Presteady-state and steady-state kinetics and turnover rate of the mouse gamma-aminobutyric acid transporter (mGAT3)

We expressed mouse gamma-aminobutyric acid (GABA) transporter (mGAT3) in Xenopus laevis oocytes and examined its steady-state and presteady-state kinetics and turnover rate by using tracer flux and electrophysiological methods. In oocytes expressing mGAT3, GABA evoked a Na+-dependent and Cl(-)-facilitated inward current. The dependence on Na+ was absolute, whereas that for Cl(-) was not. At a membrane potential of -50 mV, the half-maximal concentrations for Na+, Cl(-), and GABA were 14 mM, 5 mM, and 3 microM. The Hill coefficient for GABA activation and Cl(-) enhancement of the inward current was 1, and that for Na+ activation was > or =2. The GABA-evoked inward current was directly proportional to GABA influx (2.2 +/- 0.1 charges/GABA) into cells, indicating that under these conditions, there is tight ion/GABA coupling in the transport cycle. In response to step changes in the membrane voltage and in the absence of GABA, mGAT3 exhibited presteady-state current transients (charge movements). The charge-voltage (Q-V) relation was fitted with a single Boltzmann function. The voltage at half-maximal charge (V(0.5)) was +25 mV, and the effective valence of the moveable charge (zdelta) was 1.6. In contrast to the ON transients, which relaxed with a time constant of < or =30 msec, the OFF transients had a time constant of 1.1 sec. Reduction in external Na+ ([Na+]o) and Cl(-) ([Cl(-)]o) concentrations shifted the Q-V relationship to negative membrane potentials. At zero [Na+]o (106 mM Cl(-)), no mGAT3-mediated transients were observed, and at zero [Cl(-)]o (100 mM Na+), the charge movements decreased to approximately 30% of the maximal charge (Q(max)). GABA led to the elimination of charge movements. The half-maximal concentrations for Na+ activation, Cl(-) enhancement, and GABA elimination of the charge movements were 48 mM, 19 mM, and 5 mM, respectively. Q(max) and I(max) obtained in the same cells yielded the mGAT3 turnover rate, 1.7 sec(-1) at -50 mV. The low turnover rate of mGAT3 may be due to the slow return of the empty transporter from the internal to the external membrane surface.     

Conformational dynamics of hSGLT1 during Na+/glucose cotransport

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 tau(med) (3-20 ms, medium) and tau(slow) (15-70 ms, slow). Concurrently, SGLT1 rhodamine fluorescence intensity increased with depolarizing and decreased with hyperpolarizing voltages (DeltaF). The charge vs. voltage (Q-V) and fluorescence vs. voltage (DeltaF-V) relations (for medium and slow components) obeyed Boltzmann relations with similar parameters: zdelta (apparent valence of voltage sensor) approximately 1; and V(0.5) (midpoint voltage) between -15 and -40 mV. Sugar induced an inward current (Na(+)/glucose cotransport), and reduced maximal charge (Q(max)) and fluorescence (DeltaF(max)) with half-maximal concentrations (K(0.5)) of 1 mM. Increasing [alphaMDG](o) also shifted the V(0.5) for Q and DeltaF to more positive values, with K(0.5)'s approximately 1 mM. The major difference between Q and DeltaF was that at saturating [alphaMDG](o), the presteady-state current (and Q(max)) was totally abolished, whereas DeltaF(max) was only reduced 50%. Phlorizin reduced both Q(max) and DeltaF(max) (K(i) approximately 0.4 microM), with no changes in V(0.5)'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.     

