Role of Cl- in electrogenic Na+-coupled cotransporters GAT1 and SGLT1.

We have investigated the functional role of Cl(-) in the human Na(+)/Cl(-)/gamma-aminobutyric acid (GABA) and Na(+)/glucose cotransporters (GAT1 and SGLT1, respectively) expressed in Xenopus laevis oocytes. Substrate-evoked steady-state inward currents were examined in the presence and absence of external Cl(-). Replacement of Cl(-) by gluconate or 2-(N-morpholino)ethanesulfonic acid decreased the apparent affinity of GAT1 and SGLT1 for Na(+) and the organic substrate. In the absence of substrate, GAT1 and SGLT1 exhibited charge movements that manifested as pre-steady-state current transients. Removal of Cl(-) shifted the voltage dependence of charge movements to more negative potentials, with apparent affinity constants (K(0.5)) for Cl(-) of 21 and 115 mm for SGLT1 and GAT1, respectively. The maximum charge moved and the apparent valence were not altered. GAT1 stoichiometry was determined by measuring GABA-evoked currents and the unidirectional influx of (36)Cl(-), (22)Na(+), or [(3)H]GABA. Uptake of each GABA molecule was accompanied by inward movement of 2 positive charges, which was entirely accounted for by the influx of Na(+) in the presence or absence of Cl(-). Thus, the GAT1 stoichiometry was 2Na(+):1GABA. However, Cl(-) was transported by GAT1 because the inward movement of 2 positive charges was accompanied by the influx of one Cl(-) ion, suggesting unidirectional influx of 2Na(+):1Cl(-):1GABA per transport cycle. Activation of forward Na(+)/Cl(-)/GABA transport evoked (36)Cl(-) efflux and was blocked by the inhibitor SKF 89976A. These data suggest a Cl(-)/Cl(-) exchange mechanism during the GAT1 transport cycle. In contrast, Cl(-) was not transported by SGLT1. Thus, in both GAT1 and SGLT1, Cl(-) modulates the kinetics of cotransport by altering Na(+) affinity, but does not contribute to net charge transported per transport cycle. We conclude that Cl(-) dependence per se is not a useful criterion to classify Na(+) cotransporters.

A high affinity fungal nitrate carrier with two transport mechanisms.

We have expressed the CRNA high affinity nitrate transporter from Emericella (Aspergillus) nidulans in Xenopus oocytes and used electrophysiology to study its properties. This method was used because there are no convenient radiolabeled substrates for the transporter. Oocytes injected with crnA mRNA showed nitrate-, nitrite-, and chlorite-dependent currents. Although the gene was originally identified by chlorate selection there was no evidence for transport of this anion. The gene selection is explained by the high affinity of the transporter for chlorite, and the fact that this ion contaminates solutions of chlorate. The pH-dependence of the anion-elicited currents was consistent with H(+)-coupled mechanism of transport. At any given voltage, currents showed hyperbolic kinetics with respect to extracellular H(+), and these data could be fitted with a Michaelis-Menten relationship. But this equation did not adequately describe transport of the anion substrates. At higher concentrations of the anion substrates and more negative membrane voltages, the currents were decreased, but this effect was independent of changes in external pH. These more complicated kinetics could be fit by an equation containing two Michaelis-Menten terms. The substrate inhibition of the currents could be explained by a transport reaction cycle that included two routes for the transfer of nitrate across the membrane, one on the empty carrier and the other proton coupled. The model predicts that the substrate inhibition of transporter current depends on the cytosolic nitrate concentration. This is the first time a high affinity nitrate transport activity has been characterized in a heterologous system and the measurements show how the properties of the CRNA transporter are modified by changes in the membrane potential, external pH, and nitrate concentration. The physiological significance of these observations is discussed.

Specificity and stoichiometry of the Arabidopsis H+/amino acid transporter AAP5.

