Inositol 1,4,5-trisphosphate receptors (InsP₃Rs) are major intracellular Ca²⁺ release channels in non-excitable cells. In addition to regulation by InsP₃ and Ca²⁺, they are modulated by post-translational modification and the binding of accessory proteins. Thus, InsP₃Rs act as scaffolds, anchoring many signalling proteins to the site of Ca²⁺ release, where they either regulate, or are regulated by Ca²⁺ release through the channel (1). To identify novel InsP₃R interacting proteins, a proteomic approach was used. To this end, InsP₃Rs were immunoprecipitated from rat brain lysate, which is a rich source of type 1 InsP₃Rs. Co-immunoprecipitated proteins were subsequently eluted and subjected to 2-dimensional liquid chromatography and Matrix Assisted Laser Desorption Ionization Time-of-flight Mass Spectrometry analysis (MALDI-TOF). Using this strategy, we identified the SNARE-associated protein SNAP-25. This interaction was confirmed by subsequent immunoblotting of proteins co-immunoprecipitated with the InsP₃R (n>3). SNAP-25 is a protein involved in Ca²⁺-regulated secretory processes and associates with N- and P/Q-type voltage-operated plasma membrane Ca²⁺-channels. To test whether the expression of SNAP-25 affects Ca²⁺ signalling, we established stable HeLa cell lines that expressed YFP-SNAP-25 in a tetracycline-inducible manner. In these cells, YFP-SNAP-25 was targeted to the plasma membrane. Using fura-2 imaging, we investigated whether overexpression of YFP-SNAP-25 affected InsP₃-induced Ca²⁺ release and store operated Ca²⁺ entry. Cells overexpressing YFP were used as control. Experiments were performed on at least two different days and on three coverslides for each cell line per day. Statistical significance was determined by a Mann-Whitney U test. Data are presented as mean±SEM. To investigate the effect of SNAP-25 on InsP₃-induced calcium release, cells were stimulated with increasing doses of the InsP₃-generating agonist histamine (0.5, 1 and 100 µM). Cells overexpressing YFP-SNAP-25 exhibited decreased Ca²⁺ responses to all concentrations of histamine applied (78.6±5.7%, 74.5±6.5%, and 73.3±1.7%, p<0.01 for all three concentrations). The rate of quench of cellular fura-2 fluorescence by Mn²⁺, which was used as Ca²⁺ surrogate in these experiments, was used to measure the effects of SNAP-25 on Ca²⁺ entry. In the YFP-SNAP-25 overexpressing cells, basal and histamine-induced Ca²⁺ entry (100 µM histamine for 10 min) was significantly greater than in YFP-expressing control cells (241.9±17.2% and 357.3±12.8% respectively, both p<0.0001). The SNAP-25-enhanced Ca²⁺ entry was inhibited by 1 µM Gd³⁺, indicating that it occurred via the store-operated Ca²⁺ entry (SOCE) pathway. In summary, we have shown that SNAP-25 is a novel InsP₃R interacting protein, which affects both Ca²⁺-release from the ER and SOCE.