The anticonvulsant valproate increases the turnover rate of gamma-aminobutyric acid transporters

Valproate is an important anticonvulsant currently in clinical use for the treatment of seizures. We used electrophysiological and tracer uptake methods to examine the effect of valproate on a gamma-aminobutyric acid (GABA) transporter (mouse GAT3) expressed in Xenopus laevis oocytes. In the absence of GABA, valproate (up to 50 mm) had no noticeable effect on the steady-state electrogenic properties of mGAT3. In the presence of GABA, however, valproate enhanced the GABA-evoked steady-state inward current in a dose-dependent manner with a half-maximal concentration of 4.6 +/- 0.5 mm. Maximal enhancement of the GABA-evoked current was 275 +/- 10%. Qualitatively similar observations were obtained for human GAT1 and mouse GAT4. The valproate enhancement did not alter the Na(+) or Cl(-) dependence of the steady-state GABA-evoked currents. Uptake experiments under voltage clamp suggested that the valproate enhancement of the GABA-evoked current was matched by an enhancement in GABA uptake. Thus, despite the increase in GABA-evoked current, ion/GABA co-transport remained tightly coupled. Uptake experiments indicated that valproate is not transported by mouse GAT3 in the absence or presence of GABA. Valproate also enhanced the rate of the partial steps involved in transporter presteady-state charge movements. We propose that valproate increases the turnover rate of GABA transporters by an allosteric mechanism. The data suggest that at its therapeutic concentration, valproate may enhance the activity of neuronal and glial GABA transporters by up to 10%.     

Unified modeling of the mammalian and fish proton-dependent oligopeptide transporter PepT1

Electrophysiological and biophysical analyses were used to compare the partial and complete transport cycles of the intestinal oligopeptide transporter PepT1 among three species (seabass, zebrafish and rabbit). On the whole, the presteady-state currents of the fish transporters were similar to each other. Rabbit PepT1 differed from the fish transporters by having slower-decaying currents, and the charge vs. potential (Q/V) and time constant vs. potential (τ/V) curves shifted to more positive potentials. All of the isoforms were similarly affected by external pH, showing acidity-induced slowing of the transients and positive shifts in the Q/V and τ/V curves. Analysis of the pH-dependence of the unidirectional rates of the intramembrane charge movement suggested that external protonation of the protein limits the speed of this process in both directions. The complete cycle of the transporter was studied using the neutral dipeptide Gly-Gln. Michaelis-Menten analysis confirmed that, in all species, acidity significantly increases the apparent affinity for the substrate but does not strongly impact maximal transport current. Simulations using a kinetic model incorporating the new findings showed good agreement with experimental data for all three species, both with respect to the presteady-state and the transport currents.     

Role of the conserved lysine residue in the middle of the predicted extracellular loop between M2 and M3 in the GABA(A) receptor.

In alpha1, beta2, and gamma2 subunits of the gamma-aminobutyric acid A (GABA(A)) receptor, a conserved lysine residue occupies the position in the middle of the predicted extracellular loop between the transmembrane M2 and M3 regions. In all three subunits, this residue was mutated to alanine. Whereas the mutation in alpha1 and beta2 subunits resulted each in about a sixfold shift of the concentration-response curve for GABA to higher concentrations, no significant effect by mutation in the gamma subunit was detected. The affinity for the competitive inhibitor bicuculline methiodide was not affected by the mutations in either the alpha1 subunit or the beta2 subunit. Concentration-response curves for channel activation by pentobarbital were also shifted to higher concentrations by the mutation in the alpha and beta subunits. Binding of [3H]Ro 15-1788 was unaffected by the mutation in the alpha subunit, whereas the binding of [3H]muscimol was shifted to lower affinity. Mutation of the residue in the alpha1 subunit to E, Q, or R resulted in an about eight-, 10-, or fivefold shift, respectively, to higher concentrations of the concentration-response curve for GABA. From these observations, it is concluded that the corresponding residues on the alpha1 and beta2 subunits are involved more likely in the gating of the channel by GABA than in the binding of GABA or benzodiazepines.     

Novel Properties of a Mouse γ-Aminobutyric Acid Transporter (GAT4)

