title: Cellular cholesterol controls TRPC3 function: evidence from a novel dominant-negative knockdown strategy.
abstract: TRPC3 (canonical transient receptor potential protein 3) has been suggested to be a component of cation channel complexes that are targeted to cholesterol-rich lipid membrane microdomains. In the present study, we investigated the potential role of membrane cholesterol as a regulator of cellular TRPC3 conductances. Functional experiments demonstrated that cholesterol loading activates a non-selective cation conductance and a Ca2+ entry pathway in TRPC3-overexpressing cells but not in wild-type HEK-293 (human embryonic kidney 293) cells. The cholesterol-induced membrane conductance exhibited a current-to-voltage relationship similar to that observed upon PLC (phospholipase C)-dependent activation of TRPC3 channels. Nonetheless, the cholesterol-activated conductance lacked negative modulation by extracellular Ca2+, a typical feature of agonist-activated TRPC3 currents. Involvement of TRPC3 in the cholesterol-dependent membrane conductance was further corroborated by a novel dominant-negative strategy for selective blockade of TRPC3 channel activity. Expression of a TRPC3 mutant, which contained a haemagglutinin epitope tag in the second extracellular loop, conferred antibody sensitivity to both the classical PLC-activated as well as the cholesterol-activated conductance in TRPC3-expressing cells. Moreover, cholesterol loading as well as PLC stimulation was found to increase surface expression of TRPC3. Promotion of TRPC3 membrane expression by cholesterol was persistent over 30 min, while PLC-mediated enhancement of plasma membrane expression of TRPC3 was transient in nature. We suggest the cholesterol content of the plasma membrane as a determinant of cellular TRPC3 activity and provide evidence for cholesterol dependence of TRPC3 surface expression.
authors.affiliation: Institute of Pharmaceutical Sciences, Pharmacology and Toxicology, Karl-Franzens-University of Graz, Universitaetsplatz 2, A-8010 Graz, Austria.
authors.forename: Annarita
authors.initials: A
authors.lastname: Graziani
authors.forename: Christian
authors.initials: C
authors.lastname: Rosker
authors.forename: Sepp D
authors.initials: SD
authors.lastname: Kohlwein
authors.forename: Michael X
authors.initials: MX
authors.lastname: Zhu
authors.forename: Christoph
authors.initials: C
authors.lastname: Romanin
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Sattler
authors.forename: Klaus
authors.initials: K
authors.lastname: Groschner
authors.forename: Michael
authors.initials: M
authors.lastname: Poteser
mesh_terms: D020013:Calcium Signaling; D002217:Carbachol / Q000494:pharmacology; D002412:Cations / Q000378:metabolism; D002460:Cell Line; D002784:Cholesterol / Q000502:physiology*; D000939:Epitopes / Q000235:genetics; D018390:Gene Targeting; D005799:Genes, Dominant; D017930:Genes, Reporter; D006801:Humans; D017136:Ion Transport / Q000187:drug effects*; D007668:Kidney / Q000166:cytology; D008563:Membrane Lipids / Q000502:physiology*; D016254:Mutagenesis, Insertional; D018408:Patch-Clamp Techniques; D011993:Recombinant Fusion Proteins / Q000502:physiology; D050052:TRPC Cation Channels / Q000235:genetics / Q000502:physiology*; D014162:Transfection; D047392:beta-Cyclodextrins / Q000494:pharmacology
chemical_list: D002412:Cations; D000939:Epitopes; D008563:Membrane Lipids; D011993:Recombinant Fusion Proteins; D050052:TRPC Cation Channels; C104603:TRPC3 cation channel; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D002217:Carbachol; D002784:Cholesterol