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 ...

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...

During scrapie infection an abnormal isoform of the prion protein (PrP), designated PrPSc, accumulates and is found to copurify with infectivity; to date, no nucleic acid has been found which is scrapie-specific. Both uninfected and scrapie-infected cells synthesize a PrP isoform, denoted PrPC, which exhibits physical properties that differentiate it from PrPSc. PrPC was purified by immunoaffinity chromatography using a PrP-specific monoclonal antibody cross-linked to protein-A--Avidgel. PrPSc was purified by detergent extraction, poly(ethylene glycol) precipitation and repeated differential centrifugation of PrPSc polymers. Both PrP isoforms were found to have the same N-terminal amino acid sequence which begins at a predicted signal peptide cleavage site. The first 8 residues of PrPC were found to be KKXPKPGG and the first 29 residues of PrPSc were found to be KKXPKPGGWNTGGSXYPGQGSPGGNRYPP. Arg residues 3 and 15 in PrPSc and 3 in PrPC appear to be modified since no detectable signals (denoted X) were found at these positions during gas-phase sequencing. Both PrP isoforms were found to contain an intramolecular disulfide bond, linking Cys 179 and 214, which creates a loop of 36 amino acids containing the two N-linked glycosylation sites. Development of a purification protocol for PrPC should facilitate comparisons of the two PrP isoforms and lead to an understanding of how PrPSc is synthesized either from PrPC or a precursor.

Glucose/galactose malabsorption (GGM) is an autosomal recessive disease manifesting within the first weeks of life and characterized by a selective failure to absorb dietary glucose and galactose from the intestine. The consequent severe diarrhoea and dehydration are usually fatal unless these sugars are eliminated from the diet. Intestinal biopsies of GGM patients have revealed a specific defect in Na(+)-dependent absorption of glucose in the brush border. Normal glucose absorption is mediated by the Na+/glucose cotransporter in the brush border membrane of the intestinal epithelium. Cellular influx is driven by the transmembrane Na+ electrochemical potential gradient; thereafter the sugar moves to the blood across the basolateral membrane via the facilitated glucose carrier. We have previously cloned and sequenced a Na+/glucose cotransporter from normal human ileum and shown that this gene, SGLT1, resides on the distal q arm of chromosome 22. We have now amplified SGLT1 complementary DNA and genomic DNA from members of a family affected with GGM by the polymerase chain reaction. Sequence analysis of the amplified products has revealed a single missense mutation in SGLT1 which cosegregates with the GGM phenotype and results in a complete loss of Na(+)-dependent glucose transport in Xenopus oocytes injected with this complementary RNA.