We expressed the mouse gamma-aminobutyric acid (GABA) transporter GAT4 (homologous to rat/ human GAT-3) in Xenopus laevis oocytes and examined its functional and pharmacological properties by using electrophysiological and tracer uptake methods. In the coupled mode of transport (Na+/ Cl-/GABA cotransport), there was tight coupling between charge flux and GABA flux across the plasma membrane (2 charges/GABA). Transport was highly temperature-dependent with a temperature coefficient (Q10) of 4.3. The GAT4 turnover rate (1.5 s(-l); -50 mV, 21 degrees C) and temperature dependence suggest physiological turnover rates of 15-20 s(-1). No uncoupled current was observed in the presence of Na+. In the absence of external Na+, GAT4 exhibited two distinct uncoupled currents. (i) A Cl- leak current (ICl(leak)) was observed when Na+ was replaced with choline or tetraethylammonium. The reversal potential of (ICl(leak)) followed the Cl- Nernst potential. (ii) A Li+ leak current (ILi(leak)) was observed when Na+ was replaced with Li+. Both leak currents were inhibited by Na+, and both were temperature-independent (Q10 approximately 1). The two leak modes appeared not to coexist, as Li+ inhibited (ICl(leak)). The results suggest the existence of cation- and anion-selective channel-like pathways in GAT4. Flufenamic acid inhibited GAT4 Na+/Cl-/GABA cotransport, ILi(leak), and ICl(leak), (Ki approximately 30 microM), and the voltage-induced presteady-state charge movements (Ki approximately 440 microM). Flufenamic acid exhibited little or no selectivity for GAT1, GAT2, or GAT3. Sodium and GABA concentration jicroumps revealed that slow Na+ binding to the transporter is followed by rapid GABA-induced translocation of the ligands across the plasma membrane. Thus, Na+ binding and associated conformational changes constitute the rate-limiting steps in the transport cycle.     

Differential effect of pH on sodium binding by the various GABA transporters expressed in Xenopus oocytes

Mouse GABA transporters belong to the family of Na(+)- and Cl(-)-dependent neurotransmitter transporters. The four GABA transporters exhibit unique presteady-state currents when expressed in Xenopus oocytes. The properties of the presteady-state currents correspond to their different affinities to Na(+). In the presence of 20 microM GABA and at pH 7.5, the half-maximal uptake activity was 47, 120, 25 and 35 mM Na(+) for GAT1, GAT2, GAT3 and GAT4, respectively. The appearance of presteady-state currents at positive or negative imposed potentials was in correlation with the affinity to Na(+). Changing the external pH differentially affected the GABA uptake and the presteady-state activities of the various GABA transporters. It is suggested that protons compete with Na(+) on its binding site; however, the proton binding is not productive and is unable to drive GABA uptake.     

On the mechanism of the gamma-aminobutyric acid receptor in the mammalian (mouse) cerebral cortex. Chemical kinetic investigations with a 10-ms time resolution adapted to measurements of neuronal receptor function in single cells.

The gamma-aminobutyric acidA (GABA) receptor belongs to a superfamily of proteins involved in chemical reactions that regulate signal transmission between cells of the nervous system and is the target of some of the agents most frequently used in medicine to control disorders of the central nervous system. In contrast to the nicotinic acetylcholine receptor, which initiates signal transmission and is the best characterized member of the superfamily, the GABA receptor forms anion-specific transmembrane channels and inhibits signal transmission. The chemical kinetic experiments described here, in which fast chemical reaction techniques were used, indicate that both receptor proteins may operate by the same mechanism. Also described is the use of a chemical kinetic technique with a 10-ms time resolution that we have developed for making measurements with single cells isolated from specific areas of the nervous system, in this case the cerebral cortex of embryonic mice. A flow device was used to equilibrate receptors on the cell surface with GABA, and the concentration of open transmembrane channels in the cells was then measured by recording the whole-cell currents at pH 7.2, 21-23 degrees C, and a transmembrane voltage of -70 mV. Two different receptor forms, A alpha and A beta, were detected in cerebral cortical cells. Although the ratio of A alpha to A beta varied from cell to cell, on average 35% and 65% of the receptor-controlled current was associated with receptor forms A alpha and A beta, respectively. At saturating concentrations of GABA, the rate coefficients of desensitization, alpha and beta, associated with these two forms have maximal values of 4.4 and 0.7 s-1, respectively. The constants of a mechanism that accounts for the open transmembrane channels of both receptor forms were evaluated over a 50-fold range of GABA concentration. The dissociation constant of the site controlling channel opening was 40 microM for A alpha and 320 microM for A beta. The channel-opening equilibrium constant, phi-1, was 3.5 for A alpha and 20 for A beta. The evaluated constants allow one to calculate Po, the conditional probability that at a given concentration of GABA the receptor-channel is open. Po could also be determined in the presence of 100 microM GABA by an independent method in which different assumptions are made in the interpretation of the experimental results, the single-channel current-recording technique. The value of Po obtained (0.56) was in good agreement with the Po value (0.61) calculated for receptor form A alpha from chemical kinetic measurements at 100 microM GABA.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of sodium lithium and proton concentrations on the electrophysiological properties of the four mouse GABA transporters expressed in Xenopus oocytes

