The seven mammalian TRPC isoforms have been separated into two groups (TRPC1, 4, 5 and TRPC3, 6, 7 (Trebak et al. 2003)) based on sequence relationships, their functional properties when over-expressed in transfected cells and their ability to assemble into heteromeric channels. Among these seven isoforms, TRPC7 is probably one of the least studied. We cloned it, expressed it transiently in the HEK-293T cell line and studied its localisation and its regulation.

The full-length cDNA sequence for htrpc7 was subcloned into the mammalian expression vector pcDNA3.1(-) (Invitrogen) and HEK 293T cells were transfected with the pcDNA3.1(-)-hTRPC7 construct using Fugene^TM (Roche). An epitope-tagged version of the hTRPC7 cDNA was used to transfect HEK-293T cells and the distribution of the channel examined using an anti-FLAG antibody and immunofluorescence microscopy. As with other members of the TRPC family, some fluorescence was detected at the plasma membrane, but the majority was present in an intracellular compartment.

For the functional experiments, we used the pIRES2-EGFP (Clontech, Basingstoke, UK) vector to express the full-length htrpc7 cDNA thus allowing a clear localisation of transfected cells. Ca²⁺ concentrations of individual HEK-293T cells were monitored with Fura 2 AM. Data are expressed as means ± S.E.M. Ca²⁺ elevation was assessed by measuring the area under the Ca²⁺ transient and was expressed as sum of (340/380) ratio. When a calcium re-addition protocol was used with ATP (100µM) as an agonist, we found that there was a significant increase in Ca²⁺ entry in pIRES2-htrp7-transfected cells compared to pIRES2-EGFP transfected cells (Calcium elevation = 3.44 ± 0.54 in control cells, n = 39, 3 transfections, compared to 12.85 ± 1.19 in htrp7-transfected cells, n = 34 cells, 3 transfections, P < 0.05 unpaired Student’s t test). However, when CPA (10 µM) was used to empty the Ca²⁺ stores, there was no significant increase in calcium entry (Calcium entry = 31.92 ± 1.68 in control cells, n = 115 cells, 3 transfections compared to 34.70 ± 1.10 in pIRES2-EGFP-htrp7 transfected cells, n = 125 cells, 4 transfections. We therefore conclude that hTRPC7 is not activated by store depletion but by PLC activation.