Functional Interactions between BK_Caα‐Subunit and Annexin A5: Implications in Apoptosis

Abstract

Proteomic studies have suggested a biochemical interaction between α subunit of the large conductance, voltage- and Ca2+-activated potassium channel (BKCa α), 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 BKCa α contains 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 BKCa α expressing HEK 293 cells. The functional consequences of ANXA5 partnership with BKCa α were striking, with ANXA5 knockdown causing an increase and ANXA5 overexpression causing a decrease, in single BKCa channel 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.

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“…Annexins are usually associated with the onset of Ca 2+ -dependent apoptosis in different animal tissues. However recent studies showed that annexin action on apoptosis is induced by inhibition of K + efflux channels [ 96 ]. In resting conditions, annexin blocked Ca 2+ -activated cytoplasmic site of K + efflux channels, but increased cytosolic Ca 2+ causes relocation of annexin to the extracellular side triggering K + efflux channel activation by cytosolic Ca 2+ .…”