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Mol Pharmacol. 2019 Jul;96(1):90-98. doi: 10.1124/mol.119.115949.

Small Fluorescein Arsenical Hairpin-Based Förster Resonance Energy Transfer Analysis Reveals Changes in Amino- to Carboxyl-Terminal Interactions upon OAG Activation of Classical Transient Receptor Potential 6.

Author information

1
Walther-Straub-Institute of Pharmacology and Toxicology, Member of the German Center for Lung Research (DZL) (S.F., U.S., S.E., T.G., M.M.S., A.D.) and Institute for Cardiovascular Prevention (IPEK) (U.S.), Ludwig-Maximilians-Universitaet Muenchen, Munich, Germany; German Centre for Cardiovascular Research (DZHK), Munich Heart Alliance, Munich, Germany (T.G., M.M.S.); and Comprehensive Pneumology Center Munich (CPC-M), Munich, Germany (T.G., A.D.).
2
Walther-Straub-Institute of Pharmacology and Toxicology, Member of the German Center for Lung Research (DZL) (S.F., U.S., S.E., T.G., M.M.S., A.D.) and Institute for Cardiovascular Prevention (IPEK) (U.S.), Ludwig-Maximilians-Universitaet Muenchen, Munich, Germany; German Centre for Cardiovascular Research (DZHK), Munich Heart Alliance, Munich, Germany (T.G., M.M.S.); and Comprehensive Pneumology Center Munich (CPC-M), Munich, Germany (T.G., A.D.) Alexander.Dietrich@lrz.uni-muenchen.de.

Abstract

Although the overall structure of many classical transient receptor potential proteins (TRPC), including human and murine TRPC6, were recently resolved by cryoelectron microscopy analysis, structural changes during channel activation by 1-oleoyl-1-acetyl-sn-glycerol (OAG), the membrane-permeable analog of diacylglycerol, were not defined. Moreover, data on carboxyl- and amino-terminal interactions were not provided, as the amino-terminal regions of murine and human TRPC6 were not resolved. Therefore, we employed a Förster resonance energy transfer (FRET) approach using a small fluorescein arsenical hairpin (FlAsH) targeted to a short tetracysteine sequence at the unresolved amino-terminus and cerulean, a cyan fluorescent protein, as a tag at the carboxyl-terminus of the murine TRPC6 protein. After OAG as well as GSK-1702934A activation, FRET efficiency was simultaneously and significantly reduced, indicating a decreased interaction between the amino to carboxyl termini in the functional tagged murine TRPC6 tetramer (TRPC6 WT) heterologously expressed in human embryonic kidney 293T cells. There was a significant reduction in the FRET signal obtained from analysis of murine TRPC6 FRET constructs with homologous amino-terminal mutations (M131T, G108S) that had been identified in human patients with inherited focal segmental glomerulosclerosis, a condition that can lead to end-stage renal disease. A novel, designed loss-of-function TRPC6 mutation (N109A) in the amino-terminus in close proximity to the carboxyl-terminus produced similar FRET ratios. SIGNIFICANCE STATEMENT: Our data show for the first time that FlAsH-tagging of ion channels is a promising tool to study conformational changes after channel opening and may significantly advance the analysis of ion channel activation as well as their mutants involved in channelopathies.

PMID:
31171574
DOI:
10.1124/mol.119.115949

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