Relationship of the Donnan potential to the transmembrane pH gradient in tracheal apical membrane vesicles

Experiments were performed to determine the factors which contribute to the transmembrane pH gradient (delta pH) and the potential gradient (delta psi) in apical plasma membrane vesicles isolated from bovine tracheal epithelium. As indicated by the accumulation of 14C-methylamine, the vesicles maintained a delta pH (inside acidic) which was dependent upon the external pH. The delta pH was also proportional to the ionic strength of the suspending medium, suggesting that the H+ distribution was dictated by a Donnan potential. Measurements of the distribution of 86Rb+ demonstrated an electrical potential gradient across the vesicle membrane, inside negative which was proportional to the medium ionic strength. delta pH changed in parallel with delta psi in response to a variety of imposed conditions. These results are compatible with the existence of a H+ conductance in the vesicle membrane. Thus the endogenous electrical and proton gradients may be manipulated and used as a general experimental tool to complement kinetic analysis in investigations of transport mechanisms using isolated vesicle preparations.