The human Na+‐glucose cotransporter (hSGLT1) was expressed in Xenopus laevis oocytes. The transport activity, given by the Na+ current, was monitored as a clamp current and the concomitant flux of water followed optically as the change in oocyte volume. When glucose was added to the bathing solution there was an abrupt increase in clamp current and an immediate swelling of the oocyte. The transmembrane transport of two Na+ ions and one sugar molecule was coupled, within the protein itself, to the influx of 210 water molecules. This stoichiometry was constant and independent of the external parameters: Na+ concentrations, sugar concentrations, transmembrane voltages, temperature and osmotic gradients. The cotransport of water occurred in the presence of adverse osmotic gradients. In accordance with the Gibbs equation, energy was transferred within the protein from the downhill fluxes of Na+ and sugar to the uphill transport of water, indicative of secondary active transport of water. Unstirred layer effects were ruled out on the basis of experiments on oocytes treated with gramicidin or other ionophores. Na+ currents maintained by ionophores did not lead to any initial water movements. The finding of a molecular water pump allows for new models of cellular water transport which include coupling between ion and water fluxes at the protein level; the hSGLT1 could account for almost half the daily reuptake of water from the small intestine.

Modifications of skeletal muscle mitochondria following exposure to high altitude (HA) are generally studied by morphological examinations and biochemical analysis of expression. The aim of this study was to examine tangible measures of mitochondrial function following a prolonged exposure to HA. For this purpose, skeletal muscle biopsies were obtained from 8 lowland natives at sea level (SL) prior to exposure and again after 28 d of exposure to HA at 3454 m. High-resolution respirometry was performed on the muscle samples comparing respiratory capacity and efficiency. Exercise capacity was assessed at SL and HA. Respirometric analysis revealed that mitochondrial respiratory capacity diminished in complex I- and complex II-specific respiration in addition to a loss of maximal state-3 oxidative phosphorylation capacity from SL to HA, all independent from alterations in mitochondrial content. Leak control coupling, respiratory control ratio, and oligomycin-induced leak respiration, all measures of mitochondrial efficiency, improved in response to HA exposure. SL respiratory capacities correlated with measures of exercise capacity near SL, whereas mitochondrial efficiency correlated best with exercise capacity following HA. This data demonstrate that 1 mo of exposure to HA reduces respiratory capacity in human skeletal muscle; however, the efficiency of electron transport improves.

The dimensions of the aqueous pore in aquaporins (AQP) 0, 1, 2, 3, 4, and 5 expressed in Xenopus laevis oocytes were probed by comparing the ability of various solutes to generate osmotic flow. By improved techniques, volume flows were determined from initial rates of changes. Identical values for the osmotic water permeability (L p ) were obtained in swelling as in shrinkage experiments demonstrating, for the first time, that aquaporins are bidirectional. The reflection coefficients () of urea, glycerol, acetamide, and formamide at 23°C were: AQP0: 1, 1, 0.8, 0.6; AQP1: 1, 0.8, 1, 1; AQP2: 1, 0.8, 1, 1; AQP3: 1, 0.2, 0.7, 0.4; AQP4: 1, 0.9, 1, 1; and AQP5: 1, 1, 1, 0.8. As seen there is no clear connection between solute size and permeation. At 13°C the s for AQP3 were 1, 0.4, 1, and 0.5; functionally, this pore narrows at lower temperatures. HgCl 2 reversibly reduced the L p of AQP3 and increased glyc to 1 and form to 0.6. We conclude that the pore of the various aquaporins are structurally different and that a simple steric model is insufficient to explain solute-pore interactions. Aquaporins (AQPs)1 are a class of membrane proteins in which water transport is thought to take place via an aqueous pore by osmosis (1-3). The physical dimensions, the location, and the chemical composition of the putative pore are largely unknown. Such parameters, however, will be central to the interpretation of structural data (4 -6) and to the elucidation of the osmotic mechanism. Here we probe the pore size by testing the osmotic efficiencies of solutes of different molecular weights, dimensions, and chemical compositions; if the test solute can permeate the pore, the resulting water fluxes will be smaller than that caused by an impermeable solute (7,8).To obtain precise measurements, it was necessary first to evaluate critically and to improve the Xenopus oocyte system as an assay of osmotic water transport. So far, water transport by aquaporins expressed in Xenopus oocytes have been investigated by challenging with hyposmolar solutions creating gradients of about 100 mosM for up to 30 min. This results in rates of swelling that vary with time. Furthermore, oocytes do not shrink as well as they swell when exposed to large hypertonic gradients (9). We have improved the volume recording and the solution changing system to employ osmotic gradients as small as 2.5 mosM and as short as 5 s. In this way the transport parameters could be determined unambiguously from the initial rate of oocyte volume changes in both swelling and shrinkage experiments.We tested six aquaporins: AQP0 (10, 11), a major intrinsic protein of the lens fibers; AQP1 (12), present predominantly in red blood cells and kidney proximal tubules cells; AQP2 (13), present in the kidney collecting duct; AQP3 (14 -16), present in the kidney collecting duct and other epithelia; AQP4 (17, 18), a mercury-insensitive channel expressed in brain; AQP5 (19), present predominantly in lung.The data demonstrate that the water flow through aquaporins are bidirectional and that...

