Conformational basis for the Li+-induced leak current in the rat -aminobutyric acid (GABA) transporter-1 (original) (raw)
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Ion Binding and Permeation at the GABA Transporter GAT1
This study addresses the binding of ions and the permeation of substrates during function of the GABA transporter GAT1. GAT1 was expressed in Xenopus oocytes and studied electrophysiologically as well as with [ 3 H]GABA flux; GAT1 was also expressed in mammalian cells and studied with [ 3 H]GABA and [ 3 H]tiagabine binding. Voltage jumps, Na ϩ and Cl Ϫ concentration jumps, and exposure to high-affinity blockers (NO-05-711 and SKF-100330A) all produce capacitive charge movements. Occlusive interactions among these three types of perturbations show that they all measure the same population of charges. The concentration dependences of the charge movements reveal (1) that two Na ϩ ions interact with the transporter even in the absence of GABA, and (2) that Cl Ϫ facilitates the binding of Na ϩ . Comparison between the charge movements and the transport-associated current shows that this initial Na ϩ -transporter interaction limits the overall transport rate when [GABA] is saturating. However, two classes of manipulation-treatment with high-affinity uptake blockers and the W68L mutation-"lock" Na ϩ onto the transporter by slowing or preventing the subsequent events that release the substrates to the intracellular medium. The Na ϩ substitutes Li ϩ and Cs ϩ do not support charge movements, but they can permeate the transporter in an uncoupled manner. Our results (1) support the hypothesis that efficient removal of synaptic transmitter by the GABA transporter GAT1 depends on the previous binding of Na ϩ and Cl Ϫ , and (2) indicate the important role of the conserved putative transmembrane domain 1 in interactions with the permeant substrates.
Novel Properties of a Mouse γ-Aminobutyric Acid Transporter (GAT4
Journal of Membrane Biology, 2005
We expressed the mouse γ-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 (Q 10) of 4.3. The GAT4 turnover rate (1.5 s−1; −50 mV, 21°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 ( $ I_{{\rm leak}}^{{\rm Cl}} $ ) was observed when Na+ was replaced with choline or tetraethylammonium. The reversal potential of ( IrmleakrmClI_{{\rm leak}}^{{\rm Cl}} IrmleakrmCl ) followed the Cl− Nernst potential. (ii) A Li+ leak current ( IrmleakrmLiI_{{\rm leak}}^{{\rm{Li}}} IrmleakrmLi ) was observed when Na+ was replaced with Li+. Both leak currents were inhibited by Na+, and both were temperature-independent (Q 10 ≈ 1). The two leak modes appeared not to coexist, as Li+ inhibited ( IrmleakrmClI_{{\rm leak}}^{{\rm Cl}} IrmleakrmCl ). The results suggest the existence of cation- and anion-selective channel-like pathways in GAT4. Flufenamic acid inhibited GAT4 Na+/C1−/GABA cotransport, IrmleakrmLiI_{{\rm leak}}^{{\rm{Li}}}IrmleakrmLi , and IrmleakrmClI_{{\rm leak}}^{{\rm Cl}}IrmleakrmCl , (K i ≈ 30 μM), and the voltage-induced presteady-state charge movements (K i ≈ 440 μM). Flufenamic acid exhibited little or no selectivity for GAT1, GAT2, or GAT3. Sodium and GABA concentration jumps 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.
GABA reverse transport by the neuronal cotransporter GAT1: influence of internal chloride depletion
AJP: Cell Physiology, 2011
The role of intracellular ions on the reverse GABA transport by the neuronal transporter GAT1 was studied using voltage-clamp and [3H]GABA efflux determinations in Xenopus oocytes transfected with heterologous mRNA. Reverse transport was induced by intracellular GABA injections and measured in terms of the net outward current generated by the transporter. Changes in various intracellular ionic conditions affected the reverse current: higher concentrations of Na+ enhanced the ratio of outward over inward transport current, while a considerable decrease of the outward current and a parallel reduction of the transporter-mediated GABA efflux were observed after treatments causing a diminution of the intracellular Cl− concentration. Particularly interesting was the impairment of the reverse transport observed after depletion of internal Cl− generated by the activity of a coexpressed K+-Cl− exporter KCC2. This finding suggests that reverse GABA transport may be physiologically regulated d...