Mouse GABA transporters belong to the family of Na(+) and Cl(-) dependent neurotransmitter transporter. GABA transport, by these family members, was shown to be electrogenic and driven by sodium ions. It was demonstrated that, as in several other transporters, sodium binding and release by GAT1, GAT3 and BGT-1, the canine homolog of GAT2, resulted in the appearance of presteady-state currents. In this work we show that each of the four GABA transporters exhibit unique presteady-state currents when expressed in Xenopus oocytes. The properties of the presteady-state currents correspond to the transporters affinities to Na(+). At 100 mM GAT1 exhibited symmetric presteady-state currents at all imposed potentials, whereas GAT2 exhibited asymmetric presteady-state currents exclusively at negative imposed potentials, GAT3 or GAT4 exhibited presteady-state currents predominantly at positive imposed potentials. GABA uptake by GAT2 and GAT4 was much more sensitive to external pH than GAT1 and GAT3. Reducing the external Na(+) concentration rendered the GABA uptake activity by GAT1 and GAT3 to be sensitive to pH. Lowering the external pH reduced the Na(+) affinity of GAT1. Substitution of the external Na(+) to Li(+) resulted in the appearance of leak currents exclusively at negative potentials in Xenopus oocyte expressing GAT1 and GAT3. Low Na(+) concentrations inhibited the leak currents of GAT1 but Na(+) had little effect on the leak currents of GAT3. Washing of occluded Na(+) in GAT1 enhanced the leak currents. Similarly addition of GABA in the presence of 80 mM Li(+), that presumably accelerated the release of the bound Na(+), also induced the leak currents. Conversely, addition of GABA to GAT3 expressing oocytes, in the presence of 80 mM Li(+), inhibited the leak currents.     

Unified modeling of the mammalian and fish proton-dependent oligopeptide transporter PepT1

The partial and complete cycle of the intestinal pH-dependent oligopeptide transporter PepT1 from three species (seabass, zebrafish and rabbit) were studied using an electrophysiological approach and a biophysical analysis, in order to identify similarities and differences. On the whole the presteady state currents of the fish transporters were similar to each other, while presenting some quantitative differences with respect to rabbit PepT1: this last form showed slower decaying currents and the charge vs. potential (Q/V) and time constant vs. potential (τ/V) curves shifted to more positive potentials. All isoforms were similarly affected by external pH, showing acidity-induced slowing of the transients and positive shifts in the Q/V and τ/V curves. Analysis of the pH dependence of the unidirectional rates of the intramembrane charge movement suggested that external protonation of the protein limits the speed of this process in both directions. The complete cycle of the transporter was studied using the neutral dipeptide Gly-Gln. Michaelis-Menten analysis confirmed that in all species the apparent affinity for the substrate is significantly increased by acidity, while the maximal transport current is not strongly affected. Simulations using a kinetic model incorporating the new findings show good agreement with experimental data for all three species both with respect to the presteady-state and transport currents.     

Two novel residues in M2 of the gamma-aminobutyric acid type A receptor affecting gating by GABA and picrotoxin affinity

An amino acid residue was found in M2 of gamma-aminobutyric acid (GABA) type A receptors that has profound effects on the binding of picrotoxin to the receptor and therefore may form part of its binding pocket. In addition, it strongly affects channel gating. The residue is located N-terminally to residues suggested so far to be important for channel gating. Point mutated alpha1beta(3) receptors were expressed in Xenopus oocytes and analyzed using the electrophysiological techniques. Coexpression of the alpha(1) subunit with the mutated beta(3) subunit beta(3)L253F led to spontaneous picrotoxin-sensitive currents in the absence of GABA. Nanomolar concentrations of GABA further promoted channel opening. Upon washout of picrotoxin, a huge transient inward current was observed. The reversal potential of the inward current was indicative of a chloride ion selectivity. The amplitude of the inward current was strongly dependent on the picrotoxin concentration and on the duration of its application. There was more than a 100-fold decrease in picrotoxin affinity. A kinetic model is presented that mimics the gating behavior of the mutant receptor. The point mutation in the neighboring residue beta(3)A252V resulted in receptors that displayed an about 6-fold increased apparent affinity to GABA and an about 10-fold reduced sensitivity to picrotoxin.     

