An important class of integral membrane proteins, cotransporters, couple solute transport to electrochemical potential gradients; e.g., the Na+/glucose cotransporter uses the Na+ electrochemical potential gradient to accumulate sugar in ceils. So far, kinetic analysis of cotransporters has mostly been limited to steady-state parameters. In this study, we have examined pre-steady-state kinetics of Na+/glucose cotransport. The cloned human transporter (hSGLT1) was expressed in Xenopus oocytes, and voltageclamp techniques were used to monitor current transients after step changes in membrane potential. Transients exhibited a voltage-dependent time constant (Xr) ranging between 2 and 10 ms. The charge movement Q was fitted to a Boltzmann relation with mamal charge Q. of =20 nC, apparent valence z of 1, and potential Vo.s of -39 mV for 50% Q.. Lowering external Na+ from 100 to 10 mM reduced Q.,. 40%, shifted Vo.s from -39 to -70 mV, had no effect on z, and reduced the voltage dependence of T. Q. was independent of, but was dependent on, temperature (a 10°C increase increased X by a factor of ""2.5 at -50 mV). Addition ofsugar or phlorizin reduced Q",. Analyses of hSGLT1 pre-steady-state kinetics indicate that charge transfer upon a step of membrane potential in the absence of sugar is due to two steps in the reaction cycle: Na+ binding/dissociation (30%) and reorientation of the protein in the membrane field (70%). The rate-limiting step appears to be Na+ binding/dissociation. Qm. provides a measure of transporter density (=104/pm2). Charge transfer measurements give insight into the partdal reactions of the Na+/glucose cotransporter, and, combined with genetic engineering of the protein, provide a powerful tool for studying transport mechCotransporters are membrane transport proteins widely expressed in bacterial, plant, and animal cells (1, 2) which couple the transport of sugars, amino acids, neurotransmitters, osmolytes, and ions into cells to electrochemical potential gradients (Na+, H+, Cl-). An important example is the Na+/glucose cotransporter, which is responsible for the "active" accumulation of sugars in epithelial cells of the intestine.In recent electrophysiological experiments designed to measure steady-state kinetic properties of the cloned Na+/ glucose cotransporter (SGLT1) expressed in Xenopus oocytes we observed pre-steady-state currents (3, 4). These pre-steady-state currents were central in formulating a detailed quantitative nonrapid equilibrium six-state kinetic model of Na+/glucose transport (5). This model (see Fig. 4A (Fig. 4A).Here we have isolated the SGLT1 pre-steady-state currents, using a fast two-electrode voltage clamp, and have determined their kinetics as a function of voltage and Na+ and sugar concentrations. The results enable us to estimate the number of transporters in the membrane, the apparent valence of the voltage sensor, and rates for the voltagedependent steps in the transport reaction cycle (see Fig. 4A). Analysis ofpre-steady-state currents, therefore, represents ...

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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.

Mitochondrial acetoacetyl-CoA thiolase (T2) deficiency is an inborn error of metabolism affecting isoleucine and ketone bodies in the catabolic process. Mutation analysis and expression analysis of mutant cDNAs have facilitated the division of T2-deficient patients into two groups: those with null mutations in either allele (group 1) and those with mutation(s) retaining some residual T2 activity in at least one of two mutant alleles (group II). Among 5 Japanese T2-deficient patients, GK01 belonged to group I and the other patients (GK19, GK19B, GK30 and GK31) to group II. As we have suggested previously, the severity of ketoacidotic episodes in the group II patients was similar to that in the group I patient. However, the urinary organic acid and blood spot acylcarnitine profiles under stable conditions differed between the two groups. The group I patient had typical profiles for the T2 deficiency. In contrast, in all four patients in group II, tiglylglycine was not or was only faintly detected and the 2-methyl-3-hydroxybutyrate levels were less than the cutoff value. Their tiglylcarnitine levels were within the normal range and 2-methyl-3-hydroxy-, butyrylcarnitine was detected just around the cutoff value in our newborn screening pilot test. Hence, these analyses under stable conditions are not reliable for diagnosing the T2 deficiency in the group II patients. The T2 deficiency (group II) can be misdiagnosed as normal if these analyses are performed under nonepisodic conditions and possibly during the newborn screening for inborn errors of metabolism.

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