Basic Mechanisms of Ion Channel Function

描述(由申请人提供)：这项研究的目的是详细了解属于“ClC-通道家族”的蛋白质工作的基本机制。CLC通道广泛存在于生物界，对于哺乳动物细胞膜在许多不同的生理环境中的正常功能是必不可少的。人类基因组中九种CLC亚型中任何一种的突变或破坏都会导致许多疾病，从骨骼肌强直到肾脏对血压的控制受损，再到骨的不适当重吸收。由于最近的工作，我们现在知道CLC家族包括通过根本不同的氯离子运输机制操作的异构体；虽然长期研究的肌型CLC是被动通道，但令人惊讶的是，一个细菌同系物被发现是一种二级主动转运体，在这种转运体中，上行的氯离子运输与下行的H+运输耦合。我建议通过对细菌CLC同源物进行详细的结构-功能研究来研究这一令人困惑的发现的后果。我们将研究质子偶联到氯离子转运的机制，试图通过定向突变将活性转运蛋白转化为真正的通道，并将致力于克隆、表达和重组一组携带长C-末端结构域的原核CLCs。

DESCRIPTION (provided by applicant): This research is aimed at understanding in chemical detail the fundamental mechanisms by which proteins belonging to the "CLC family of Cl- channels" work. CLC channels are widespread throughout the biological world and are essential to the proper functioning of mammalian cell membranes in many different physiological contexts. Mutations or disruptions of any of the nine CLC isoforms inhabiting the human genome lead to numerous diseases, from skeletal muscle myotonias to compromised renal control of blood pressure to improper reabsorption of bone. As a result of recent work we now know that the CLC family includes isoforms that operate by fundamentally different Cl- transport mechanisms; while the long-studied muscle type CLCs are passive channels, a bacterial homolog was surprisingly found to be a secondary active transporter in which uphill Cl- transport is coupled to downhill H+ transport. I propose to investigate the ramifications of this perplexing discovery by carrying out a detailed structure-function study of the bacterial CLC homolog. We will investigate the mechanism of proton coupling to Cl- transport, will attempt to transform, by directed mutagenesis, the active transporter into a true channel, and will work to clone, express, and reconstitute a set of prokaryotic CLCs carrying a long c-terminal domain.