Hilgemann DW and Lu CC (1999), GAT1 (GABA:Na+:Cl-) cotransport function. Datab...

XB-ART-12414

J Gen Physiol 1999 Sep 01;1143:459-75.

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GAT1 (GABA:Na+:Cl-) cotransport function. Database reconstruction with an alternating access model.

Hilgemann DW , Lu CC .

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We have developed an alternating access transport model that accounts well for GAT1 (GABA:Na+:Cl-) cotransport function in Xenopus oocyte membranes. To do so, many alternative models were fitted to a database on GAT1 function, and discrepancies were analyzed. The model assumes that GAT1 exists predominantly in two states, Ein and E(out). In the Ein state, one chloride and two sodium ions can bind sequentially from the cytoplasmic side. In the Eout state, one sodium ion is occluded within the transporter, and one chloride, one sodium, and one gamma-aminobutyric acid (GABA) molecule can bind from the extracellular side. When Ein sites are empty, a transition to the Eout state opens binding sites to the outside and occludes one extracellular sodium ion. This conformational change is the major electrogenic GAT1 reaction, and it rate-limits forward transport (i.e., GABA uptake) at 0 mV. From the Eout state, one GABA can be translocated with one sodium ion to the cytoplasmic side, thereby forming the *Ein state. Thereafter, an extracellular chloride ion can be translocated and the occluded sodium ion released to the cytoplasm, which returns the transporter to the Ein state. GABA-GABA exchange can occur in the absence of extracellular chloride, but a chloride ion must be transported to complete a forward transport cycle. In the reverse transport cycle, one cytoplasmic chloride ion binds first to the Ein state, followed by two sodium ions. One chloride ion and one sodium ion are occluded together, and thereafter the second sodium ion and GABA are occluded and translocated. The weak voltage dependence of these reactions determines the slopes of outward current-voltage relations. Experimental results that are simulated accurately include (a) all current-voltage relations, (b) all substrate dependencies described to date, (c) cis-cis and cis-trans substrate interactions, (d) charge movements in the absence of transport current, (e) dependencies of charge movement kinetics on substrate concentrations, (f) pre-steady state current transients in the presence of substrates, (g) substrate-induced capacitance changes, (h) GABA-GABA exchange, and (i) the existence of inward transport current and GABA-GABA exchange in the nominal absence of extracellular chloride.

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Species referenced: Xenopus laevis
Genes referenced: slc6a1 tbx2

