The influence of transmembrane ion gradients on the flux of weak acids and bases across liposomal systems

A primary role of biological membranes is to provide a permeability barrier to ion translocation. This thesis examines how ion gradients and in particular pH gradients influence the net flux of weak acids and bases across membranes. This first requires an understanding of the relationship between transmembrane electrical potentials (ΔΨ) and transmembrane pH gradients (ΔpH). It is demonstrated that an imposed ΔΨ or an imposed ΔpH will drive the net transport of protons across large unilamellar vesicles (LUVs). However, transport of protons does not result in the establishment of the expected electrochemical equilibrium over the time course investigated (8 hr). The results confirm that the stability of the transmembrane electrical potential is primarily influenced by the presence of permeable counterions and not to the high intrinsic proton permeability of the LUVs. The second area of investigation demonstrates that the presence of a high concentration of the weak base TRIS (2-amino-2-hydroxy-methylpropane-1,3-diol) in the external buffer of large unilamellar vesicles exhibiting an imposed ΔΨ (inside negative) decreases the apparent ΔΨ induced pH gradient. This model membrane system is utilized to examine the mechanism of transport of several lipophilic amines which are of biological interest. Transport of the amines at neutral pH is demonstrated to occur primarily via the neutral unprotonated molecule. In addition, it is demonstrated that the amines do not act as proton lonophores except in the presence of the lipophilic anion tetraphenylboron. The third area of investigation examines influence of transmembrane pH gradients on the net flux of acidic phospholipids. It is demonstrated that a transmembrane pH gradient (inside basic) will drive the net transbilayer transport of phosphatidylglycerol (PG) and phosphatidic acid (PA) but not phosphatidylserine (PS) across LUVs. The observed rate of PG transbilayer transport is several orders of magnitude faster than that previously observed in other model membrane systems. The pH dependence of the kinetics of transport is consistent with a model in which the neutral protonated form of PG is transported in response to pH gradients. The primary energetic barrier to transport of the neutral form of PG is the acyl chain component of the model membrane system.Medicine, Faculty ofBiochemistry and Molecular Biology, Department ofGraduat