Functional Interactions between BK-Subunit and Annexin A5: Implications in Apoptosis.

Proteomic studies have suggested a biochemical interaction between subunit of the large conductance, voltage- and Ca-activated potassium channel (BK), 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 BK contains both Ca-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 BK expressing HEK 293 cells. The functional consequences of ANXA5 partnership with BK were striking, with ANXA5 knockdown causing an increase and ANXA5 overexpression causing a decrease, in single BK channel Ca-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 Ca influx and augmented activation of caspases.