Aim: 

Stored-operated calcium entry (SOCE), a mechanism regulated by the filling state of intracellular calcium stores, implies the activation of plasma membrane Ca²⁺ channels and the massive entry of Ca²⁺ into the cytoplasm. This mechanism is mediated by STIM1, a Ca²⁺ sensor in the membrane of stores. hTRPC1 has been largely suggested as a component of store-operated channels, and recently we demonstrated that STIM1-hTRPC1 interaction, and Ca²⁺ entry, requires Orai1 (Jardín et al. 2008). We have investigated the role of lipid raft domains in the assembly of the complex STIM1-Orai1-hTRPC1.

Methods: 

Intracellular free Ca²⁺ concentration ([Ca²⁺]_c) was determined by spectrofluorimetry and protein-protein interactions by Western blotting.

Results: 

In a Ca²⁺-free medium thapsigargin evoked a prolonged increase in [Ca²⁺]_c and increased the association between hTRPC1 and both STIM1 and Orai1. The subsequent addition of Ca²⁺ (1 mM) to the external medium induces a sustained increase in [Ca²⁺]_c indicative of SOCE. Disturbance of lipid raft domains by platelet incubation with methyl-b-cyclodextrin (10 mM) for 30 min resulted in attenuation of thapsigargin-stimulated interaction between hTRPC1, STIM1 and Orai1 and significantly reduced SOCE.

Conclusion: 

Our results suggest that the cholesterol-rich lipid raft domains are necessary for STIM1-hTRPC1-Orai1 complex formation, which is very relevant for SOCE in platelets.

References: Jardin, I., Lopez, J.J., Salido, G.M. & Rosado, J.A. 2008. Orai1 mediates the interaction between STIM1 and hTRPC1 and regulates the mode of activation of hTRPC1-forming Ca²⁺ channels. J Biol Chem 283, 25296-25304.

Supported by BFU2007-60104; I.J. was supported by BES-2008-002875