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	Figure 1. GAT1 cotransport model. The transporter cartoons are all oriented with the extracellular side facing upward. Substrate dependencies of each reaction are indicated in a box. (â) A reaction can occur only when the given substrate is not bound; (+) a reaction can occur only when the given substrate is bound. The model assumes the existence of two stable GAT1 states (Ein and Eout) and two transitional GAT1 states (Ein and Eout). In the Ein state, one Clâ (Cl) and two Na+ (N) can bind sequentially from the cytoplasmic side. When Ein binding sites are empty, a Na+ binding site can open to the extracellular side to form the Eout state. One extracellular Na+ ion can be bound in the Eout state and occluded into the transporter, thereby forming the Eout state. The Eout state can bind sequentially one Na+, one Clâ, and one GABA (G). When the Eout binding sites are fully occupied, a transition to the *Ein state can occur in which GABA and one Na+ can dissociate to the cytoplasmic side. Further opening of binding sites returns transporters to the Ein state. Conformational changes of the transitional states (1b, 2b, 3b, and 4b) are assumed to take place at infinite rates. See text for further details.
	Figure 2. Hypothetical mechanisms of GAT1 electrogenicity. (A) Binding site opening reaction of an empty transporter. It is assumed that opening of the binding site changes the membrane potential profile across the transporter, such that the membrane electrical field moves across the charged binding site. The charge of the binding site, while open, remains outside the electrical field. Therefore, the binding site opening rate (Î±) will be more strongly voltage dependent than the closure rate (Î²). (B) Hypothetical mechanism of electrogenicity during Na+o occlusion by the GAT1 transporter. In the E1 state, binding sites are closed to the extracellular side. When they open, one Na+ can bind in the *E state, and the transporter binding sites close to the E2 state with an occluded Na+. In the opening reaction, the membrane field moves partially across the negatively charged binding site, and in the closing reaction, the membrane field moves in the opposite direction across the bound Na+. See text for further explanations.
	Figure 3. Currentâvoltage relations of the fully activated GAT cotransport currents. Here, and in subsequent figures, the steady state transport rate is plotted on the y axis. The data points are scaled membrane current values from experiments described previously (Lu and Hilgemann 1999a,Lu and Hilgemann 1999b). Solid curves are model predictions. The reverse (outward) currentâvoltage relation (âª) is with 120 mM cytoplasmic NaCl, 20 mM cytoplasmic GABA, and 20 mM extracellular Clâ. The forward (inward) currentâvoltage relation (â¢) is with 120 mM extracellular NaCl, 0.2 mM extracellular GABA, and no cytoplasmic substrates. The mixed currentâvoltage relation (dashed line) is with 120 mM NaCl on both membrane sides, 2 mM cytoplasmic GABA, and 0.2 mM extracellular GABA.
	Figure 5. Measured and predicted characteristics of GABAo-stimulated GABA efflux. (A) GABA efflux from outside-out synaptic vesicles (Kanner et al. 1983). Vesicles were loaded with GAT1 substrates by forward transport. GABA efflux was monitored as loss of labeled GABA from the vesicles after diluting them into solutions containing Na+. Extracellular GABA (20 Î¼M) stimulates GABA efflux by about twofold in the presence of 100 mM Clâo, as well as in its nominal absence (<5 mM). Stimulation of GABA efflux by GABAo required the presence of extracellular Na+. (B) Model predictions: extracellular GABA dependence of GABA efflux in the presence of 40 mM cytoplasmic NaCl, 20 mM cytoplasmic GABA, and 100 mM extracellular Na+ (0 mV). GABA efflux is increased about fourfold by GABAo in the presence of 100 mM Clâo, and by about twofold in the presence of 5 mM Clâo.
	Figure 4. (A) GABA dependence of the outward transport current at 0 mV in the presence of 120 mM cytoplasmic Clâ, with and without 120 mM extracellular Na+. See text for details. (B) GABA dependence of the outward transport current at 0 mV in the presence of 120 mM extracellular NaCl, with 120 and 3 mM cytoplasmic Clâ.
	Figure 7. Extracellular substrate dependence of inward GAT1 current. (A) Extracellular Na+ dependence of current at â140 and â60 mV. (B) Extracellular Clâ dependence of current at â140 and â40 mV. (C) Extracellular GABA dependence of current at â140 and â40 mV. The experimental results are whole-oocyte currents (Mager et al. 1993). See text for further details.
	Figure 6. Currentâvoltage relations of outward GAT1 current. Fully activated outward current (120 mM NaCl and 20 mM GABA) is plotted in each case (â¢). (A) Inhibition of outward current by 100 mM extracellular Na+. (B) Inhibition of outward current by reducing the cytoplasmic Na+ concentration from 120 to 20 mM. (C) Inhibition of outward current by reducing cytoplasmic GABA from 20 to 0.5 mM. (D) Inhibition of outward current by reducing cytoplasmic Clâ from 120 to 15 mM. See text for further details.
	Figure 9. Currentâvoltage relations of the inward GAT1 current. (A) Inward GAT1 current in a giant patch with 0, 30, and 120 mM cytoplasmic Clâ. Results in BâD are whole-oocyte experiments (Mager et al. 1993), and the corresponding simulations assume 25 mM cytoplasmic Clâ and 12 mM cytoplasmic Na+. (B) Inward current with 96 and 29 mM extracellular Na+. (C) Inward current with 96 mM and nominally zero extracellular Clâ; the simulation is with 1 Î¼M Clâ. (D) Inward current with 100 and 10 Î¼M extracellular GABA. See text for further details.
	Figure 8. Cytoplasmic substrate dependence of inward GAT1 current. (A) Inhibition of inward GAT1 current by cytoplasmic Clâ. (B) Weak inhibition of inward GAT1 current by cytoplasmic Na+. (C) Lack of effect of GABAi on inward GAT1 current. (D) Weak inhibition of inward GAT1 current by GABAi in the presence of 120 mM cytoplasmic Na+ and no Clâ. See text for further details.
	Figure 11. Rateâ (K/V) and chargeâvoltage (Q/V) relations. (A and B) Results from an excised oocyte patch with 40 mM extracellular NaCl, simulated with 80 mM NaCl. (C) Effect of 120 mM cytoplasmic Clâ on Q/V relation of a patch with 120 mM extracellular NaCl. The simulated charge is calibrated as elementary charges (e) moved through membrane field per single transporter. See text for details.
	Figure 10. Simulated and measured GAT1 charge movements. The simulation is calibrated as elementary charges (e) moved through membrane field per single transporter. The experimental results are with 40 mM extracellular NaCl; the simulation is with 80 mM extracellular NaCl. As indicated by the cartoons, binding sites are expected to be open to the cytoplasmic side at positive potentials and to the extracellular side at negative potentials. See text for more explanations.
	Figure 16. Simulation of GAT1-mediated membrane capacitance changes with application of cytoplasmic Clâ. Measured and predicted capacitance changes on application of cytoplasmic Clâ in the absence of cosubstrates (â¢), in the presence of 120 mM cytoplasmic Na+ (â), and in the presence of 120 mM cytoplasmic Na+ and 20 mM GABA (âª).
	Figure 12. Temperature dependence of inward and outward GAT1 currents at 0 mV in excised oocyte patches. Inward current was recorded with 120 mM NaCl and 0.2 mM GABA on the extracellular side. Outward current was recorded with 120 mM NaCl and 20 mM GABA on the cytoplasmic side.
	Figure 13. Voltage dependence of charge-movement rates in whole-oocyte voltage clamp with 96, 58, 12, and 3 mM extracellular Na+. The results are from Mager et al. 1996. See text for details.
	Figure 14. Voltage and extracellular Na+ dependence of GAT1 charge movements in whole-oocyte voltage clamp. The simulations are calibrated as elementary charges (e) moved through membrane field per single transporter. The results are from Mager et al. 1996. (A) Voltage dependence of charge moved at 96 (â¦), 77 (â¡), 48 (âª), 24 (â), and 12 (â¢) mM extracellular Na+. (B) Na+i dependence of charge moved at â80 mV by application of 96 mM extracellular Na+.
	Figure 15. Predicted GAT1 preâsteady state current transients. The voltage protocol is given below the figure. These model results are comparable to experimental results described previously (Lu and Hilgemann 1999b). (A) Outward current condition without extracellular Na+: 120 mM cytoplasmic NaCl, 20 mM cytoplasmic GABA, and 20 mM extracellular Clâ. (B) Outward current condition as in A with additional 120 mM extracellular Na+. (C) Inward current condition: 120 mM extracellular NaCl, 0.2 mM extracellular GABA, and no cytoplasmic substrates. (D) Charge movement condition: 120 mM extracellular NaCl and no other GAT1 substrates. (E) Current reversal condition: 120 mM NaCl and 2 mM GABA on the extracellular side; 120 mM Na+, 6 mM Clâ, and 2 mM GABA on the cytoplasmic side. See text for complete details.

References [+] :

Cammack, A GABA transporter operates asymmetrically and with variable stoichiometry. 1994, Pubmed