The H+-dependent AAP5 amino acid transporter from Arabidopsis thaliana was expressed in Xenopus oocytes, and we used radiotracer flux and electrophysiology methods to investigate its substrate specificity and stoichiometry. Inward currents of up to 9 microA were induced by a broad spectrum of amino acids, including anionic, cationic, and neutral amino acids. The apparent affinity of AAP5 for amino acids was influenced by the position of side chain branches, bulky ring structures, and charged groups. The maximal current was dependent on amino acid charge, but was relatively independent of amino acid structure. A detailed kinetic analysis of AAP5 using lysine, alanine, glutamate, and histidine revealed H+-dependent differences in the apparent affinity constants for each substrate. The differences were correlated to the effect of H+ concentration on the net charge of each amino acid and suggested that AAP5 transports only the neutral species of histidine and glutamate. Stoichiometry experiments, whereby the uptake of 3H-labeled amino acid and net inward charge were simultaneously measured in voltage-clamped oocytes, showed that the charge:amino acid stoichiometry was 2:1 for lysine and 1:1 for alanine, glutamate, and histidine. The results confirm that histidine is transported in its neutral form and show that the positive charge on lysine contributes to the magnitude of its inward current. Thus, the transport stoichiometry of AAP5 is 1 H+:1 amino acid irrespective of the net charge on the transported substrate. Structural features of amino acid molecules that are involved in substrate recognition by AAP5 are discussed.

Transport mechanism of the cloned potato H+/sucrose cotransporter StSUT1.

The transport mechanism of the potato StSUT1 H+/sucrose cotransporter expressed in Xenopus oocytes was investigated using the 2-electrode voltage clamp and radiotracer flux methods. Sucrose induced inward currents through the transporter that were dependent on the extracellular sucrose and H+ concentrations and the membrane voltage. The activation of StSUT1 by H+ and sucrose displayed Michaelis-Menten-type kinetics suggestive of a 1:1 H+:sucrose stoichiometry. This was confirmed by simultaneously measuring inward currents and sucrose flux in voltage-clamped oocytes. The apparent affinities K0.5 for H+ and sucrose were voltage-dependent. At -150 mV Ksuc0.5 was 0.5 +/- 0.07 mM at 10 microM H+o, and KH0.5 was 0.1 +/- 0.05 microM at 20 mM sucroseo. StSUT1 exhibited presteady-state transient currents, which relaxed with time constants between <1 and 4 ms and fitted to the Boltzmann equation: maximum charge transfer Qmax approximately 1.8 nanocoulombs; apparent valence z approximately 1; potential for 50% charge transfer V0.5 approximately -15 mV at 0.032 microM H+o and -45 mV at 10 microM H+o. The steady-state data were used to formulate a kinetic model for sucrose transport, and computer simulations were performed to obtain rate constants for the partial reaction steps. Our model is consistent with protons binding to StSUT1 before sucrose with both ligands transported simultaneously across the membrane.

Kinetics and specificity of a H+/amino acid transporter from Arabidopsis thaliana.

The amino acid transporter AAP1/NAT2 recently cloned from Arabidopsis thaliana was expressed in Xenopus oocytes, and we used electrophysiological, radiotracer flux, and electron microscopic methods to characterize the biophysical properties, kinetics, and specificity of the transporter. Uptake of alanine was H(+)-dependent increasing from 14 pmol/oocyte/h at 0.032 microM H+ to 370 pmol/oocyte/h at 10 microM H+. AAP1 was electrogenic; there was an amino acid-induced depolarization of the oocyte plasma membrane and net inward currents through the transporter due to the transport of amino acids favoring neutral amino acids with shortside chains. The maximal current (imax) for alanine, proline, glutamine, histidine, and glutamate was voltage and [H+]o-dependent. Similarly, the imaxH was voltage and [amino acid]o-dependent. The imax for both H+ and amino acid were dependent on the concentrations of their respective cosubstrates, suggesting that both ligands bind randomly to the transporter. The K0.5 of the transporter for amino acids decreased as [H+]o increased and was lower at negative membrane potentials. The K0.5 for H+ was relatively voltage-independent and decreased as [amino acid]o increased. This positive cooperativity suggests that the transporter operates via a simultaneous mechanism. The Hill coefficients n for amino acids and H+ were > 1, suggesting that the transporter has more than one binding site for both H+ and amino acid. Freeze-fracture electron microscopy was used to estimate the number of transporters expressed in the plasma membrane of oocytes. The density of particles on the protoplasmic face of the plasma membrane of oocytes expressing AAP1 increased approximately 5-fold above water-injected controls and corresponded to a turnover number 350 to 800 s-1.