Cotransporters are membrane proteins which couple the downhill transport of cations (Na¤ or H¤) to the uphill transport of substrates (sugars, amino acids, neurotransmitters, vitamins and ions) into cells. In addition to the secondary active transport of solutes, many cotransporters exhibit a cationic leak current (uniporter mode), which is the passive transport of Na¤ (or H¤) in the absence of substrates (see Wright et al. 1996). We have previously suggested that cotransporters can function as passive (osmotic) and secondary active water transporters. For example, the Na¤glucose cotransporter SGLT1 exhibits an osmotic water permeability that is blocked by the inhibitor phlorizin (Zampighi et al. 1995;Loo et al. 1996;Loike et al. 1996;Meinild et al. 1998a). Under sugar-transporting conditions, SGLT1 transports 200-260 HµO molecules along with two sodium ions and one glucose molecule for every transport cycle. This solute-coupled water transport occurs in the absence of, and even against, an osmotic gradient (Loo et al. 1996;Zeuthen et al. 1997;Meinild et al. 1998a;Wright et al. 1998). Similar observations on the K¤-Cl¦, H¤lactate, Na¤-Cl¦-GABA, Na¤-iodide and H¤-amino acid transporters indicate that water transport may be a general phenomenon in this class of membrane transport proteins with coupling coefficients varying from 50 to 500 water molecules per substrate molecule (

Conformational changes in the human Na(+)/glucose cotransporter (hSGLT1) were examined using hSGLT1 Q457C expressed in Xenopus laevis oocytes and tagged with tetramethylrhodamine-6-maleimide (TMR6M). Na(+)/glucose cotransport is abolished in the TMR6M-labeled mutant, but the protein binds Na(+) and sugar [Loo et al. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 7789-7794]. Under voltage clamp the fluorescence of labeled Q457C was dependent on external cations. Increasing [Na(+)] increased fluorescence with a Hill coefficient of 2 and half-maximal concentration (K(Na)(0.5)) of 49 mM at -90 mV. Li(+) also increased fluorescence, whereas choline, tetraethylammonium, and N-methyl-D-glucamine did not. Fluorescence was increased by sugars with specificity: methyl alpha-D-glucopyranoside > D-glucose > D-galactose >> D-mannitol. Voltage-jump experiments (in 100 mM NaCl buffer in absence of sugar) elicited parallel changes in pre-steady-state charge movement and fluorescence. Charge vs voltage and fluorescence vs voltage curves followed Boltzmann relations with the same median voltage (V(0.5) = -50 mV), but the apparent valence was 1 for charge movement and 0.4 for fluorescence. V(0.5) for fluorescence and charge movement was shifted by -100 mV per 10-fold decrease in [Na(+)]. Under Na(+)-free conditions, there was a voltage-dependent change in fluorescence. Voltage-jump experiments showed that the maximal change in fluorescence increased 20% with sugar. These results indicate that Na(+), sugar, and membrane voltage change the local environment of the fluorophore at Q457C. Our interpretation of these results is (1) the conformational change of the empty transporter is voltage dependent, (2) two Na(+) ions can bind cooperatively to the protein before sugar, and (3) sugar binding induces a conformational change.

This study examines the conformations of the Na+/glucose cotransporter (SGLT1) during sugar transport using charge and fluorescence measurements on the human SGLT1 mutant G507C expressed in Xenopus oocytes. The mutant exhibited similar steady-state and presteady-state kinetics as wild-type SGLT1, and labeling of Cys507 by tetramethylrhodamine-6-maleimide had no effect on kinetics. Our strategy was to record changes in charge and fluorescence in response to rapid jumps in membrane potential in the presence and absence of sugar or the competitive inhibitor phlorizin. In Na+ buffer, step jumps in membrane voltage elicited presteady-state currents (charge movements) that decay to the steady state with time constants τmed (3–20 ms, medium) and τslow (15–70 ms, slow). Concurrently, SGLT1 rhodamine fluorescence intensity increased with depolarizing and decreased with hyperpolarizing voltages (ΔF). The charge vs. voltage (Q-V) and fluorescence vs. voltage (ΔF-V) relations (for medium and slow components) obeyed Boltzmann relations with similar parameters: zδ (apparent valence of voltage sensor) ≈ 1; and V0.5 (midpoint voltage) between −15 and −40 mV. Sugar induced an inward current (Na+/glucose cotransport), and reduced maximal charge (Qmax) and fluorescence (ΔFmax) with half-maximal concentrations (K0.5) of 1 mM. Increasing [αMDG]o also shifted the V0.5 for Q and ΔF to more positive values, with K0.5's ≈ 1 mM. The major difference between Q and ΔF was that at saturating [αMDG]o, the presteady-state current (and Qmax) was totally abolished, whereas ΔFmax was only reduced 50%. Phlorizin reduced both Qmax and ΔFmax (Ki ≈ 0.4 μM), with no changes in V0.5's or relaxation time constants. Simulations using an eight-state kinetic model indicate that external sugar increases the occupancy probability of inward-facing conformations at the expense of outward-facing conformations. The simulations predict, and we have observed experimentally, that presteady-state currents are blocked by saturating sugar, but not the changes in fluorescence. Thus we have isolated an electroneutral conformational change that has not been previously described. This rate-limiting step at maximal inward Na+/sugar cotransport (saturating voltage and external Na+ and sugar concentrations) is the slow release of Na+ from the internal surface of SGLT1. The high affinity blocker phlorizin locks the cotransporter in an inactive conformation.

7. The data suggest that SGLT1 has three roles in isotonic water transport: it cotransports water directly, it supplies a passive pathway for osmotic water transport, and it generates an osmotic driving force that can be employed by other pathways, for example aquaporins.

Binding of Zn2؉ to an endogenous binding site in the dopamine transporter (DAT) leads to inhibition of dopamine (DA) uptake and enhancement of carrier-mediated substrate efflux.