Conformational changes couple Na ⁺ and glucose transport

Abstract

The mechanism by which cotransport proteins couple their substrates across cell membranes is not known. A commonly proposed model is that cotransport results from ligand-induced conformational transitions that change the accessibility of ligand-binding sites from one side of the membrane to the other. To test this model, we have measured the accessibility of covalent probes to a cysteine residue (Q457C) placed in the putative sugar-translocation domain of the Na ؉ ͞glucose cotransporter (SGLT1). The mutant protein Q457C was able to transport sugar, but transport was abolished after alkylation by methanethiosulfonate reagents. Alkylation blocked sugar translocation but not sugar binding. Accessibility of Q457C to alkylating reagents required external Na ؉ and was blocked by external sugar and phlorizin. The voltage dependence of accessibility was directly correlated with the presteady-state charge movement of SGLT1. Voltage-jump experiments with rhodamine-6-maleimide-labeled Q457C showed that the time course and level of changes in f luorescence closely followed the presteadystate charge movement. We conclude that conformational changes are responsible for the coupling of Na ؉ and sugar transport and that Q457 plays a critical role in sugar translocation by SGLT1.Cotransporters are a major class of membrane proteins that are formed by members of several gene families. They share the common property of being able to couple the electrochemical potential gradient of a cation (Na ϩ or H ϩ ) to transport of organic solutes, ions, and water uphill into cells (see refs. 1 and 2). Although the mechanism of energy transduction is unknown, cotransporters share several common functional properties. For example, they exhibit presteady-state currents with step changes in membrane potential, which suggests the existence of a common mechanism.Kinetic models of cotransport have been proposed. Most popular are: alternating access models in which binding of multiple substrates at distinct sites that are only accessible on one side of the membrane at a time; transport then occurs via ligand and voltage induced conformational changes (3, 4); and channel-like models with multiple substrate occupancy without conformational changes (5). Mathematical simulations of each type of model can account for many experimental observations. In the alternating access model, the presteadystate currents are caused by both relaxations of charged or polar residues in the protein in response to voltage perturbations and movement of the transported ions in the membrane field (4, 6). In contrast, in the channel model, presteady-state currents are strictly caused by the transported ions (5).The advent of cysteine mutagenesis and derivatization with probe reagents has proved to be a useful tool for structure͞ function studies of ion channels and transporters (7-9), and membrane voltage has been found to affect the accessibility of residues in the transmembrane domain (8, 10). The dependence of cotransport function on both substrates and membrane vo...

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

“…6, Parent et al, 1992b). The model is supported by a wealth of experimental data and it, in turn, quantitatively predicts the steady-state kinetics of Na + /glucose cotransport in the forward direction, and qualitatively the presteady-state kinetic behavior of the transporter (Parent et al, 1992a,b;Loo et al, 1993Loo et al, ,1998Loo et al, ,2002Hazama et al, 1997;Meinild et al, 2002). In this model, the protein is negatively charged (valence − 2) with 6 kinetic states, consisting of the empty (C 1 , C 6 ), the Na + -bound (C 2 , C 5 ) and the Na + -and sugar-bound (C 3 , C 4 ) forms on the external and internal membrane surfaces.…”

“…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.…”

Functional identification and characterization of sodium binding sites in Na symporters

“…Given the structure of the Na1 and Na2 binding sites and the data presented here, it is no longer necessary to assume that the valence of SGLT1 is 2, especially as a valence of 1 now accounts for the charge movements as a function of [Na + ] o . However, because two Na + ions are coupled to each glucose molecule transported by WT hSGLT1 (20,21,26,27,31), this raises questions about what other step of the overall transport cycle is voltage dependent (41). Clues may come from closer inspection of the kinetics of Y290F, W291F, and hSGLT2, where the coupling ratio is 1/1 (21,42).…”

“…Substituted cysteine accessibility method (SCAM) studies on hSGLT1 show that external Na + binding triggers a conformational change to open the sugar binding pocket to the external membrane surface (20,26,27). Identifying the Na + binding sites and the coupling between Na + and glucose transport is crucial to our understanding of the transport mechanism.…”

“…Mutants used (H83C, F101C, D204E,Y290C/S/F, W291C/F, S392A/C, F453C, Q457C, and G507C) were described previously (20,21,23,26). The same procedures were used to generate a new mutant, T287C.…”