时，荧光发生了戏剧性和可逆的变化，可能反映了显著的构象变化。各种对照表明，分离的荧光团的荧光不随pH变化，并且我们可以特异性地修饰感兴趣的半胱氨酸。我们已经确定构象变化对转运蛋白的两种天然底物氯和质子很敏感。缺乏关键的“门控谷氨酸”的转运蛋白的荧光变化持续存在，这表明其他可滴定基团参与了转运体的运输过程。此外，标记的突变体转运体是活跃的，表明荧光探针不会干扰转运过程。我们目前正在研究更多的突变体，以确定构象变化的程度，以及旨在测试有关其机制的假说的双重突变体。

This was the third year for this project, using biochemical methods to study conformational changes in ClC-type chloride channel proteins. The ClC family of chloride-conducting ion channels is involved in a host of biological processes; these channels maintain the resting membrane potential in skeletal muscle, modulate excitability in central neurons, and are involved in the homeostasis of pH in a variety of intracellular compartments. Despite their physiological importance, the mechanisms by which these channels function are poorly understood. We are attempting to understand the functional properties of these proteins by examining the function of several family members, including both eukaryotic and prokaryotic homologs. In this project, we are using the prokaryotic ClC's for biochemical studies of conformational changes upon activation of the transport process. Toward this goal, we have constructed a series of cysteine mutants in a critical region of the transport protein and can covalently attach fluorescent probes to these sites. We then examine the fluorescence properties these proteins after reconstitution into liposomes; we find that there are dramatic and reversible changes in fluorescence upon activation of transport at low pH, presumeably reflecting a significant conformational change. Various controls demonstrate no change in fluorescence of the isolated fluorophore with pH, and that we can specifically modify the cysteine of interest. We have established that the conformational change is sensitive to chloride and protons, the two native subtrates of the transporter. Fluorescence changes persist in transporters lacking a critical 'gating glutamtate,' suggesting that other titratable groups contribute to the transport process. In addition, the labeled mutant transporters are active, showing that the fluorescent probe does not disrupt the transport process. We are currently examining further mutants to establish the extent of the conformational change, as well as double mutants designed to test hypotheses regarding its mechanism.