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...
We mutated residue 166, located in the putative Na(+) transport pathway between transmembrane segments 4 and 5 of human Na(+)/glucose cotransporter (hSGLT1), from alanine to cysteine (A166C). A166C was expressed in Xenopus laevis oocytes, and electrophysiological methods were used to assay function. The affinity for Na(+) was unchanged compared to that of hSGLT1, whereas the sugar affinity was reduced and sugar specificity was altered. There was a reduction in the turnover rate of the transporter, and in contrast to that of hSGLT1, the turnover rate depended on the sugar molecule. Exposure of A166C to MTSEA and MTSET, but not MTSES, abolished sugar transport. Accessibility of A166C to alkylating reagents was independent of protein conformation, indicating that the residue is always accessible from the extracellular surface. Sugar and phlorizin did not protect the residue from being alkylated, suggesting that residue 166 is not located in the sugar pathway. MTSEA, MTSET, and MTSES all changed the pre-steady-state kinetics of A166C, independent of pH, and sugars altered these kinetics. The inability of MTSEA-labeled A166C to transport sugar was reversed (with no major change in Na(+) and sugar affinity) if the positive charge on MTSEA was neutralized by increasing the external pH to 9.0. These studies suggest that the residue at position 166 is involved in the interaction between the Na(+) and sugar transport pathways.
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