Biophysics Relaxation kinetics of the Na + / glucose cotransporter ( Na + / glucose symporter / pre - steady - state current / charge movement ) DONALD

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 ofNa+/glucose transport (5). This model (see Fig. 4A) assumes a valence of -2 for the transporter and that translocation of the empty transporter and Na+ binding are both voltage dependent. The kinetics are ordered, with two Na+ ions binding prior to the binding of sugar. The fully loaded transporter then reorients in the membrane and releases sugar and Na+ to the cytoplasm, and the cycle is completed The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. by the reorientation of the empty sugar and Na+ binding sites back to the external face of the membrane. According to this model, pre-steady-state currents observed after a voltage step are due to the charge transfer involved in the dissociation ofexternal Na+ and the reorientation of the unloaded SGLT1 protein (C, for carrier) in the membrane: [CNa2]' a [C]' k [C]" (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 a powerful experimental approach to understanding the mechanisms for the transport of solutes into cells by cotransport-