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

Carolina L. Trkulja (Institutionen för kemi- och bioteknik, Fysikalisk kemi) ; Erik T. Jansson (Institutionen för kemi- och bioteknik, Fysikalisk kemi) ; Kent Jardemark (Institutionen för kemi- och bioteknik, Fysikalisk kemi) ; O. Orwar
Journal of the American Chemical Society (0002-7863). Vol. 136 (2014), 42, p. 14875-14882.
[Artikel, refereegranskad vetenskaplig]

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.



Denna post skapades 2014-12-01.
CPL Pubid: 206898

 

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Institutioner (Chalmers)

Institutionen för kemi- och bioteknik, Fysikalisk kemi (2005-2014)

Ämnesområden

Fysikalisk kemi

Chalmers infrastruktur

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Methods for elucidating membrane protein structure and function