Molecular mechanism and cellular function of channel-like SLC26 proteins

SLC26家族的多功能阴离子转运体包括偶联阴离子交换剂和阴离子依赖的运动蛋白，但也包括在宏观传导特性上类似阴离子通道的异构体（通道样SLC26）。有些同工异构体在介导这两种功能时是混杂的。目前，这种功能变异的结构基础和生理作用都不为人所知。为了了解阴离子类通道功能的机制，并将其与二级活性阴离子交换联系起来，我们将重点研究SLC26A7、SLC26A9和SLC26A11这三种主要以类通道模式起作用的异构体。我们将定义SLC26A7、SLC26A9和SLC26A11的所有输运模式，并利用电生理和荧光光谱对它们进行深入的功能分析。通道样slc26可能通过阴离子选择性渗透途径传导阴离子，也可能具有快速构象变化的单转运体功能。为了区分单转运体和通道机制，我们将采用半胱氨酸可及性策略和电压钳形荧光测定法以及电压相关位移（“感应”）电流探针。为了揭示通道样SLC26的细胞功能，我们将研究SLC26A11在异源表达系统以及肾插层细胞（其天然位置之一）中的功能。SLC26A11似乎在原生细胞的质膜中完成其功能，但在细胞培养中的异源表达导致主要溶酶体定位。我们将结合蛋白质组学方法和光学显微镜分析新的辅助亚基和异源二聚化在控制SLC26A11亚细胞分布中的作用。我们将研究SLC26A11在培养的哺乳动物细胞和插层细胞系中设置细胞质和细胞器内[Cl-]和pH中的作用，同时通过过表达、敲除或基因组编辑来操纵A11或鉴定的相互作用物的存在。

The SLC26 family of multifunctional anion transporters encompasses coupled anion exchanger and anion-dependent motor proteins, but also isoforms that resemble anion channels in macroscopic conduction properties (channel-like SLC26s). Some isoforms are promiscuous in mediating both functions. At present, neither the structural basis nor the physiological role of this functional variability is known. To understand the mechanisms underlying anion channel-like function and to relate it to secondary-active anion exchange we will focus on three isoforms, SLC26A7, SLC26A9 and SLC26A11, that predominantly function in a channel-like mode. We will define all transport modes of SLC26A7, SLC26A9 and SLC26A11 and compare them with an in-depth functional analysis using electrophysiology and fluorescence spectroscopy. Channel-like SLC26s might conduct anions through an anion-selective aqueous permeation pathway or function as uniporter with fast conformational changes. To distinguish between uniporter and channel mechanisms we will employ cysteine accessibility strategies and voltage clamp fluorometry and probe for voltage-dependent displacement ('sensing') currents. To uncover cellular functions of channel-like SLC26 we will study SLC26A11 in heterologous expression systems as well as in renal intercalated cells, one of its native locations. SLC26A11 appears to fulfill its functions in the plasma membrane of native cells, but heterologous expression in cell culture results in predominant lysosomal localization. We will analyze the role of novel accessory subunits and heterodimerization in controlling the SLC26A11 subcellular distribution with a combination of proteomic approaches and light microscopy. We will investigate the role of SLC26A11 in setting cytosolic and intra-organelle [Cl-] and pH in cultured mammalian cells and in intercalated cell lines using fluorescent chloride and pH indicators while manipulating the presence of A11 or identified interactors by overexpression, knock-down, or genome editing.