Donald D. F. Loo

Biology of Human Sodium Glucose Transporters

There are two classes of glucose transporters involved in glucose homeostasis in the body, the facilitated transporters or uniporters (GLUTs) and the active transporters or symporters (SGLTs). The energy for active glucose transport is provided by the sodium gradient across the cell membrane, the Na(+) glucose cotransport hypothesis first proposed in 1960 by Crane. Since the cloning of SGLT1 in 1987, there have been advances in the genetics, molecular biology, biochemistry, biophysics, and structure of SGLTs. There are 12 members of the human SGLT (SLC5) gene family, including cotransporters for sugars, anions, vitamins, and short-chain fatty acids. Here we give a personal review of these advances. The SGLTs belong to a structural class of membrane proteins from unrelated gene families of antiporters and Na(+) and H(+) symporters. This class shares a common atomic architecture and a common transport mechanism. SGLTs also function as water and urea channels, glucose sensors, and coupled-water and urea transporters. We also discuss the physiology and pathophysiology of SGLTs, e.g., glucose galactose malabsorption and familial renal glycosuria, and briefly report on targeting of SGLTs for new therapies for diabetes.

Active sugar transport in health and disease

Secondary active glucose transport occurs by at least four members of the SLC5 gene family. This review considers the structure and function of two premier members, SGLT1 and SGLT2, and their role in intestinal glucose absorption and renal glucose reabsorption. Genetics disorders of SGLTs include Glucose‐Galactose Malabsorption, and Familial Renal Glucosuria. SGLT1 plays a central role in Oral Rehydration Therapy used so effectively to treat secretory diarrhoea such as cholera. Increasing attention is being focused on SGLTs as drug targets for the therapy of diabetes.

A method for determining the unitary functional capacity of cloned channels and transporters expressed in Xenopus laevis oocytes

The Xenopus laevis oocyte is widely used to express exogenous channels and transporters and is well suited for functional measurements including currents, electrolyte and nonelectrolyte fluxes, water permeability and even enzymatic activity. It is difficult, however, to transform functional measurements recorded in whole oocytes into the capacity of a single channel or transporter because their number often cannot be estimated accurately. We describe here a method of estimating the number of exogenously expressed channels and transporters inserted in the plasma membrane of oocytes. The method is based on the facts that the P (protoplasmic) face in water-injected control oocytes exhibit an extremely low density of endogenous particles (212±48 particles/μm2, mean, sd) and that exogenously expressed channels and transporters increased the density of particles (up to 5,000/μm2) only on the P face. The utility and generality of the method were demonstrated by estimating the “gating charge” per particle of the Na+/ glucose cotransporter (SGLT1) and a nonconducting mutant of the Shaker K+ channel proteins, and the single molecule water permeability of CHIP (Channel-like Intramembrane Protein) and MIP (Major Intrinsic Protein). We estimated a “gating charge” of ∼3.5 electronic charges for SGLT1 and ∼9 for the mutant Shaker K+ channel from the ratio of Qmax to density of particles measured on the same oocytes. The “gating charges” were 3-fold larger than the “effective valences” calculated by fitting a Boltzmann equation to the same charge transfer data suggesting that the charge movement in the channel and cotransporter occur in several steps. Single molecule water permeabilities (pfs) of 1.4 × 10−14 cm3/ sec for CHIP and of 1.5 × 10−16 cm3/sec for MIP were estimated from the ratio of the whole-oocyte water permeability (Pf) to the density of particles. Therefore, MIP is a water transporter in oocytes, albeit ∼100-fold less effective than CHIP.

Electrogenic properties of the cloned Na+/glucose cotransporter: I. Voltage-clamp studies.

