Electrogenic properties of the cloned Na+/glucose cotransporter: II. A transport model under nonrapid equilibrium conditions

Summary The results of the accompanying electrophysiological study of the cloned Na⁺/glucose cotransporter from small intestine (Parent, L., Supplisson, S., Loo, D.D.F., Wright, E.M. (1992) J. Membrane Biol. 125:49–62) were evaluated in terms of a kinetic model. The steady-state and presteady-state cotransporter properties are described by a 6-state ordered kinetic model (mirror symmetry) with a Na⁺:MDG stoichiometry of 2. Carrier translocation in the membrane as well as Na⁺ and sugar binding and dissociation are treated as a function of their individual rate constants. Empty carrier translocation and Na⁺ binding/ dissociation are the only steps considered to be voltage dependent. Currents were associated with the translocation of the negatively charged carrier in the membrane. Negative membrane potential facilitates sugar transport. One numerical solution was found for the 14 rate constants that account quantitatively for our experiment observations: i.e., (i) sigmoidal shape of the sugar-specific current-voltage curves (absence of outward currents and inward current saturation at high negative potentials), (ii) Na⁺ and voltage dependence of K _0.5 ^sugar and i _max ^sugar , (iii) sugar and voltage dependence of K _0.5 ^Na and i _max ^Na , (iv) presteady-state currents and their dependence on external Na⁺, MDG and membrane potential, and (v) and carrier Na⁺ leak current. We conclude that the main voltage effect is on carrier translocation. Na⁺ ions that migrate from the extracellular medium to their binding sites sense 25 to 35% of the transmembrane voltage, whereas charges associated with the carrier translocation experiences 60 to 75% of the membrane electrical field. Internal Na⁺ ion binding is not voltage dependent. In our nonrapid equilibrium model, the rate-limiting step for sugar transport is a function of the membrane potential, Na]₀ and MDG]₀. At 0 mV and at saturating Na]₀ and MDG]₀, the rate-limiting step for sugar transport is the empty carrier translocation (5 sec^–1). As the membrane potential is made more negative, the empty carrier translocation gets faster and the internal Na⁺ dissociation becomes increasingly rate limiting. However, as Na]₀ is decreased to less than 10 mm, the rate-limiting step is the external Na⁺ ions binding in the 0 to –150 mV potential range. At 0 mV, the external Na⁺ dissociation constant KNa is 80 mm and decreases to 24 mm at –150 mV. The external sugar dissociation constant KNaS is estimated to be 200 m and voltage independent. Finally, the internal leak pathway (CNa₂ translocation) is insignificant. While we cannot rule out a more complex kinetic model, the electrical properties of the cloned Na⁺/glucose cotransporter are found to be adequately described by this 6-state kinetic model.We are grateful to Drs. A. Berteloot, S. Ciani, and J.-Y. Lapointe for stimulating discussions and thank our colleagues for comments. L.P. was recipient of a post-doctoral fellowship from the Medical Research Council of Canada. This work was supported by a grant from the U.S. Public Health Service DK 19567.