Medline/PubMed Citation | Related Articles in PubMed | Download to Citation Manager

Biochem. J. (2006) 396 (147–155) (Printed in Great Britain)

Enhanced Full Text

PDF

Legacy HTML

Other papers of interest

Send to a friend

Add a comment

Cellular cholesterol controls TRPC3 function: evidence from a novel dominant-negative knockdown strategy

Annarita GRAZIANI*¹, Christian ROSKER*¹, Sepp D. KOHLWEIN†, Michael X. ZHU‡, Christoph ROMANIN§, Wolfgang SATTLER, Klaus GROSCHNER*² and Michael POTESER*

*Institute of Pharmaceutical Sciences, Pharmacology and Toxicology, Karl-Franzens-University of Graz, Universitaetsplatz 2, A-8010 Graz, Austria, †Institute of Molecular Biosciences, Karl-Franzens-University of Graz, A-8010 Graz, Austria, ‡Department of Neuroscience and Center for Molecular Neurobiology, The Ohio State University, Columbus, OH 43210, U.S.A., §Department of Biophysics, University of Linz, A-4020 Linz, Austria, and Institute of Molecular Biology and Biochemistry, Center of Molecular Medicine, Medical University Graz, A-8010 Graz, Austria

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.

Key words: canonical transient receptor potential protein 3 (TRPC3), Ca²⁺ signalling, dominant-negative knockdown, lipid microdomain, membrane cholesterol, non-selective cation channel.

Abbreviations used: BFA, brefeldin A; DMEM, Dulbecco's modified Eagle's medium; DPBS, Dulbecco's PBS; FCS, fetal calf serum; fura 2/AM, fura 2 acetoxymethyl ester; HA, haemagglutinin; HEK-293 cells, human embryonic kidney 293 cells; HEK-TSA cells, T-cell surface antigen 201-expressing HEK-293 cells; MbCD, methyl-b-cyclodextrin; PLC, phospholipase C; TRP, transient receptor potential; TRPC, canonical TRP protein.

¹These authors have contributed equally to this work.

²To whom correspondence should be addressed (email klaus.groschner@uni-graz.at).

Received 1 August 2005/19 January 2006; accepted 31 January 2006

Published as BJ Immediate Publication 31 January 2006, doi:10.1042/BJ20051246

The Biochemical Society, London ©2006