title: 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 (Vm), the external myo-inositol concentration and the external Na+ concentration, yielding the kinetic parameters: KMI0.5, KNa0.5, and the Hill coefficient n. At 100 mM NaCl, KMI0.5 was about 50 microM and was independent of Vm. At 0.5 mM myo-inositol, KNa0.5 ranged from 76 mM at Vm = -50 mV to 40 mM at Vm = -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 KI of 64 microM at Vm = -50 mV and 130 microM at Vm = -150 mV. To examine sugar specificity, sugar-induced steady-state currents (at Vm = -150 mV) were recorded for a series of sugars, each at an external concentration of 50 mM. The substrate selectivity series was myo-inositol, scylloinositol > 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 Vm = -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 > myo-inositol, 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 3H-D-glucose and 3H-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 Vm. The kinetics of SMIT is accounted for by an ordered six-state nonrapid equilibrium model.
authors.affiliation: Department of Physiology, UCLA School of Medicine 90024-1751, USA.
authors.forename: K
authors.initials: K
authors.lastname: Hager
authors.forename: A
authors.initials: A
authors.lastname: Hazama
authors.forename: H M
authors.initials: HM
authors.lastname: Kwon
authors.forename: D D
authors.initials: DD
authors.lastname: Loo
authors.forename: J S
authors.initials: JS
authors.lastname: Handler
authors.forename: E M
authors.initials: EM
authors.lastname: Wright
mesh_terms: D000818:Animals; D050260:Carbohydrate Metabolism; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D004285:Dogs; D004594:Electrophysiology; D015870:Gene Expression; D006360:Heat-Shock Proteins / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D017136:Ion Transport / Q000502:physiology; D007668:Kidney / Q000378:metabolism; D007700:Kinetics; D008565:Membrane Proteins; D009865:Oocytes / Q000378:metabolism*; D012313:RNA / Q000378:metabolism; D011817:Rabbits; D027981:Symporters; D014981:Xenopus
chemical_list: D002352:Carrier Proteins; D006360:Heat-Shock Proteins; D008565:Membrane Proteins; D027981:Symporters; C073884:SLC5A3 protein, human; D012313:RNA