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

Summary

Methods are described which demonstrate the use of unidirectional influx of¹⁴C-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 (Δψ) and Na⁺-dependent sugar influx was determined for these cells at various Na⁺ and α-methylglucoside (α-MG) concentrations. The results show a high degree of Δψ dependence for the transport Michaelis constant but a maximum velocity for transport which is independent of Δψ. No transinhibition by intracellular sugar (40mm) can be detected. Sugar influx in the absence of Na⁺ is insensitive to 1.3mm phlorizin and independent of Δψ. 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.