Electrogenic uptake of gamma-aminobutyric acid by a cloned transporter expressed in Xenopus oocytes.

GAT-1, a gamma-aminobutyric acid (GABA) transporter cloned from rat brain, was expressed in Xenopus oocytes. Voltage-clamp measurements showed concentration-dependent, inward currents in response to GABA (K0.5 4.7 microM). The transport current required extracellular sodium and chloride ions; the Hill coefficient for chloride was 0.7, and that for sodium was 1.7. Correlation of current and [3H]GABA uptake measurements indicate that flux of one positive charge occurs per molecule of GABA transported. Membrane hyperpolarization from -40 to -100 mV increased the transport current approximately 3-fold. The results indicate that the transport of one molecule of GABA involves the co-transport of two sodium ions and one chloride ion.     

Sucrose- and H+-Dependent Charge Movements Associated with the Gating of Sucrose Transporter ZmSUT1

Background

In contrast to man the majority of higher plants use sucrose as mobile carbohydrate. Accordingly proton-driven sucrose transporters are crucial for cell-to-cell and long-distance distribution within the plant body. Generally very negative plant membrane potentials and the ability to accumulate sucrose quantities of more than 1 M document that plants must have evolved transporters with unique structural and functional features.

Methodology/Principal Findings

To unravel the functional properties of one specific high capacity plasma membrane sucrose transporter in detail, we expressed the sucrose/H⁺ co-transporter from maize ZmSUT1 in Xenopus oocytes. Application of sucrose in an acidic pH environment elicited inward proton currents. Interestingly the sucrose-dependent H⁺ transport was associated with a decrease in membrane capacitance (C_m). In addition to sucrose C_m was modulated by the membrane potential and external protons. In order to explore the molecular mechanism underlying these C_m changes, presteady-state currents (I_pre) of ZmSUT1 transport were analyzed. Decay of I_pre could be best fitted by double exponentials. When plotted against the voltage the charge Q, associated to I_pre, was dependent on sucrose and protons. The mathematical derivative of the charge Q versus voltage was well in line with the observed C_m changes. Based on these parameters a turnover rate of 500 molecules sucrose/s was calculated. In contrast to gating currents of voltage dependent-potassium channels the analysis of ZmSUT1-derived presteady-state currents in the absence of sucrose (I = Q/τ) was sufficient to predict ZmSUT1 transport-associated currents.

Conclusions

Taken together our results indicate that in the absence of sucrose, ‘trapped’ protons move back and forth between an outer and an inner site within the transmembrane domains of ZmSUT1. This movement of protons in the electric field of the membrane gives rise to the presteady-state currents and in turn to C_m changes. Upon application of external sucrose, protons can pass the membrane turning presteady-state into transport currents.     

Point mutation in the first transmembrane region of the beta 2 subunit of the gamma--aminobutyric acid type A receptor alters desensitization kinetics of gamma--aminobutyric acid- and anesthetic-induced channel gating

