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的阴离子交换器和阴离子依赖的马达蛋白，但也包括宏观传导特性上类似于阴离子通道的异构体(通道样SLC26)。一些异构体在调节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尚不清楚。为了了解阴离子通道样功能的潜在机制，并将其与次级活性阴离子交换联系起来，我们将重点介绍三种主要作用于通道样模式的亚型：SLC26A7、SLC26A9和SLC26A11。我们将定义SLC26A7、SLC26A9和SLC26A11的所有转运模式，并利用电生理和荧光光谱对它们进行深入的功能分析。通道型SLC26可能通过阴离子选择性的水渗透途径传导阴离子，或者作为构象变化较快的单转运体发挥作用。为了区分单转运体和通道机制，我们将使用半胱氨酸可及性策略和电压钳荧光测定法以及电压相关位移(‘传感’)电流的探针。为了揭示通道样SLC26的细胞功能，我们将研究SLC26A11在异源表达系统中以及在其原发部位之一的肾间质细胞中的表达。SLC26A11似乎在天然细胞的质膜上完成其功能，但在细胞培养中的异源表达导致了溶酶体的主要定位。我们将结合蛋白质组学和光学显微镜的方法，分析新的辅助亚基和异源二聚在控制SLC26A11亚细胞分布中的作用。我们将使用荧光氯化物和pH指示剂研究SLC26A11在设定培养的哺乳动物细胞和嵌入细胞系中的胞浆和细胞器内[Cl-]和pH方面的作用，同时通过过度表达、敲除或基因组编辑来操纵A11或已确定的相互作用因子的存在。

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.