Structure and Gating Dynamics of Na+/Cl– Coupled Neurotransmitter Transporters
Frontiers in Molecular Biosciences
Neurotransmitters released at the neural synapse through vesicle exocytosis are spatiotemporally controlled by the action of neurotransmitter transporters. Integral membrane proteins of the solute carrier 6 (SLC6) family are involved in the sodium and chloride coupled uptake of biogenic amine neurotransmitters including dopamine, serotonin, noradrenaline and inhibitory neurotransmitters including glycine and γ-amino butyric acid. This ion-coupled symport works through a well-orchestrated gating of substrate through alternating-access, which is mediated through movements of helices that resemble a rocking-bundle. A large array of commercially prescribed drugs and psychostimulants selectively target neurotransmitter transporters thereby modulating their levels in the synaptic space. Drug-induced changes in the synaptic neurotransmitter levels can be used to treat depression or neuropathic pain whereas in some instances prolonged usage can lead to habituation. Earlier structural studies of bacterial neurotransmitter transporter homolog LeuT and recent structure elucidation of the Drosophila dopamine transporter (dDAT) and human serotonin transporter (hSERT) have yielded a wealth of information in understanding the transport and inhibition mechanism of neurotransmitter transporters. Computational studies based on the structures of dDAT and hSERT have shed light on the dynamics of varied components of these molecular gates in affecting the uphill transport of neurotransmitters. This review seeks to address structural dynamics of neurotransmitter transporters at the extracellular and intracellular gates and the effect of inhibitors on the ligand-binding pocket. We also delve into the effect of additional factors including lipids and cytosolic domains that influence the translocation of neurotransmitters across the membrane.
Glutamate101 is critical for the function of the sodium and chloride-coupled GABA transporter GAT1
Febs Letters, 1995
We have investigated the possible role of selected negatively-charged amino acids of the sodium and chloride-coupled GABA transporter GAT-I on sodium binding. These residues located adjacent to putative transmembrane domains and which are conserved throughout the large superfamily of neurotransmitter transporters were changed by site-directed mutagenesis. The functional consequences were that one of the residues, glutamat¢~ 101, was critical for transport. Its replacement by aspartate left only 1% of the activity, and no activity could be detected when it was replaced by other residues. Expression levels and targeting to the plasma membrane of the mutant transporters appeared normal. Transient sodium currents were not observed in the mutants, and increased sodium concentrations did not affect the percentage of wild type transport of the E101D mutant. It is concluded that residue glutamate-101 is critical for one or more of the conformational changes of GAT-1 during its transport cycle.
Journal of Biological Chemistry, 2002
Na ؉ /Cl ؊-dependent neurotransmitter transporters form constitutive oligomers, the significance of which is not known. In soluble proteins, leucine heptad repeats drive dimerization; the rat ␥-aminobutyric acid transporter GAT-1 (rGAT) contains a motif reminiscent of a leucine heptad repeat in the second transmembrane helix (TM2). We substituted leucine residues in TM2 of rGAT by alanine and tested the ability of the resulting mutants to form oligomers by three methods of Fö rster resonance energy transfer (FRET) microscopy. Replacement of one leucine (L97A) resulted in considerable loss of energy transfer, replacing two or more ablated it completely. Furthermore, intracellular trapping increased with the number of leucine substitutions. Only rGAT-L97A reached the cell surface to a sufficient amount such that, in intact cells, it was indistinguishable from wild type rGAT with respect to substrate transport, binding of inhibitors, and regulation by protein kinase C. However, in membrane vesicles prepared from transfected cells, all mutants were still functional. In addition, FRET was readily detected during maturation of wild type rGAT, when the bulk of the protein resided in the endoplasmic reticulum. Hence, our findings strongly argue for a role of oligomer formation during biosynthesis and subsequent delivery of the multimer from the endoplasmic reticulum to the plasma membrane. Na ϩ /Cl Ϫ-dependent neurotransmitter transporters (e.g. the transporters for dopamine, serotonin, or GABA) 1 retrieve neurotransmitters from the synaptic cleft into the presynaptic specialization (1). The medical relevance of these proteins is obvious; for instance, it has long been known that antidepressant drugs block the transporter for norepinephrine and serotonin (2). Likewise, tiagabine, an inhibitor of GABA transport, is used as an anticonvulsant in the treatment of epileptic seizures (3). Transporters support bidirectional flux of sub
Pre-steady-state and reverse transport currents in the GABA transporter GAT1
American Journal of Physiology-Cell Physiology, 2011
The role of internal substrates in the biophysical properties of the GABA transporter GAT1 has been investigated electrophysiologically in Xenopus oocytes heterologously expressing the cotransporter. Increments in Cl− and/or Na+ concentrations caused by intracellular injections did not produce significant effects on the pre-steady-state currents, while a positive shift of the charge-voltage ( Q–V) and decay time constant (τ)-voltage (τ- V) curves, together with a slowing of τ at positive potentials, was observed following treatments producing cytosolic Cl− depletion. Activation of the reverse transport mode by injections of GABA caused a reduction in the displaced charge. In the absence of external Cl−, a stronger reduction in the displaced charge, together with a significant increase in reverse transport current, was observed. Therefore, complementarity between pre-steady-state and transport currents, observed in the forward mode, is preserved in the reverse mode. All these finding...
