Relaxation kinetics of the Na+/glucose cotransporter.

Abstract

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 ...

Transport model of the human Na+-coupled l-ascorbic acid (vitamin C) transporter SVCT1

“…Maximal charge (Q max ) was conserved upon progressive removal of Na ϩ , whereas the voltage midpoint (V 0.5 ) shifted to more hyperpolarized potentials. Pre-steady-state currents have been observed for several other Na ϩ -coupled (1,9,14,17,19,27) or H ϩ -coupled (8,12,16,21,22,25) solute transporters. The results of model simulation have led investigators to attribute pre-steady-state currents to two steps in the transport cycle, namely reorientation of the empty charged carrier and binding/dissociation of the driving ion partway within the plane of the membrane electric field (and in some cases also to the binding/dissociation of the ion at the internal face).…”

“…In contrast, a simulation (data not shown) in which we set ␣Ј Ͻ ␦ (␣Ј ϭ 0.32) closely predicted the /V m and Q/V m relationships at 100 mM Na ϩ but underestimated the Na ϩ -dependent shift in V 0.5 (only Ϫ27 mV/decade) and predicted instead a one-sixth reduction in Q max between 100 and 5 mM Na ϩ . Q max effects of the driving-ion concentration were observed for the Na ϩ -glucose cotransporter SGLT1 (14) and the H ϩ -peptide cotransporter PEPT1 (16) and were accounted for by models in which ␣Ј Ͻ ␦ (␣Ј Ϸ 0.3).…”

“…We did not consider the inward-facing Na ϩ -bound configuration C7 ϭ [CNa]Љ since intracellular Na ϩ concentration ([Na ϩ ]) is generally low. Our partial-reaction model is essentially identical to that used to describe the Na ϩ -glucose cotransporters SGLT1 (9,14,28) and SGLT3 (formerly pSGLT2) (17) and similar to that used to describe a myo-[H ϩ ]inositol cotransporter (12). The rate of change in concentration of each carrier state is given by the difference of forward and reverse reactions…”

Kinetics of the Reverse Mode of the Na+/Glucose Cotransporter

“…These protocols were approved by the UCLA Animal Research Committee, and meet all the NIH guidelines for the humane treatment of animals used for research. The oocytes were defolliculated, injected with 50 ng of rabbit SGLT1 cRNA, and maintained in Barth's medium (in mM: 88 NaCl, 1 KCl, 0.33 Ca(NO 3 ) 2 , 0.41 CaCl 2 , 0.82 MgSO 4 , 2.4 NaHCO 3 , 10 HEPES, pH 7.4) supplemented with 5 µg/ml gentamycin at 18°C for 3-8 days until use (Birnir, Loo & Wright, 1991;Parent et al, 1992a;Loo et al, 1993).…”

“…The cloning of SGLT1 and its over-expression in Xenopus laevis oocytes allowed for a detailed characterization of the kinetics of cotransport (Hediger et al, 1987;Parent et al, 1992a,b;Loo et al, 1993;Hazama et al, 1997;Loo et al, 1998). Electrophysiological and tracer uptake experiments in intact oocytes expressing SGLT1 focused on the forward mode, where the substrates are transported from the extracellular to intracellular compartments.…”