FUNCTIONAL CHARACTERIZATION OF THE SLC26A9 ION CHANNEL PERMEATION PATHWAY

该项目的长期目标是对SLC26A9阴离子通道进行详细的分子理解。SLC26A9属于SLC26膜转运蛋白家族，该家族存在于从原核生物到哺乳动物的各种生物中。SLC26家族成员在营养物质和许多阴离子底物（如SO42、Cl、HCO3或I）的分泌和吸收中发挥核心作用，这些底物对细胞的生理过程（如肿胀和盐稳态）至关重要。与其他家族成员一样，SLC26A9具有模块化结构，由两半组成，每半具有七个跨膜片段，随后是细胞质硫酸盐转运蛋白和抗sigma因子拮抗剂（STAT）结构域。在功能水平上，研究提出SLC26A9作为Cl /HCO3转运体和Cl通道；因此，SLC26A9被认为具有相反的功能性质，分别介导主动和被动转运。SLC26A9在功能上的这些差异对其实际功能模式提出了几个重要的问题。我们的初步数据表明，SLC26A9是一个Cl通道，我们的主要目标是通过研究其运输机制来表征其功能。在这个项目中，我们还旨在确定形成Cl转运途径的蛋白质部分，以及支持其作为通道而不是转运体功能所必需的关键结构特征。

The long-term goal of this project is to develop a detailed molecular understanding of the SLC26A9 anion channel. SLC26A9 belongs to the SLC26 family of membrane transporters which are found in various organisms, from prokaryotes to mammals. The members of the SLC26 family play central roles in the secretion and absorption of nutrients and many anionic substrates such as SO42, Cl, HCO3 or I which are essential for the cell's physiological processes (e.g. swelling and salt homeostasis). As with other members of its family, SLC26A9 has a modular architecture consisting of two halves, each possessing seven transmembrane segments followed by a cytoplasmic sulfate transporter and an antisigma factor antagonist (STAT) domain. At the functional level, studies have proposed that SLC26A9 functions as a Cl /HCO3 transporter as well as a Cl channel; therefore, SLC26A9 is thought to have opposite functional properties, mediating active and passive transport, respectively. These discrepancies in the function of SLC26A9 raise several significant questions about its actual functional mode. Our preliminary data demonstrates that SLC26A9 is a Cl channel and our main goal is to characterise its function through the investigation of its transport mechanism. In this project, we also aim to identify the parts of the protein that form the Cl transport pathway and the key structural features essential to support its function as a channel rather than a transporter. During the term of this research proposal, we will start by identifying the best functional probes of the SLC26A9 permeation pathway. We will then use these probes to define the functional characteristics of anion permeation. Furthermore, taking advantage of the high-resolution structure of the prokaryotic SLC26 homologue (SLD26Dg; D. geothermalis), we will begin to identify key amino acid residues that form the permeation pathway and evaluate changes in their accessibility. Overall Impact: Revealing the functional and molecular details of the SLC26A9 ion channel mechanism of action will help us understand the key structural changes that makes this transporter's scaffold act as a channel. The success of this proposal will uncover the underlying mechanisms of membrane transport at the blurred boundary between channels and transporters. As an electrophysiologist, I have been studying ion channel function and structure for the last 7 years, applying sophisticated techniques and approaches to explore their mechanism of function. The breadth of my research program is thus unique, especially for such a small starting laboratory currently funded only by my Department's Startup funds. It is my hope that the NSERC committee can judge this research program for its potential within the field of ion channel structure and function.