Effect of Ionic Strength on Bax:TBID Mediated Mitochondrial Outer Membrane Permeabilization (MOMP)

Abstract

Fluctuations in ionic strength have been documented in apoptosis. While the exact molecular identity of the channels through which proteins permeate across the outer mitochondrial membrane is unclear, it is fairly certain that the channel is large in size and sustained over time. We sought to determine the effect of salt concentration on Bax and tBid in inducing MOMP. While high salt buffer (60mM) facilitated more release of inter-membrane space (IMS) proteins (Adenylate Kinase and Sulfite Oxidase) with Bax and tBid than low salt (5mM HEPES buffer), low salt conditions induced more sustained, real-time permeabilization, as measured by accessibility of exogenous cytochrome c to complex IV. There was a dichotomy between the kinetics of IMS protein release and real-time permeabilization at high salt conditions, while under low salt conditions, they were congruent. The effect of ionic strength on sustained permeabilization is biphasic, with maxima at 10mM. The sustained permeabilization is inducible by diluting the salt concentration to that of low salt, but irreversible by incorporating high salt conditions. This observation is inconsistent with a dynamic channel model sensitive to ionic strength. It seems likely that under low salt conditions, there is a co-operative interaction between Bax molecules in the membrane and those in solution, but the growth of the channel by membrane-bound Bax molecules itself is independent of ionic strength. Supported by a grant from NSF (MCB-0641208).