SummaryThe cloned rabbit intestinal Na+/glucose cotransporter was expressed in Xenopus laevis oocytes. Presteady-state and steady-state currents associated with cotransporter activity were measured with the two-electrode voltage-clamp technique. Steady-state sugar-dependent currents were measured between −150 and +90 mV as a function of external Na+ ([Na]0) and α-methyl-d-glucopyranoside concentrations ([αMDG]0). K0.5αMDGwas found to be dependent upon [Na]0 and the membrane potential. At Vm=−50 mV, increasing [Na]0 from 10 to 100 mm decreased K0.5αMDGfrom 1.5 mm to 180 μm. Increasing membrane potential toward negative values decreased K0.5αMDGat nonsaturating [Na]0. For instance, at 10 mM [Na]0, K0.5αMDGdecreased from 1.5 mm to 360 μm on increasing the membrane potential from −50 to −150 mV. The imaxαMDGwas relatively insensitive to [Na]0 between 10 and 100 mm and weakly voltage dependent (e-fold increase per 140 mV). K0.5Naand imaxNawere found to be dependent upon membrane potential and [sugar]0. In the presence of 1 mm [αMDG]0, K0.5Nadecreased from 50 to 5 mm between 0 and −150 mV and imaxNaincreased twofold between −30 and −200 mV. The voltage dependence of K0.5Nais consistent with an effect of potential on Na+ binding (Na+-well effect), whereas the voltage dependence of imaxNais compatible with the translocation step being voltage dependent. It is concluded that voltage influences both Na+ binding and translocation. Presteady-state currents were observed for depolarization pulses in the presence of 100 mm [Na]0. The transient current relaxed with a half time of =10 msec, and both the half time and magnitude of the transient varied with the holding potential and the size of depolarization pulse. Presteady-state currents were not observed after the addition of phlorizin or αMDG to the external Na+ solution and were not observed for water-injected control oocytes. We conclude that presteady-state currents are due to the activity of the carrier and that they may give a novel insight to the transport mechanism of the Na+/ glucose cotransporter.

Electrogenic properties of the cloned Na+/glucose cotransporter: II. A transport model under nonrapid equilibrium conditions

SummaryThe results of the accompanying electrophysiological study of the cloned Na+/glucose cotransporter from small intestine (Parent, L., Supplisson, S., Loo, D.D.F., Wright, E.M. (1992) J. Membrane Biol.125:49–62) were evaluated in terms of a kinetic model. The steady-state and presteady-state cotransporter properties are described by a 6-state ordered kinetic model (“mirror” symmetry) with a Na+:αMDG stoichiometry of 2. Carrier translocation in the membrane as well as Na+ and sugar binding and dissociation are treated as a function of their individual rate constants. Empty carrier translocation and Na+ binding/ dissociation are the only steps considered to be voltage dependent. Currents were associated with the translocation of the negatively charged carrier in the membrane. Negative membrane potential facilitates sugar transport. One numerical solution was found for the 14 rate constants that account quantitatively for our experiment observations: i.e., (i) sigmoidal shape of the sugar-specific current-voltage curves (absence of outward currents and inward current saturation at high negative potentials), (ii) Na+ and voltage dependence of K0.5sugarand imaxsugar, (iii) sugar and voltage dependence of K0.5Naand imaxNa, (iv) presteady-state currents and their dependence on external Na+, αMDG and membrane potential, and (v) and carrier Na+ leak current. We conclude that the main voltage effect is on carrier translocation. Na+ ions that migrate from the extracellular medium to their binding sites sense 25 to 35% of the transmembrane voltage, whereas charges associated with the carrier translocation experiences 60 to 75% of the membrane electrical field. Internal Na+ ion binding is not voltage dependent. In our nonrapid equilibrium model, the rate-limiting step for sugar transport is a function of the membrane potential, [Na]0 and [αMDG]0. At 0 mV and at saturating [Na]0 and [αMDG]0, the rate-limiting step for sugar transport is the empty carrier translocation (5 sec−1). As the membrane potential is made more negative, the empty carrier translocation gets faster and the internal Na+ dissociation becomes increasingly rate limiting. However, as [Na]0 is decreased to less than 10 mm, the rate-limiting step is the external Na+ ions binding in the 0 to −150 mV potential range. At 0 mV, the external Na+ dissociation constant KNa′ is 80 mm and decreases to 24 mm at −150 mV. The external sugar dissociation constant KNaS′ is estimated to be 200 μm and voltage independent. Finally, the internal leak pathway (CNa2 translocation) is insignificant. While we cannot rule out a more complex kinetic model, the electrical properties of the cloned Na+/glucose cotransporter are found to be adequately described by this 6-state kinetic model.

