title: Probing structure and function of ion channels using limited proteolysis and microfluidics.
abstract: 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-function 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 "shotgun" method for chemical truncation of a membrane protein, which allows for functional assessments of various peptide regions.
authors.affiliation: Department of Chemical and Biological Engineering, Chalmers University of Technology , SE-412 96 Göteborg, Sweden.
authors.forename: Carolina L
authors.initials: CL
authors.lastname: Trkulja
authors.forename: Erik T
authors.initials: ET
authors.lastname: Jansson
authors.forename: Kent
authors.initials: K
authors.lastname: Jardemark
authors.forename: Owe
authors.initials: O
authors.lastname: Orwar
mesh_terms: D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D015640:Ion Channel Gating; D056656:Lab-On-A-Chip Devices / Q000379:methods*; D008958:Models, Molecular; D011487:Protein Conformation; D059748:Proteolysis; D013499:Surface Properties; D050916:TRPV Cation Channels / Q000737:chemistry* / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism
chemical_list: D050916:TRPV Cation Channels; C494482:TRPV1 protein, human; D014357:Trypsin