title: Functional Interactions between BKCaα-Subunit and Annexin A5: Implications in Apoptosis.
abstract: Proteomic studies have suggested a biochemical interaction betweenαsubunit of the large conductance, voltage- and Ca2+-activated potassium channel (BKCaα), and annexin A5 (ANXA5), which we verify here by coimmunoprecipitation and double labelling immunocytochemistry. The observation that annexin is flipped to the outer membrane leaflet of the plasma membrane during apoptosis, together with the knowledge that the intracellular C-terminal of BKCaαcontains both Ca2+-binding and a putative annexin-binding motif, prompted us to investigate the functional consequences of this protein partnership to cell death. Membrane biotinylation demonstrated that ANXA5 was flipped to the outer membrane leaflet of HEK 293 cells early in serum deprivation-evoked apoptosis. As expected, serum deprivation caused caspase-3/7 activation and this was accentuated in BKCaαexpressing HEK 293 cells. The functional consequences of ANXA5 partnership with BKCaαwere striking, with ANXA5 knockdown causing an increase and ANXA5 overexpression causing a decrease, in single BKCachannel Ca2+-sensitivity, measured in inside-out membrane patches by patch-clamp. Taken together, these data suggest a novel model of the early stages of apoptosis where membrane flippage results in removal of the inhibitory effect of ANXA5 on K+channel activity with the consequent amplification of Ca2+influx and augmented activation of caspases.
authors.affiliation: Division of Pathophysiology and Repair, School of Biosciences, Sir Martin Evans Building, Museum Avenue, Cardiff University, Cardiff CF10 3AX, UK.
authors.forename: Stephen P
authors.identifier: 0000-0002-6644-4718
authors.initials: SP
authors.lastname: Brazier
authors.affiliation: Division of Pathophysiology and Repair, School of Biosciences, Sir Martin Evans Building, Museum Avenue, Cardiff University, Cardiff CF10 3AX, UK.
authors.forename: Vsevolod
authors.identifier: 0000-0002-5054-8774
authors.initials: V
authors.lastname: Telezhkin
authors.affiliation: Division of Pathophysiology and Repair, School of Biosciences, Sir Martin Evans Building, Museum Avenue, Cardiff University, Cardiff CF10 3AX, UK.
authors.forename: Paul J
authors.identifier: 0000-0003-2773-973X
authors.initials: PJ
authors.lastname: Kemp
mesh_terms: D017304:Annexin A5 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D017209:Apoptosis; D002118:Calcium / Q000378:metabolism; D053148:Caspase 3 / Q000378:metabolism; D053179:Caspase 7 / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D051036:Large-Conductance Calcium-Activated Potassium Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018613:Microscopy, Confocal; D018408:Patch-Clamp Techniques; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism
chemical_list: D017304:Annexin A5; D051036:Large-Conductance Calcium-Activated Potassium Channels; D021122:Protein Subunits; D034741:RNA, Small Interfering; D053148:Caspase 3; D053179:Caspase 7; D002118:Calcium