The large conductance calcium-activated potassium channel affects extrinsic and intrinsic mechanisms of apoptosis

摘要

The large conductance calcium-activated K⁺ or BK channel underlies electrical signals in a number of different cell types. Studies show that BK activity can also serve to regulate cellular homeostasis by protecting cells from apoptosis due to events such as ischemia. Recent coimmunoprecipitation studies combined with mass spectrometry suggest putative protein partners that interact with BK to regulate intrinsic and extrinsic apoptotic pathways. Here, we test two of those partners to determine the effects on these two signaling pathways. Through reciprocal coimmunoprecipitation (coIP) experiments, we show that BK interacts with p53 and FADD both in mouse brain and when overexpressed in a heterologous expression system, such as HEK293 cells. Moreover, coIP experiments using N- and C-terminal fragments reveal that FADD interacts with the C-terminus of BK, whereas p53 interacts with either the N- or C-terminus. Immunolocalization studies show that BK colocalizes with p53 and FADD in the mitochondrion and plasmalemma, respectively. HEK293 cells that stably express BK are more resistant to apoptosis when p53 or FADD are overexpressed or when their intrinsic and extrinsic pathways are stimulated via Mitomycin C or TNF-related apoptosis-inducing ligand (TRAIL), respectively. Moreover, when stimulating with TRAIL, caspase 8 activation decreases in BK expressing cells. These data suggest that BK may be part of a larger complex of proteins that protect against apoptosis by interacting with pro-apoptotic proteins such as p53 and FADD.