KINETICS AND SPECIFICITY OF THE RENAL NA+/MYO-INOSITOL COTRANSPORTER EXPRESSED IN XENOPUS OOCYTES

Abstract

The two-microelectrode voltage clamp technique was used to examine the kinetics and substrate specificity of the cloned renal Na+/myo-inositol cotransporter (SMIT) expressed in Xenopus oocytes. The steady-state myo-inositol-induced current was measured as a function of the applied membrane potential (V-m), the external myo-inositol concentration and the external Na+ concentration, yielding the kinetic parameters: K-0.5(MI), K-0.5(Na), and the Hill coefficient n. At 100 mM NaCl, K-0.5(MI) was about 50 mu M and was independent of V-m. At 0.5 mM myo-inositol, K-0.5(Na) ranged from 76 mM at V-m = -50 mV to 40 mM at V-m = -150 mV. n was voltage independent with a value of 1.9 +/- 0.2, suggesting that two Na+ ions are transported per molecule of myo-inositol. Phlorizin was an inhibitor with a voltage-dependent apparent K-I of 64 mu M at V-m = -50 mV and 130 mu M at V-m = -150 mV. To examine sugar specificity, sugar-induced steady-state currents (at V-m= -150 mV) were recorded for a series of sugars, each at an external concentration of 50 mM. The substrate selectivity series was myo-inositol, scyllo-inositol > L-fucose > L-xylose > L-glucose, D-glucose, alpha-methyl-D-glucopyranoside > D-galactose, D-fucose, 3-O-methyl-D-glucose, 2-deoxy-D-glucose > D-xylose. For comparison, oocytes were injected with cRNA for the rabbit intestinal Na+/glucose cotransporter (SGLT1) and sugar-induced steady-state currents (at V-m = -150 mV) were measured. For oocytes expressing SGLT1, the sugar selectivity was: D-glucose, alpha-methyl-D-glucopyranoside, D-galactose, D-fucose, 3-O-methyl-D-glucose > D-xylose, L-xylose, 2-deoxy-D-glucose > myoinositol, L-glucose, L-fucose. The ability of SMIT to transport glucose and SGLT1 to transport myo-inositol was independently confirmed by monitoring the Na+ dependent uptake of H-3-D-glucose and H-3-myo-inositol, respectively. In common with SGLT1, SMIT gave a relaxation current in the presence of 100 mM Na+ that was abolished by phlorizin (0.5 mM). This transient current decayed with a voltage-sensitive time constant between 10 and 14 msec. The presteady-state current is apparently due to the reorientation of the cotransporter protein in the membrane in response to a change in V-m. The kinetics of SMIT is accounted for by an ordered six-state nonrapid equilibrium model.