title: Molecular interaction and functional coupling between type 3 inositol 1,4,5-trisphosphate receptor and BKCa channel stimulate breast cancer cell proliferation.
abstract: BACKGROUND
  The implication of ion channels and inositol 1,4,5-trisphosphate (IP3)-induced Ca(2+) 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 Ca(2+)-dependent K(+) channels BKCa channels are involved in human breast cancer cell proliferation. In this context, we investigated the probable interaction between these two proteins (IP3R3 and BKCa channel) in normal and in breast cancer cells.
  
  
  METHODS
  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 BKCa and IP3R3 and their molecular interaction. Finally, whole cell patch-clamp and Ca(2+) imaging were performed to assess the functional interaction.
  
  
  RESULTS
  Our results are in favour of a functional and a molecular coupling between IP3R3 and BKCa channel that is involved in MCF-7 proliferation. Indeed, ATP increased MCF-7 cell proliferation and this effect was impaired when the expression of BKCa 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, BKCa 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 Ca(2+) 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 BKCa. In the same way, intracellular application of Ins(2,4,5)P3 triggered an IbTx-sensitive membrane hyperpolarisation. Moreover, intracellular Ca(2+) chelation with 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) prevented ATP-induced BKCa activation. BKCa 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 BKCa silencing affected cell proliferation. Furthermore, IP3R3 and BKCa did not co-immunoprecipitate, suggesting the absence of a molecular coupling between BKCa and IP3R3 in the MCF-10A normal cell line.
  
  
  CONCLUSIONS
  Altogether, our results suggest a molecular and functional link between BKCa channel and IP3R3 in cancer cells. Our findings led us to propose this coupling between BKCa and IP3R3 as an important mechanism for tumour cell proliferation.
authors.affiliation: Laboratory of Cellular and Molecular Physiology (EA-4667), 'Ion Channels in Breast Cancer', SFR CAP-SANTE (FED-4231), University of Amiens, UFR Sciences, 33 Rue Saint-Leu, 80039 Amiens, France.
authors.forename: Abdallah
authors.initials: A
authors.lastname: Mound
authors.forename: Lise
authors.initials: L
authors.lastname: Rodat-Despoix
authors.forename: Salim
authors.initials: S
authors.lastname: Bougarn
authors.forename: Halima
authors.initials: H
authors.lastname: Ouadid-Ahidouch
authors.forename: Fabrice
authors.initials: F
authors.lastname: Matifat
mesh_terms: D000230:Adenocarcinoma / Q000378:metabolism / Q000473:pathology*; D001943:Breast Neoplasms / Q000378:metabolism / Q000473:pathology*; D002453:Cell Cycle / Q000502:physiology; D049109:Cell Proliferation; D002478:Cells, Cultured; D005260:Female; D006801:Humans; D047468:Immunoprecipitation; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D053496:Inositol 1,4,5-Trisphosphate Receptors / Q000378:metabolism*; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits / Q000378:metabolism*; D061986:MCF-7 Cells; D011485:Protein Binding
chemical_list: C506929:ITPR3 protein, human; D053496:Inositol 1,4,5-Trisphosphate Receptors; C494841:KCNMA1 protein, human; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; D015544:Inositol 1,4,5-Trisphosphate
keywords: BK(Ca) channel; Breast cancer; Ca(2+); Proliferation; Type 3 inositol 1,4,5-trisphosphate receptor; voltage- and Ca(2+)-dependent K(+) channels