Conformational changes in CIC chloride channels

这是该项目的第三年，使用生物化学方法研究ClC型氯离子通道蛋白的构象变化。氯离子传导离子通道的CLC家族参与许多生物过程;这些通道维持骨骼肌中的静息膜电位，调节中枢神经元的兴奋性，并参与各种细胞内隔室中pH的稳态。尽管它们的生理重要性，这些通道的功能机制知之甚少。我们正试图通过研究几个家族成员的功能来了解这些蛋白质的功能特性，包括真核和原核同源物。在这个项目中，我们使用原核CLC来进行转运过程激活后构象变化的生化研究。为了实现这一目标，我们在转运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点。然后，我们检查这些蛋白质的荧光特性后，重组成脂质体，我们发现，有显着的和可逆的荧光变化后，激活运输在低pH值，大概反映了显着的构象变化。各种对照证明分离的荧光团的荧光随pH没有变化，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，构象变化是敏感的氯离子和质子，两个天然substrates的转运。荧光变化持续缺乏一个关键的“门控谷氨酸转运，”这表明其他可滴定的集团有助于运输过程。此外，标记的突变转运蛋白是有活性的，表明荧光探针不会破坏转运过程。我们目前正在研究进一步的突变体，以建立构象变化的程度，以及双突变体设计来测试有关其机制的假设。

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.