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/microns2, mean, SD) and that exogenously expressed channels and transporters increased the density of particles (up to 5,000/microns2) 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 In-tramembrane Protein) and MIP (Major Intrinsic Protein). We estimated a "gating charge" of approximately 3.5 electronic charges for SGLT1 and approximately 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 x 10(-14) cm3/sec for CHIP and of 1.5 x 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 approximately 100-fold less effective than CHIP.

Steady-state and presteady-state kinetics of the H+/hexose cotransporter (STP1) from Arabidopsis thaliana expressed in Xenopus oocytes.

We have investigated the steady-state and presteady-state kinetics of the cloned H+/hexose cotransporter from Arabidopsis thaliana (STP1) expressed in Xenopus oocytes using the two-electrode voltage-clamp method. Steady-state sugar-dependent currents were measured between -150 and +50 mV as a function of external [3-O-methyl-D-glucose] (3OMG) and [H+]. At pH 6.5 (316 nM H+) the maximal current for sugar, i3OMGmax, was voltage-dependent, increasing from 40 nA at -30 mV to 95 nA at -150 mV. The apparent affinity of sugar, K3OMG0.5, at pH 6.5 decreased from 30 microM at -30 mV to 11 microM at -70 mV and was then voltage-independent between -70 and -150 mV. Increasing the extracellular [H+] to 3160 nM (pH 5.5) increased i3OMGmax to 65 nA at -30 mV and 212 nA at -150 mV. K3OMG0.5 at pH 5.5 also increased and was voltage-dependent: 15 microM at -50 mV rising to 25 microM at -150 mV. At pH 6.5 and 5.5, the Hill coefficient n for 3OMG was voltage-independent and averaged 1.2 and 1.4, respectively. At saturating [3OMG] iHmax was voltage-dependent and KH0.5 was voltage-independent, averaging 370 nM (pH 6.4). The Hill coefficient n for H+ was voltage-independent and averaged 1. The sugar specificity of STP1 was D-mannose > or = 2-deoxyglucose > D-galactose > or = 3OMG > D-xylose > D-glucose > D-fucose > D-fructose > L-glucose > L-arabinose > D-arabinose, demonstrating that STP1 has a low substrate specificity. Transient currents recorded after rapid steps in membrane potential relaxed with time constants tau, between 3 and 14 ms. The charge movement Q (the integral of the current transients) fitted to a Boltzmann relation with maximal charge Qmax of 3.4 nanocoulombs and an apparent valence z approximately 1 corresponding to a transporter density of 2 x 10(10)/oocyte. Potential for 50% Qmax (V0.5) was -28 mV. At saturating 3OMG and at low external [H+] (pH 7.5), the transient STP1 currents were eliminated. The presteady-state data indicate that STP1 can bind H+ in the absence of sugar, and the steady-state data suggest that H+/hexose cotransport occurs via a sequential mechanism.

Functional expression of a plant plasma membrane transporter in Xenopus oocytes.

A full-length cDNA clone for the H+/hexose co-transporter (STP1) from Arabidopsis thaliana has been transcribed in vitro and the mRNA injected into Xenopus oocytes. Under optimized conditions, oocytes injected with the STP1 mRNA accumulated 3-O-[methyl-14C]glucose at rates of more than a 1000-fold greater than water-injected control oocytes. A hexose-elicited depolarization of the oocyte membrane potential was demonstrated, and uptake was shown to be stimulated by low external pH, confirming the activity of a H+/hexose co-transport system. This is the first example of the functional expression of a plant membrane transporter in oocytes.