The mechanistic nature of the membrane potential dependence of sodium-sugar cotransport in small intestine

Abstract

Methods are described which demonstrate the use of unidirectional influx of 14C-tetraphenylphosphonium (14C-TPP+) into isolated intestinal epithelial cells as a quantitative sensor of the magnitude of membrane potentials created by experimentally imposed ion gradients. Using this technique the quantitative relationship between membrane potential (delta psi) and Na+-dependent sugar influx was determined for these cells at various Na+ and alpha-methylglucoside (alpha-MG) concentrations. The results show a high degree of delta psi dependence for the transport Michaelis constant but a maximum velocity for transport which is independent of delta psi. No transinhibition by intracellular sugar (40 mM) can be detected. Sugar influx in the absence of Na+ is insensitive to 1.3 mM phlorizin and independent of delta psi. The mechanistic implications of these results were evaluated using the quality of fit between calculated and experimentally observed kinetic constants for rate equations derived from several transport models. The analysis shows that for models in which translocation is the potential-dependent step the free carrier cannot be neutral. If it is anionic, the transporter must be functionally asymmetric. A model in which Na+ binding is the potential-dependent step (Na+ well concept) also provides an appropriate kinetic fit to the experimental data, and must be considered as a possible mechanistic basis for function of the system.