title: TRPC5 activation kinetics are modulated by the scaffolding protein ezrin/radixin/moesin-binding phosphoprotein-50 (EBP50).
abstract: TRPC1-7 proteins are members of a family of mammalian non-specific cation channels that mediate receptor-operated, phospholipase Cbeta/Cgamma dependent Ca(2+) influx in various cell types. TRPC4 and TRPC5 form a subfamily within TRPCs. Uniquely in the TRPC family, these channels possess a C-terminal "VTTRL" motif that binds to PDZ-domains of the scaffolding protein, EBP50 (NHERF1; Tang et al., J Biol Chem 275:37559-37564). The functional effects of EBP50 on TRPC4/5 activity have not been investigated. We have cloned rat TRPC5 (rTRPC5), functionally expressed it in HEK293 cell, and studied channel regulation with patch-clamp techniques. Both rTRPC5 and its VTTRL deletion mutant (r5dV) were localized to the plasma membrane. rTRPC5 did not display any significant basal activity in unstimulated HEK293 cells. In cells co-expressing rTRPC5 and H1 histamine receptor, rTRPC5 current evoked by GTPgammaS or histamine developed in two phases: a slowly developing, small inward current was followed by a rapidly developing, transient, large inward current. Each phase had a characteristic non-linear current-voltage (I-V) relationship. Deletion of the VTTRL motif had no detectable effect on the biophysical properties of the channel. Co-expression of EBP50 with rTRPC5 caused a significant delay in the time-to-peak of the histamine-evoked, transient large inward current. EBP50 did not modify the activation kinetics of the VTTRL-deletion mutant. We conclude that the VTTRL motif is not necessary for activation of TRPC5, but may mediate the modulatory effect of EBP50 on TRPC5 activation kinetics.
authors.affiliation: Department of Pharmacology and Physiology, UMDNJ, New Jersey Medical School, Newark, New Jersey 07101-1709, USA.
authors.forename: Alexander G
authors.initials: AG
authors.lastname: Obukhov
authors.forename: Martha C
authors.initials: MC
authors.lastname: Nowycky
mesh_terms: D000818:Animals; D001798:Blood Proteins / Q000378:metabolism; D015220:Calcium Channels / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D027682:Cation Transport Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D003598:Cytoskeletal Proteins / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006632:Histamine / Q000494:pharmacology; D006801:Humans; D007150:Immunohistochemistry; D007700:Kinetics; D008564:Membrane Potentials / Q000187:drug effects; D008565:Membrane Proteins / Q000378:metabolism; D008840:Microfilament Proteins / Q000378:metabolism; D008954:Models, Biological; D008969:Molecular Sequence Data; D018408:Patch-Clamp Techniques; D010750:Phosphoproteins / Q000378:metabolism; D051381:Rats; D011969:Receptors, Histamine H1 / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction / Q000502:physiology*; D017923:Sodium-Hydrogen Exchangers / Q000187:drug effects / Q000378:metabolism*; D050052:TRPC Cation Channels; D014162:Transfection; D000099149:Ezrin
chemical_list: D001798:Blood Proteins; D015220:Calcium Channels; D027682:Cation Transport Proteins; D003598:Cytoskeletal Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006632:Histamine; D008565:Membrane Proteins; D008840:Microfilament Proteins; D010750:Phosphoproteins; D011969:Receptors, Histamine H1; D017923:Sodium-Hydrogen Exchangers; D050052:TRPC Cation Channels; D000099149:Ezrin; C491780:TRPC5 protein, human; C093376:sodium-hydrogen exchanger regulatory factor; C071058:moesin; C071776:radixin