ISSN:
1432-1424
Keywords:
Na+/myo
;
inositol cotransport
;
Na+/glucose cotransport
;
Kinetics
;
Electrophysiology
;
Xenopus oocytes
Source:
Springer Online Journal Archives 1860-2000
Topics:
Biology
,
Chemistry and Pharmacology
Notes:
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 μm 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 μm at V m =−50 mV and 130 μm 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, α-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, α-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 V m . The kinetics of SMIT is accounted for by an ordered six-state nonrapid equilibrium model.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/BF00234656
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