The Family of Na+/Cl− Neurotransmitter Transporters
Journal of Neurochemistry, 2002
The termination of neurotransmission is achieved by rapid uptake of the released neurotransmitter by specific high-affinity neurotransmitter transporters. Most of these transporters are encoded by a family of genes (Na~ICI transporters) having a similar membrane topography of 12 transmembrane helices. An evolutionary tree revealed fivedistinct subfamilies: y-aminobutyric acid transporters, monoamine transporters, amino acid transporters, "orphan" transporters, and the recently discovered bacterial transporters. The bacterial transporters that belong to this family may help to develop heterologous expression systems with the aim of solving the threedimensional structure of these membrane proteins. Some of the neurotransmitter transporters have been implicated as important sites for drug action. Monoamine transporters, for example, are targeted by major classes of antidepressants, psychostimulants, and antihypertensive drugs. Localization of individual transporters in specific cells and brain areas is pertinent to understanding their contribution to neurotransmission and their potential as targets for drugs. The most important questions in the field include resolving the mechanism of neurotransmitter transport, the structure of the transporters, and the interaction of each transporter in complex neurological activities.
Turnover Rate of the γ-Aminobutyric Acid Transporter GAT1
Journal of Membrane Biology, 2007
We combined electrophysiological and freeze-fracture methods to estimate the unitary turnover rate of the γ-aminobutyric acid (GABA) transporter GAT1. Human GAT1 was expressed in Xenopus laevis oocytes, and individual cells were used to measure and correlate the macroscopic rate of GABA transport and the total number of transporters in the plasma membrane. The twoelectrode voltage-clamp method was used to measure the transporter-mediated macroscopic current evoked by GABA ( ), macroscopic charge movements (Q NaCl ) evoked by voltage pulses and whole-cell capacitance. The same cells were then examined by freeze-fracture and electron microscopy in order to estimate the total number of GAT1 copies in the plasma membrane. GAT1 expression in the plasma membrane led to the appearance of a distinct population of 9-nm freeze-fracture particles which represented GAT1 dimers. There was a direct correlation between Q NaCl and the total number of transporters in the plasma membrane. This relationship yielded an apparent valence of 8 ± 1 elementary charges per GAT1 particle. Assuming that the monomer is the functional unit, we obtained 4 ± 1 elementary charges per GAT1 monomer. This information and the relationship between and Q NaCl were used to estimate a GAT1 unitary turnover rate of 15 ± 2 s −1 (21°C, −50 mV). The temperature and voltage dependence of GAT1 were used to estimate the physiological turnover rate to be 79-93 s −1 (37°C, −50 to −90 mV).
The Journal of Physiology
The effects of pH on the properties of the neurotransmitter cotransporters may be useful in defining regions with functional relevance. In fact, variations in extracellular pH induce diverse consequences on the functional properties of several cotransporters: in rat 5HT transporter (rSERT) acidic pH enhances the current in the absence of organic substrate (uncoupled current) and the transport-associated current, while inhibiting the pre-steady-state charge movements upon voltage jumps (Cao et al. 1997). Similar effects can also be observed in GABA (GAT1), glucose (SLGT1) and dopamine (DAT) transporters, but not in a glycine (GLYT1) transporter (Hirayama et al. 1994; Cao et al. 1997; Sonders et al. 1997), while little or no potentiation of the substrate-induced current was seen in GAT1 and SGLT. Finally, in GLYT1 acidic pH actually reduced the glycine-induced current (Cao et al. 1997). Of particular interest are the results from the human 5HT transporter (hSERT) which, although 92 % ...