Probing Structure and Function of Ion Channels Using Limited Proteolysis and Microfluidics

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• 作者：Carolina L. Trkulja ; Erik T. Jansson ; Kent Jardemark ; Owe Orwar
• 刊名：Journal of the American Chemical Society
• 出版年：2014
• 出版时间：October 22, 2014
• 年：2014
• 卷：136
• 期：42
• 页码：14875-14882
• 全文大小：641K
• ISSN：1520-5126

文摘

Even though gain, loss, or modulation of ion channel function is implicated in many diseases, both rare and common, the development of new pharmaceuticals targeting this class has been disappointing, where it has been a major problem to obtain correlated structural and functional information. Here, we present a microfluidic method in which the ion channel TRPV1, contained in proteoliposomes or in excised patches, was exposed to limited trypsin proteolysis. Cleaved-off peptides were identified by MS, and electrophysiological properties were recorded by patch clamp. Thus, the structure鈥揻unction relationship was evaluated by correlating changes in function with removal of structural elements. Using this approach, we pinpointed regions of TRPV1 that affect channel properties upon their removal, causing changes in current amplitude, single-channel conductance, and EC₅₀ value toward its agonist, capsaicin. We have provided a fast 鈥渟hotgun鈥?method for chemical truncation of a membrane protein, which allows for functional assessments of various peptide regions.