A conserved glycine residue in the first transmembrane (TM1) domain of the beta2 subunit has been identified to be involved with desensitization induced by gamma-aminobutyric acid (GABA) and anesthetics. Recombinant GABA(A) receptors expressed in Sf9 cells were recorded using semi-fast agonist application. Upon direct activation by GABA or anesthetics, the main effect of the TM1 point mutation on the beta2 subunit (G219F) was to slow the time constant (tau) of desensitization. At GABA concentrations eliciting maximum currents, the corresponding median tau values were 0.87 s (25-75% interval (0.76; 1.04 s)), 0.93 s (0.76; 1.23 s), and 1.36 s (1.17; 1.57 s) for alpha1beta2gamma2, alpha1(G223F)beta2gamma2, and alpha1beta2(G219F)gamma2, respectively. The tau value for the beta2-mutant receptor was significantly longer than alpha1beta2gamma2 (p < 0.01) and alpha1(G223F)beta2gamma2 (p < 0.05). For pentobarbital-induced currents (500 microm), the corresponding median tau values were 1.36 s (0.81; 1.41 s), 1.47 s (1.31; 2.38 s), and 2.82 s (2.21; 5.56 s) for alpha1beta2gamma2, alpha1(G223F)beta2gamma2, and alpha1beta2(G219F)gamma2, respectively. The tau value for the beta2-mutant receptor was significantly longer than that for alpha1beta2gamma2 (p < 0.01). The present findings suggest that this TM1 glycine residue is critical for the rate at which desensitization occurs and that both GABA and intravenous anesthetics implement an analogous pathway for generating desensitization.     

Temperature Dependence of Steady-State and Presteady-State Kinetics of a Type IIb Na+/Pi Cotransporter

The temperature dependence of the transport kinetics of flounder Na(+)-coupled inorganic phosphate (P(i)) cotransporters (NaPi-IIb) expressed in Xenopus oocytes was investigated using radiotracer and electrophysiological assays. (32)P(i) uptake was strongly temperature-dependent and decreased by approximately 80% at a temperature change from 25 degrees C to 5 degrees C. The corresponding activation energy (E (a)) was approximately 14 kcal mol(-1) for the cotransport mode. The temperature dependence of the cotransport and leak modes was determined from electrogenic responses to 1 mM P(i) and phosphonoformic acid (PFA), respectively, under voltage clamp. The magnitude of the P(i)- and PFA-induced changes in holding current decreased with temperature. E (a) at -100 mV for the cotransport and leak modes was approximately 16 kcal mol(-1) and approximately 11 kcal mol(-1), respectively, which suggested that the leak is mediated by a carrier, rather than a channel, mechanism. Moreover, E (a) for cotransport was voltage-independent, suggesting that a major conformational change in the transport cycle is electroneutral. To identify partial reactions that confer temperature dependence, we acquired presteady-state currents at different temperatures with 0 mM P(i) over a range of external Na(+). The relaxation time constants increased, and the peak time constant shifted toward more positive potentials with decreasing temperature. Likewise, there was a depolarizing shift of the charge distribution, whereas the total available charge and apparent valency predicted from single Boltzmann fits were temperature-independent. These effects were explained by an increased temperature sensitivity of the Na(+)-debinding rate compared with the other voltage-dependent rate constants.     

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.     

Engineered Zn(2+) switches in the gamma-aminobutyric acid (GABA) transporter-1. Differential effects on GABA uptake and currents

Two high affinity Zn(2+) binding sites were engineered in the otherwise Zn(2+)-insensitive rat gamma-aminobutyric acid (GABA) transporter-1 (rGAT-1) based on structural information derived from Zn(2+) binding sites engineered previously in the homologous dopamine transporter. Introduction of a histidine (T349H) at the extracellular end of transmembrane segment (TM) 7 together with a histidine (E370H) or a cysteine (Q374C) at the extracellular end of TM 8 resulted in potent inhibition of [3H]GABA uptake by Zn(2+) (IC(50) = 35 and 44 microM, respectively). Upon expression in Xenopus laevis oocytes it was similarly observed that Zn(2+) was a potent inhibitor of the GABA-induced current (IC(50) = 21 microM for T349H/E370H and 51 microM for T349H/Q374C), albeit maximum inhibition was only approximately 40% in T349H/E370H versus approximately 90% in T349H/Q374C. In the wild type, Zn(2+) did not affect the Na(+)-dependent transient currents elicited by voltage jumps and thought to reflect capacitive charge movements associated with Na(+) binding. However, in both mutants Zn(2+) caused a reduction of the inward transient currents upon jumping to hyperpolarized potentials as reflected in rightward-shifted Q/V relationships. This suggests that Zn(2+) is inhibiting transporter function by stabilizing the outward-facing Na(+)-bound state. Translocation of lithium by the transporter does not require GABA binding and analysis of this uncoupled Li(+) conductance revealed a potent inhibition by Zn(2+) in T349H/E370H, whereas surprisingly the T349H/Q374C leak was unaffected. This differential effect supports that the leak conductance represents a unique operational mode of the transporter involving conformational changes different from those of the substrate translocation process. Altogether our results support both an evolutionary conserved structural organization of the TM 7/8 domain and a key role of this domain in GABA-dependent and -independent conformational changes of the transporter.     