The human Na+–glucose cotransporter is a molecular water pump

1 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. 2 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. 3 This stoichiometry was constant and independent of the external parameters: Na+ concentrations, sugar concentrations, transmembrane voltages, temperature and osmotic gradients. 4 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. 5 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. 6 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.

Kinetics of Steady-state Currents and Charge Movements Associated with the Rat Na+/Glucose Cotransporter

The rat Na+/glucose cotransporter (SGLT1) was expressed in Xenopus oocytes and steady-state and transient currents were measured using a two-electrode voltage clamp. The maximal glucose induced Na+-dependent inward current was ∼300-500 nA. The apparent affinity constants for sugar (α-methyl-D-glucopyranoside; αMDG) (KαMDG0.5) and sodium (KNa0.5) at a membrane potential of −150 mV were 0.2 mM and 4 mM. The KαMDG0.5 increased continuously with depolarizing potentials reaching 40 mM at −30 mV. KαMDG0.5 was steeply voltage dependent, 0.46 mM at −30 mV and 1 mM at −10 mV. From all tested monovalent cations only Li+ could substitute for Na+, but with lower affinity. The relative substrate specificity was D-glucose > αMDG ≈ D-galactose > 3-O-Me-Glc β-naphthyl-D-glucoside uridine. Phlorizin (Pz), the specific blocker of sugar transport, showed an extremely high affinity for the rat cotransporter with an inhibitor constant (KPzi) of 12 nM. SGLT1 charge movements in the absence of sugar were fitted by the Boltzmann equation with an apparent valence of the movable charge of ∼1, a potential for 50% maximal charge transfer (VαMDG) of −43 mV, and a maximal charge (Qmax) of 9 nanocoulombs. The apparent turnover number for the rat SGLT1 was 30 s−1. Model simulations showed that the kinetics of the rat SGLT1 are described by a six-state ordered nonrapid equilibrium model, and comparison of the kinetics of the rat, rabbit and human cotransporters indicate that they differ mainly in their presteady-state kinetic parameters.

Mechanisms of the Human Intestinal H-coupled Oligopeptide Transporter hPEPT1

The hPEPT1 cDNA cloned from human intestine (Liang, R., Fei, Y.-J., Prasad, P. D., Ramamoorthy, S., Han, H., Yang-Feng, T. L., Hediger, M. A., Ganapathy, V., and Leibach, F. H. (1995) J. Biol. Chem. 270, 6456-6463) encodes a H/oligopeptide cotransporter. Using two-microelectrode voltage-clamp in Xenopus oocytes expressing hPEPT1, we have investigated the transport mechanisms of hPEPT1 with regard to voltage dependence, steady-state kinetics, and transient charge movements. The currents evoked by 20 mM glycyl-sarcosine (Gly-Sar) at pH 5.0 were dependent upon membrane potential (V) between −150 mV and +50 mV. Gly-Sar-evoked currents increased hyperbolically with increasing extracellular [H], with Hill coefficient ≈1, and the apparent affinity constant (K0.5) for H was in the range of 0.05-1 μM. K0.5 for Gly-Sar (K0.5) was dependent upon V and pH; at −50 mV, K0.5 was minimal (≈0.7 mM) at pH 6.0. Following step-changes in V, in the absence of Gly-Sar, hPEPT1 exhibited H-dependent transient currents with characteristics similar to those of Na-coupled transporters. These charge movements (which relaxed with time constants of 2-10 ms) were fitted to Boltzmann relations with maximal charge (Q) of up to 12 nC; the apparent valence was determined to be ≈1. Q is an index of the level of transporter expression which for hPEPT1 was in the order of 10/oocyte. In general our data are consistent with an ordered, simultaneous transport model for hPEPT1 in which H binds first.

