NA+ BINDING TO THE NA+-GLUCOSE COTRANSPORTER IS POTENTIAL DEPENDENT

Activity of the Na+-glucose cotransporter in LLC-PK1 epithelial cells was assayed by measuring sugar-induced currents (I(AMG)) using whole cell recording techniques. I(AMG) was compared among cells by standardizing the measured currents to cell size using cell capacitance measurements. I(AMG) at a given membrane potential was measured as a function of alpha-methylglucoside (AMG) concentration and can be fit to Michaelis-Menten kinetics. I(AMG) at varying Na+ concentrations can be described by the Hill equation with a Hill coefficient of 1.6 at all tested potentials. At high external Na+ levels (155 mM), Na+ is at least 90% saturating at all tested potentials. Maximal currents at a given membrane potential (I(m)) are calculated from the Michaelis-Menten equation fit to data measuring I(AMG) vs. AMG concentration at a constant Na+ concentration. I(m) showed potential dependence under all conditions. Potential-dependent Na+ binding rate(s) cannot alone explain the observed potential dependence of I(m) under saturating Na+ conditions. Therefore, because I(m) is potential dependent, at least one step of the transport cycle other than external Na+ binding must be potential dependent. I(m) was also calculated from data taken at 40 mM external Na+. At all potentials studied, I(m) at 155 mM Na+ is greater than I(m) calculated at 40 mM Na+. This implies that the rate of external Na+ binding to the transporter at 40 mM also affects the maximal transport rate. Furthermore, I(m) at 40 mM external Na+ increases with hyperpolarization faster than I(m) at 155 mM Na+. Together, these facts indicate that the rate at which Na+ binds to the transporter is also potential dependent. Therefore, a minimum of two steps of the transport cycle are potential dependent, one of which is the rate of external Na+ binding. The data are consistent with a model in which Na+ binding and dissociation rates and the rate of translocation of the free carrier across the membrane are potential dependent.