Activation and deactivation rates of recombinant GABA(A) receptor channels are dependent on alpha-subunit isoform.

The role of subunit composition in determining intrinsic maximum activation and deactivation kinetics of GABA(A) receptor channels is unknown. We used rapid ligand application (100-micros solution exchange) to examine the effects of alpha-subunit composition on GABA-evoked activation and deactivation rates. HEK 293 cells were transfected with human cDNAs encoding alpha1beta1gamma2- or alpha2beta1gamma2-subunits. Channel kinetics were similar across different transfections of the same subunits and reproducible across several GABA applications in the same patch. Current rise to peak was at least twice as fast for alpha2beta1gamma2 receptors than for alpha1beta1gamma2 receptors (reflected in 10-90% rise times of 0.5 versus 1.0 ms, respectively), and deactivation was six to seven times slower (long time constants of 208 ms versus 31 ms) after saturating GABA applications. Thus alpha-subunit composition determined activation and deactivation kinetics of GABA(A) receptor channels and is therefore likely to influence the kinetics and efficacy of inhibitory postsynaptic currents.     

缓激肽抑制大鼠DRG分离神经元的GABA激活电流

本文应用全细胞膜片箝技术在新鲜分离的大鼠背根神经节（ＤＲＧ）神经元上研究缓激肽（ＢＫ）对γ－氨基丁酸（ＧＡＢＡ）反应的调制作用。结果发现：在３４个对ＧＡＢＡ反应的细胞中有３１个细胞对ＢＫ敏感。在对ＢＫ敏感并引起内向电流的２７个细胞中预加ＢＫ,对ＧＡＢＡ－激活电流具有明显的抑制作用,如１０＾－６ｍｏｌ／Ｌ的ＢＫ可抑制ＧＡＢＡ（１０＾－４ｍｏｌ／Ｌ）激活电流３０％。ＢＫ可将ＧＡＢＡ量效曲线明显下移,并     

Position 170 of Rabbit Na+/glucose cotransporter (rSGLT1) lies in the Na+ pathway; modulation of polarity/charge at this site regulates charge transfer and carrier turnover

Positions 163, 166, and 173, within the putative external loop joining transmembrane segments IV and V of rabbit Na(+)/glucose cotransporter, form part of its Na(+) interaction and voltage-sensing domain. Since a Q170C mutation within this region exhibits anomalous behavior, its function was further investigated. We used Xenopus oocytes coinjected with mouse T-antigen to enhance Q170C expression, and the two-microelectrode voltage-clamp technique. For Q170C, alpha-methyl D-glucopyranoside, phloridzin, and Na(+) affinity values are equivalent to those of wild-type; but turnover is reduced approximately 50%. Decreased [Na(+)] reduces Q170C, but not wild-type, charge transfer. Q170C presteady-state currents exhibit three time constants, tau, identical to wild-type. MTSES decreases maximal alpha-methyl D-glucopyranoside-induced currents by approximately 64% and Na(+) leak by approximately 55%; phloridzin and Na(+) affinity are unchanged. MTSES also reduces charge transfer (dithiothreitol-reversible) and Q170C turnover by approximately 60-70%. MTSEA and MTSET protect against MTSES, but neither affect Q170C function. MTSES has no obvious effect on the tau-values. Q170A behaves the same as Q170C. The mutation Q170E affects voltage sensitivity and reduces turnover, but also appears to influence Na(+) interaction. We conclude that 1), glutamine 170 lies in the Na(+) pathway in rabbit Na(+)/glucose cotransporter and 2), altered polarity and charge at position 170 affect a cotransporter conformational state and transition, which is rate-limiting, but probably not associated with empty carrier reorientation.