The implication of ion channels and inositol 1,4,5-trisphosphate (IP₃)-induced Ca²⁺ signalling (IICS) in the carcinogenesis processes, including deregulation of cell proliferation, migration and invasion, is increasingly studied. Studies from our laboratory have shown that type 3 IP₃ receptor (IP₃R3) and voltage- and Ca²⁺-dependent K⁺ channels BK_Ca channels are involved in human breast cancer cell proliferation. In this context, we investigated the probable interaction between these two proteins (IP₃R3 and BK_Ca channel) in normal and in breast cancer cells.

MCF-7 and MCF-10A cell viability was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-assay in the presence or absence of adenosine triphosphate (ATP). Furthermore, cell-cycle analysis was carried out and cell cycle protein expression was examined by Western blotting. Immunocytochemistry and co-immunoprecipitation assays were used to check co-localisation of BK_Ca and IP₃R3 and their molecular interaction. Finally, whole cell patch-clamp and Ca²⁺ imaging were performed to assess the functional interaction.

Our results are in favour of a functional and a molecular coupling between IP₃R3 and BK_Ca channel that is involved in MCF-7 proliferation. Indeed, ATP increased MCF-7 cell proliferation and this effect was impaired when the expression of BK_Ca and/or IP₃R3 has been reduced by specific small interfering RNAs (siRNAs). Flow cytometry experiments showed that both siRNAs led to cell cycle arrest in the G0/G1 phase and these results were confirmed by the analysis of cell cycle protein expression. Specifically, BK_Ca and IP₃R3 silencing decreased both cyclin-D1 and cyclin-dependent kinase 4 (CDK4) expression levels. Furthermore, ATP elicited a phospholipase C (PLC)-dependent elevation of internal Ca²⁺ that triggered in turn an iberiotoxin (IbTx)- and a tetra-ethyl-ammonium (TEA)-sensitive membrane hyperpolarisation that was strongly reduced in the cells with silenced IP₃R3 or BK_Ca. In the same way, intracellular application of Ins(2,4,5)P₃ triggered an IbTx-sensitive membrane hyperpolarisation. Moreover, intracellular Ca²⁺ chelation with 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) prevented ATP-induced BK_Ca activation. BK_Ca and IP₃R3 also co-immunoprecipitated and this interaction seemed to occur in cholesterol-enriched microdomains. Conversely, in the normal breast cell line MCF-10A, neither ATP application nor BK_Ca silencing affected cell proliferation. Furthermore, IP₃R3 and BK_Ca did not co-immunoprecipitate, suggesting the absence of a molecular coupling between BK_Ca and IP₃R3 in the MCF-10A normal cell line.

Altogether, our results suggest a molecular and functional link between BK_Ca channel and IP₃R3 in cancer cells. Our findings led us to propose this coupling between BK_Ca and IP₃R3 as an important mechanism for tumour cell proliferation.