The exact nature of the calcium channels involved in capacitative and receptor-activated calcium influx has yet to be determined. However, the TRP polypeptides are candidate components of these channels. Seven different TRP proteins have been identified in mammalian cells (Harteneck et al. 2000).
We have transfected HEK293 cells with an epitope (myc)-tagged htrp3 cDNA in order to study the localisation of the polypeptide by immunofluorescence. Using a monoclonal anti-myc antibody, wild-type HEK293 only showed a very weak and diffuse background staining. A much stronger specific signal was observed at the plasma membrane of an hTRP3-over-expressing cell line. By contrast, in cells transiently over-expressing hTRP3 a punctate staining was observed near the nucleus and at the plasma membrane.
To study receptor-mediated calcium signalling in cell lines that were stably over-expressing hTRP3, intracellular calcium was measured in cells loaded with fura-2 AM (excitation 340 and 380 nm, emission 510 nm). Calcium influx was measured using a calcium re-addition protocol that dissociates intracellular calcium release from calcium influx. In control cells, in a calcium-free medium containing 100 µM EGTA, ATP (100 µM) caused a transient increase of intracellular calcium of 458 ± 33 nM (mean ± S.E.M.; n = 118 cells, 3 separate experiments) while in hTRP3-transfected cells, the Ca2+ concentration increased by 505 ± 127 nM (n = 77 cells in 3 separate experiments); these increases were not significantly different (P = 0.74, Student’s unpaired t test). Upon re-addition of 2 mM calcium, intracellular calcium increased by 65 ± 26 nM in control cells while in hTRP3-transfected cells calcium increased by 193 ± 9 nM (P = 0.009). These experiments show that while the intracellular calcium pool released by ATP was similar in the two cell types, ATP-induced calcium entry was more than tripled in the hTRP3-transfected cell line. Furthermore, the percentage of cells with a calcium entry increased from 56 ± 12 % in the control cells to 94 ± 3 % (P < 0.001; x2 test) in the transfected cells. These data suggest that calcium influx, but not store release, in response to stimulation of the G-protein-coupled P2Y receptors is increased in HEK293 cells overexpressing hTRP3.
- Harteneck, C., Plant, T.D. & Schultz, G. (2000). Trends Neurosci. 23, 159-166.