We have presented evidence that in human platelets store-mediated Ca²⁺ entry (SMCE) involves secretion-like coupling between the human homologue of the Drosophila transient receptor potential channel, hTrp1, and the type II inositol 1,4,5-trisphosphate receptor (Rosado et al. 2000; Rosado & Sage, 2000; Rosado et al. 2002). In other cells Trp1 has been reported to be associated with cholesterol-rich lipid raft domains (LRDs) in the plasma membrane, which form the basis for its assembly into signalling complexes (e.g. Lockwich et al. 2000). LRDs have recently been demonstrated in platelets (Gousset et al. 2002) and here we have investigated whether hTrp1 is functionally associated with these domains.

Blood was drawn from healthy, drug-free volunteers with local ethical committee approval. Platelets were prepared, fluorescence measured and hTrp1 detected as previously described (Rosado & Sage, 2000). Platelet membranes were prepared as described by Gousset et al. (2002). Thapsigargin (TG)-induced Ca²⁺ entry was estimated as the integral of the rise in [Ca²⁺]_i for 2.5 min after addition of Ca²⁺. Thrombin-evoked Ca²⁺ influx was estimated as the integral of the rise in [Ca²⁺]_i above basal for 2 min after addition of thrombin, corrected by subtraction of the response in the absence of external Ca²⁺ (1.2 mM EGTA added).

Trp1 was found to be associated with platelet membranes and remained associated with membranes to a large degree after detergent (1 % Triton X-100) treatment for 30 min at 37 °C, although Triton released some hTrp1 into the solubilizate. After cholesterol depletion of the membrane by treatment with methyl-β-cyclodextrin (MBCD; 10 mM for 30 min at 37 °C), detergent treatment decreased the amount of hTrp1 associated with the membrane fraction and increased that in the solubilizate.

Cholesterol depletion using MBCD (10 mM) reduced Ca²⁺ release evoked by thrombin (1 U ml^-1) to 70.3 ± 3.8 % of control (mean ± S.E.M.; n = 13). Under the same conditions the inhibition of Ca²⁺ entry was significantly greater (P < 0.001; Student’s unpaired t test), being reduced to 48.2 ± 4.5 % of control (mean ± S.E.M.; n = 13). To investigate the effect of cholesterol depletion on SMCE alone, cells were treated with TG (200 nM) for 3 min in the presence of 200 µM EGTA to deplete the Ca²⁺ stores. SMCE was then assessed by raising external [Ca²⁺] to 300 µM. Cholesterol depletion using MBCD inhibited SMCE in a concentration-dependent manner, reducing it to 38.1 ± 4.1 % of control (mean ± S.E.M.; n = 13) after treatment with 10 mM MBCD. TG-evoked Ca²⁺ release was unaffected by MBCD.

These results suggest that hTrp1 is present in LRDs in human platelets and that these domains are involved in SMCE in these cells.

This work was supported by vacation studentships from the Nuffield Foundation (A.G.S.H.) and The Wellcome Trust (M.T.H.).