The "canonical" Transient Receptor Potential cation channels, TRPCs, are molecular candidates for receptor-operated calcium channels. Receptor-mediated activation of TRPC6 channels is well-documented (Boulay et al., 1997, Hofmann et al., 1999,). The aim of this study was to develop an assay suitable for evaluating the effects of modulators of TRPC6 channels in a human TRPC6-transfected cell line.

A FLIPR-based assay for TRPC6 was developed using a proprietary membrane potential sensitive dye obtained from Molecular Devices Corporation (Sunnyvale, CA). In this study, either NaCl assay buffer containing (in mM) 140 NaCl, 0.15 CaCl₂, 3.3 KH₂PO₄, 0.8 K₂HPO₄, 1.2 MgCl₂, 10.0 D-glucose, 20.0 HEPES (pH 7.4) or Na⁺-free NMDG-Cl assay buffer containing (in mM) 140 NMDG-Cl, 0.15 CaCl₂, 3.3 KH₂PO₄, 0.8 K₂HPO₄, 1.2 MgCl₂, 10.0 D-glucose, 20.0 HEPES (pH 7.4) was used. HEK 293 transfectants (TRPC6 clone 14 or lacZ control) were plated at 6x10⁴ per well in black 96-well clear bottom poly-D-lysine coated plates (Biocoat, Becton Dickenson), incubated overnight (37ºC, 5%CO₂) and washed once with 100µl assay buffer prior to loading with the 100µl membrane potential dye in assay buffer (supplemented with 30µM BAPTA-AM, 37ºC, 5% CO₂, 20 min) before using in the FLIPR. In some experiments, the cells were mixed with the dye in suspension prior to plating (1.5x10⁵ cells per well, 100µl), centrifuging (5 min, 200g) prior to incubation as described above. After baseline recordings for 10s, carbachol (CCh) in either NaCl or NMDG-Cl assay buffer was added and changes in fluorescence were recorded for 300s. The effect of compounds (10 min preincubation prior to stimulation with 10µM CCh) was evaluated in TRPC6 HEK cells loaded with dye after plating only. All reagents were purchased from Sigma-Aldrich except for LOE 908 which was synthesised by QuChem, Northern Ireland. All experiments were performed at room temperature. Results are expressed as mean values ± s.e.m.

The muscarinic receptor agonist CCh stimulated an increase in fluorescence consistent with membrane depolarisation only in TRPC6-expressing HEK 293 cells, no increase in fluorescence was observed in the HEK lacZ control cell line in response to CCh up to a concentration of 100µM (n=5) suggesting that this response was due to TRPC6-expression. The CCh-stimulated membrane depolarisation in TRPC6-HEK cells was mediated by Na⁺ influx, as no response was observed in Na⁺-free NMDG-Cl assay buffer (n=5). CCh appeared to be more potent when the TRPC6-HEK cells were loaded with dye after plating, CCh EC₅₀ value 1.9±0.4µM (n=5), compared with dye-loading in suspension CCh EC₅₀=22.3µM±6.1µM (n=3), therefore all subsequent experiments were conducted using cells loaded with dye post-plating. The CCh (10µM) response in TRPC6-HEK cells was concentration-dependently-inhibited by the receptor-operated channel blocker SK&F 96365 (IC₅₀=4.9±0.8µM, n=4) and the nonselective cation channel blocker LOE 908 (IC50=37.3±4.8µM, n=4). However, the L-type voltage-operated calcium channel (L-VOCC) blocker nifedipine was not an effective inhibitor of the CCh response (IC₅₀>30µM, n=3).

The potency of SK&F 96365 in this study is consistent with published electrophysiological studies with murine TRPC6 channels (Inoue et al., 2001), and the lack of effect of nifedipine suggests that the CCh response is not mediated via L-VOCC channels. The ability of LOE 908 to inhibit the TRPC6 response suggests this may be a useful pharmacological tool to study TRPC6 function. Therefore, these data suggest that the novel assay described in this study may be useful for evaluating modulators of TRPC6 channel function.

HEK 293 transfectants (TRPC6 clone 14 and lacZ control) were generated by W.P. Schilling and W.G. Sinkins, Cleveland, Ohio, US.

Boulay, G. et al., (1997) J. Biol. Chem. 272(47), 29672-29680.
Hofmann, T. et al., (1999) Nature 397, 259-263.
Inoue, R. et al., (2001) Circ. Res. 88, 325-332.