valinomycin and methylglucoside

Topics: Biological Transport, Active; Carbohydrate Metabolism; Cell Membrane; Cell Membrane Permeability; Electrochemistry; Indicators and Reagents; Kinetics; Membrane Potentials; Methylglucosides; Onium Compounds; Organophosphorus Compounds; Potassium; Sodium; Valinomycin

Zero-trans kinetics of Na+-sugar cotransport were investigated. Sugar influx was measured at various sodium and sugar concentrations in K+-loaded cells treated with rotenone and valinomycin. Sugar influx follows Michaelis-Menten kinetics as a function of sugar concentration but not as a function of Na+ concentration. Nine models with 1:1 or 2:1 sodium:sugar stoichiometry were considered. The flux equations for these models were solved assuming steady-state distribution of carrier forms and that translocation across the membrane is rate limiting. Classical enzyme kinetic methods and a least-squares fit of flux equations to the experimental data were used to assess the fit of the different models. Four models can be discarded on this basis. Of the remaining models, we discard two on the basis of the trans sodium dependence and the coupling stoichiometry [G. A. Kimmich and J. Randles, Am. J. Physiol. 247 (Cell Physiol. 16): C74-C82, 1984]. The remaining models are terter ordered mechanisms with sodium debinding first at the trans side. If transfer across the membrane is rate limiting, the binding order can be determined to be sodium:sugar:sodium.

Topics: Animals; Carbohydrate Metabolism; Chickens; Intestinal Mucosa; Kinetics; Mathematics; Membrane Potentials; Methylglucosides; Models, Biological; Sodium; Valinomycin

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.

Topics: Animals; Biological Transport, Active; Cell Membrane; Chickens; Epithelium; Intestinal Mucosa; Intestine, Small; Intracellular Fluid; Kinetics; Membrane Potentials; Methylglucosides; Methylglycosides; Onium Compounds; Organophosphorus Compounds; Phlorhizin; Potassium; Sodium; Valinomycin

Sodium-dependent sugar transport systems involve the function of membrane components that couple the transmembrane flow of Na+ to the concomitant flow of certain sugar molecules. The coupling stoichiometry between Na+ and sugar fluxes via these systems must be measured under conditions in which the membrane potential does not change due to the induction of transport or during the interval of flux measurement. This can be accomplished by utilizing gradients of highly permeant ions (NO-3 and K+ plus valinomycin) to create diffusion potentials of sufficient magnitude that the sugar-induced Na+ flux does not introduce an appreciable change in the imposed potential. Under these conditions, the coupling stoichiometry for chicken intestinal cells proves to be 2 Na+:1 sugar as reported earlier for studies performed in the absence of a membrane potential. When control of the potential is not maintained, a coupling ratio of 1:1 is observed. The stoichiometry does not change as a function of Na+ concentration, which suggests that carrier forms with only one Na+ bound do not contribute to the carrier-mediated Na+ or sugar fluxes. When no potential is present, the stoichiometry is modified by the level of intracellular Na+ and sugar in a manner indicative of a transport mechanism in which Na+ must dissociate from the "loaded" carrier at the inward facing membrane surface before the sugar molecule dissociates.

Topics: Adenosine Triphosphate; Animals; Biological Transport, Active; Carbohydrate Metabolism; Cell Membrane Permeability; Chickens; Epithelial Cells; In Vitro Techniques; Intestinal Mucosa; Intestines; Ion Channels; Membrane Potentials; Methylglucosides; Potassium; Sodium; Valinomycin