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

TRPC proteins have been described as non-selective cation channels and are thought to be involved in the regulation of Ca(2+) 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 Ca(2+) movement. Thus, TRPC6 channels cannot be defined as simple Ca(2+) 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, Ca(2+) 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.