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 (IAMG) using whole cell recording techniques. IAMG was compared among cells by standardizing the measured currents to cell size using cell capacitance measurements. IAMG at a given membrane potential was measured as a function of alpha-methylglucoside (AMG) concentration and can be fit to Michaelis-Menten kinetics. IAMG 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 (Im) are calculated from the Michaelis-Menten equation fit to data measuring IAMG vs. AMG concentration at a constant Na+ concentration. Im showed potential dependence under all conditions. Potential-dependent Na+ binding rate(s) cannot alone explain the observed potential dependence of Im under saturating Na+ conditions. Therefore, because Im is potential dependent, at least one step of the transport cycle other than external Na+ binding must be potential dependent. Im was also calculated from data taken at 40 mM external Na+. At all potentials studied, Im at 155 mM Na+ is greater than Im 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, Im at 40 mM external Na+ increases with hyperpolarization faster than Im at 155 mM Na+. Together, these facts indicate that the rate at which Na+ binds to the transporter is also potential dependent.(ABSTRACT TRUNCATED AT 250 WORDS)
Bennett
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E
Department of Biophysics, University of Rochester, School of Medicine and Dentistry, New York 14642.
Kimmich
G A
GA
D002460:Cell Line; D004594:Electrophysiology; D007700:Kinetics; D008564:Membrane Potentials; D008757:Methylglucosides / Q000378:metabolism; D008954:Models, Biological; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*; D009994:Osmolar Concentration; D012964:Sodium / Q000378:metabolism*
D008757:Methylglucosides; D009004:Monosaccharide Transport Proteins; C027020:methylglucoside; D012964:Sodium