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TRPC6 silencing in primary airway smooth muscle cells inhibits protein expression without affecting OAG-induced calcium entry

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Abstract

TRPC proteins have been described as non-selective cation channels and are thought to be involved in the regulation of Ca2+ movement in various cells, including airway smooth muscle (ASM) cells. In order to study the role of these channels in ASM cells, transfection of a small interfering RNA (siRNA) designed against the TRPC6 channel was performed in guinea pig primary ASM cells. This specific siRNA was complexed with the new X-TremeGene (X-TG) chemical transfection reagent, whose efficiency and low cytotoxicity were determined by the use of a non-silencing rhodamine-tagged siRNA. It was found that more than 95% of cells were transfected by an optimized protocol. Verification of TRPC6 transcript down-regulation was determined by RT-PCR while Western blot analysis attested to lower protein content in the microsomal fraction. Micro-spectrofluorimetry measurements of control and siRNA-treated cells revealed that lower TRPC6 expression did not affect OAG-induced intracellular Ca2+ movement. Thus, TRPC6 channels cannot be defined as simple Ca2+ transporters but more likely as protein complexes supporting monovalent cation conductance in ASM cells. These conductances would in turn facilitate membrane depolarization of high input resistance cells, Ca2+ channel activation and tone increase. In conclusion, this study defines a valuable model of RNA interference study in primary cultures of ASM cells, eventually allowing for silencing of other target proteins for which no pharmacological modulators are currently available.

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Acknowledgments

The authors wish to thank Claude Roberge for constructive comments and Pierre Pothier for the critical reading of the manuscript. This work was supported by a CIHR grant MOP-57677 to ER. Dr Rousseau is a member of the Health Respiratory Network of the FRSQ: http://www.rsr.chus.qc.ca

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Authors and Affiliations

  1. Le Bilarium, Department of Physiology and Biophysics, Faculty of Medicine and Health Sciences, Université de Sherbrooke, 3001, 12th avenue north, J1H 5N4, Sherbrooke, QC, Canada

    Nicolas Godin & Eric Rousseau

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  1. Nicolas Godin
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  2. Eric Rousseau
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Correspondence to Eric Rousseau.

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Godin, N., Rousseau, E. TRPC6 silencing in primary airway smooth muscle cells inhibits protein expression without affecting OAG-induced calcium entry. Mol Cell Biochem 296, 193–201 (2007). https://doi.org/10.1007/s11010-006-9309-1

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  • DOI: https://doi.org/10.1007/s11010-006-9309-1

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