Phosphatidylinositol 4,5-bisphosphate (PIP2) has been presented as a versatile regulator of transient receptor potential (TRP) channels. Breakdown of PIP2 has been shown to gate TRPV1 independently of its hydrolysis products. In contrast, PIP2 activates TRPM proteins by interaction with a C-terminal PIP2-binding site. We have investigated the role of PIP2 in the regulation of store-operated Ca²⁺ entry (SOCE) through TRPC channels in human platelets. Inclusion of a water soluble PIP2 analogue, PIP2 1,2-dioctanoyl, enhanced Ca²⁺

entry induced by the physiological agonist thrombin or selective depletion of the Ca²⁺ stores that have been described in human platelets, i.e.: the dense tubular system, using 10 nM thapsigargin (TG), and the acidic stores, using 2,5-di-(tert-butyl)-1,4- hydroquinone (20 mM; TBHQ). PIP2 1,2-dioctanoyl did not alter TG- or TBHQ-induced Ca²⁺ release from the stores or the ability of platelets to remove Ca²⁺ from the cytosol. Reduction of PIP2 levels, either by blocking PIP2 resynthesis with Li⁺ or by introducing a monoclonal anti-PIP2 antibody, attenuates Ca²⁺ entry induced by thrombin, TG or TBHQ. Finally, incubation with the anti-hTRPC1 antibody, which was effective reducing SOCE did not alter the enhancing effect of PIP2 on agonist-induced Ca²⁺ entry whilst introduction of an anti-hTRPC6 antibody, directed towards the C-terminus of hTRPC6, reduced Ca²⁺ entry by thrombin, TG or TBHQ, which strongly suggest a role for hTRPC6 in SOCE in platelets, and abolished PIP2-induced increase in Ca²⁺ entry. We conclude that PIP2 positively regulates hTRPC6-mediated SOCE in human platelets. Supported by Junta de Extremadura-Consejería de Sanidad y Consumo (SCSS0619).