TRPC3 and TRPC4 associate to form a redox-sensitive cation channel. Evidence for expression of native TRPC3-TRPC4 heteromeric channels in endothelial cells.
Canonical transient receptor potential proteins (TRPC) have been proposed to form homo- or heteromeric cation channels in a variety of tissues, including the vascular endothelium. Assembly of TRPC multimers is incompletely understood. In particular, heteromeric assembly of distantly related TRPC isoforms is still a controversial issue. Because we have previously suggested TRPC proteins as the basis of the redox-activated cation conductance of porcine aortic endothelial cells (PAECs), we set out to analyze the TRPC subunit composition of endogenous endothelial TRPC channels and report here on a redox-sensitive TRPC3-TRPC4 channel complex. The ability of TRPC3 and TRPC4 proteins to associate and to form a cation-conducting pore complex was supported by four lines of evidence as follows: 1) Co-immunoprecipitation experiments in PAECs and in HEK293 cells demonstrated the association of TRPC3 and TRPC4 in the same complex. 2) Fluorescence resonance energy transfer analysis demonstrated TRPC3-TRPC4 association, involving close proximity between the N terminus of TRPC4 and the C terminus of TRPC3 subunits. 3) Co-expression of TRPC3 and TRPC4 in HEK293 cells generated a channel that displayed distinct biophysical and regulatory properties. 4) Expression of dominant-negative TRPC4 proteins suppressed TRPC3-related channel activity in the HEK293 expression system and in native endothelial cells. Specifically, an extracellularly hemagglutinin (HA)-tagged TRPC4 mutant, which is sensitive to blockage by anti-HA-antibody, was found to transfer anti-HA sensitivity to both TRPC3-related currents in the HEK293 expression system and the redox-sensitive cation conductance of PAECs. We propose TRPC3 and TRPC4 as subunits of native endothelial cation channels that are governed by the cellular redox state.
Poteser
Michael
M
Institute of Pharmaceutical Sciences, Pharmacology and Toxicology, Karl-Franzens-University of Graz, Universitaetsplatz 2, A-8010 Graz, Austria.
Graziani
Annarita
A
Institute of Pharmaceutical Sciences, Pharmacology and Toxicology, Karl-Franzens-University of Graz, Universitaetsplatz 2, A-8010 Graz, Austria.
Rosker
Christian
C
Institute of Pharmaceutical Sciences, Pharmacology and Toxicology, Karl-Franzens-University of Graz, Universitaetsplatz 2, A-8010 Graz, Austria.
Eder
Petra
P
Institute of Pharmaceutical Sciences, Pharmacology and Toxicology, Karl-Franzens-University of Graz, Universitaetsplatz 2, A-8010 Graz, Austria.
Derler
Isabella
I
Institute of Biophysics, University of Linz, A-4020 Linz, Austria.
Kahr
Heike
H
Institute of Biophysics, University of Linz, A-4020 Linz, Austria.
Zhu
Michael X
MX
Department of Neuroscience and Center for Molecular Neurobiology, The Ohio State University, Columbus, Ohio 43210.
Romanin
Christoph
C
Institute of Biophysics, University of Linz, A-4020 Linz, Austria.
Groschner
Klaus
K
Institute of Pharmaceutical Sciences, Pharmacology and Toxicology, Karl-Franzens-University of Graz, Universitaetsplatz 2, A-8010 Graz, Austria. Electronic address: klaus.groschner@uni-graz.at.
D000818:Animals; D002460:Cell Line; D042783:Endothelial Cells / Q000378:metabolism*; D005786:Gene Expression Regulation; D006801:Humans; D008564:Membrane Potentials; D051379:Mice; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D013552:Swine; D050052:TRPC Cation Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D050052:TRPC Cation Channels; C104603:TRPC3 cation channel; C489571:TRPC4 ion channel