Glucose transport by human renal Na+/D-glucose cotransporters SGLT1 and SGLT2

The human Na(+)/D-glucose cotransporter 2 (hSGLT2) is believed to be responsible for the bulk of glucose reabsorption in the kidney proximal convoluted tubule. Since blocking reabsorption increases urinary glucose excretion, hSGLT2 has become a novel drug target for Type 2 diabetes treatment. Glucose transport by hSGLT2 was studied at 37°C in human embryonic kidney 293T cells using whole cell patch-clamp electrophysiology. We compared hSGLT2 with hSGLT1, the transporter in the straight proximal tubule (S3 segment). hSGLT2 transports with surprisingly similar glucose affinity and lower concentrative power than hSGLT1: Na(+)/D-glucose cotransport by hSGLT2 was electrogenic with apparent glucose and Na(+) affinities of 5 and 25 mM, and a Na(+):glucose coupling ratio of 1; hSGLT1 affinities were 2 and 70 mM and coupling ratio of 2. Both proteins showed voltage-dependent steady-state transport; however, unlike hSGLT1, hSGLT2 did not exhibit detectable pre-steady-state currents in response to rapid jumps in membrane voltage. D-Galactose was transported by both proteins, but with very low affinity by hSGLT2 (≥100 vs. 6 mM). β-D-Glucopyranosides were either substrates or blockers. Phlorizin exhibited higher affinity with hSGLT2 (K(i) 11 vs. 140 nM) and a lower Off-rate (0.03 vs. 0.2 s⁻¹) compared with hSGLT1. These studies indicate that, in the early proximal tubule, hSGLT2 works at 50% capacity and becomes saturated only when glucose is ≥35 mM. Furthermore, results on hSGLT1 suggest it may play a significant role in the reabsorption of filtered glucose in the late proximal tubule. Our electrophysiological study provides groundwork for a molecular understanding of how hSGLT inhibitors affect renal glucose reabsorption.

Inhibition of gap junction hemichannels by chloride channel blockers

Electrophysiological methods were used to assess the effect of chloride-channel blockers on the macroscopic and microscopic currents of mouse connexin50 (Cx50) and rat connexin46 (Cx46) hemichannels expressed in Xenopus laevis oocytes. Oocytes were voltage-clamped at -50 mV and hemichannel currents (ICx50 or ICx46) were activated by lowering the extracellular Ca2+ concentration ([Ca2+]o) from 5 mM to 10 microM. Ion-replacement experiments suggested that ICx50 is carried primarily (>95%) by monovalent cations (PK : PNa : PCl = 1.0 : 0.74 : 0.05). ICx50 was inhibited by 18beta-glycyrrhetinic acid (apparent Ki, 2 microM), gadolinium (3 microM), flufenamic acid (3 microM), niflumic acid (11 microM), NPPB (15 microM), diphenyl-2-carboxylate (26 microM), and octanol (177 microM). With the exception of octanol, niflumic acid, and diphenyl-2-carboxylate, the above agents also inhibited ICx46. Anthracene-9-carboxylate, furosemide, DIDS, SITS, IAA-94, and tamoxifen had no inhibitory effect on either ICx50 or ICx46. The kinetics of ICx50 inhibition were not altered at widely different [Ca2+]o (10-500 microM), suggesting that drug-hemichannel interaction does not involve the Ca2+ binding site. In excised membrane patches, application of flufenamic acid or octanol to the extracellular surface of Cx50 hemichannels reduced single channel-open probability without altering the single-channel conductance, but application to the cytoplasmic surface had no effect on the channels. We conclude that some chloride-channel blockers inhibit lens-connexin hemichannels by acting on a site accessible only from the extracellular space, and that drug-hemichannel interaction involves a high-affinity site other than the Ca2+ binding site.