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.
Institute of Pharmaceutical Sciences, Pharmacology and Toxicology, Karl-Franzens-University of Graz, Universitaetsplatz 2, A-8010 Graz, Austria.
Michael
M
Poteser
Institute of Pharmaceutical Sciences, Pharmacology and Toxicology, Karl-Franzens-University of Graz, Universitaetsplatz 2, A-8010 Graz, Austria.
Annarita
A
Graziani
Institute of Pharmaceutical Sciences, Pharmacology and Toxicology, Karl-Franzens-University of Graz, Universitaetsplatz 2, A-8010 Graz, Austria.
Christian
C
Rosker
Institute of Pharmaceutical Sciences, Pharmacology and Toxicology, Karl-Franzens-University of Graz, Universitaetsplatz 2, A-8010 Graz, Austria.
Petra
P
Eder
Institute of Biophysics, University of Linz, A-4020 Linz, Austria.
Isabella
I
Derler
Institute of Biophysics, University of Linz, A-4020 Linz, Austria.
Heike
H
Kahr
Department of Neuroscience and Center for Molecular Neurobiology, The Ohio State University, Columbus, Ohio 43210.
Michael X
MX
Zhu
Institute of Biophysics, University of Linz, A-4020 Linz, Austria.
Christoph
C
Romanin
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.
Klaus
K
Groschner
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