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

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.     

Amino Acid Neurotransmitters in the CNS: Properties of Diaminobutyric Acid Transport

Uptake of L-2,4-diaminobutyric acid (DABA), a positively charged analogue of gamma-aminobutyric acid (GABA), by a synaptosomal fraction isolated from rat brain occurred with a Km of 54 +/- 12 microM and a Vmax of 1.3 +/- 0.2 nmol/min/mg protein. The transport of DABA was inhibited competitively by GABA whereas that of GABA was affected in the same manner by addition of DABA. The maximal accumulation of DABA ([DABA]i/[DABA]c) was observed to increase as the second power of the transmembrane electrical potential ([K+]i/[K+]e) and the first power of the sodium ion concentration gradient. These findings indicate that DABA is transported on the GABA carrier with a net charge of +2, where one charge is provided by the cotransported Na+ and the second is contributed by the amino acid itself. Since uptake of GABA, an electroneutral molecule, is accompanied by transfer of two sodium ions, the results obtained with DABA suggest that one of the sodium binding sites on the GABA transporter is in proximity to the amino acid binding site.     

The ionic mechanism associated with the action of putative transmitters on identified neurons of the snail, Helix aspersa

Intracellular recordings were made from identified neurons in the suboesophageal ganglionic mass of the snail, Helix aspersa. The ionic mechanisms associated with acetylcholine excitation and inhibition, dopamine excitation and inhibition, gamma-aminobutyric acid (GABA) excitation and inhibition and serotonin excitation were investigated. Acetylcholine excitation was found to involve an initial increase in sodium conductance while acetylcholine inhibition was a pure chloride event which reversed at membrane potentials more negative than the chloride equilibrium potential. Dopamine excitation appeared to involve only an increase in sodium conductance while serotonin excitation involved an increase in conductance to both sodium and calcium ions. Dopamine inhibition was associated with an increase in potassium conductance but failed to reverse at membrane potentials more negative than the potassium equilibrium potential. GABA excitation involved conductance increases to both sodium and chloride ions while GABA inhibition was a pure chloride event. An attempt was made to estimate the degree of co-operativity of the putative transmitters with their receptors using log-log and Hill plots. The slopes of the line for the log-log plots for acetylcholine excitation and inhibition were 0.88 and 1.1, respectively, suggesting the interaction of one molecule of acetylcholine with the receptor. The slope of the log-log plot for dopamine inhibition was 0.46 while that for serotonin excitation was 0.75. The Hill plots for GABA excitation and inhibition were 1.64 and 1.42, respectively, suggesting that two molecules of GABA are required for receptor activation.     

beta-Amino acid transport across the renal brush-border membrane is coupled to both Na and Cl

Sodium-dependent beta-alanine uptake into dog renal brush-border membrane vesicles was studied. Kinetic analysis indicated a single transport system, highly specific for beta-amino acids, with Km = 35 microM at 100 mM NaCl. Sodium-dependent beta-alanine transport was markedly anion-dependent, being highest in the presence of chloride (Cl greater than Br greater than SCN greater than NO3 approximately I greater than F) and virtually nonexistent in the presence of gluconate and other nonphysiological chloride substitutes. In addition, it was observed that beta-alanine uptake could be driven against a concentration gradient by a chloride gradient. Similar results were found for sodium. Taken together, these observations provide strong evidence that beta-alanine transport across the renal brush-border membrane is coupled to both sodium and chloride. Studies of the dependence of beta-alanine flux on chloride and sodium concentrations indicated that one chloride ion and multiple sodium ions were involved in the beta-alanine transport event. beta-Alanine flux on chloride found to involve the net transfer of positive charge, consistent with these stoichiometric assignments. The hallucinogen harmaline inhibited beta-alanine uptake in a 1:1 fashion, presumably by acting at a single site on the transport molecule. The ability of harmaline to inhibit beta-alanine uptake was decreased when the chloride concentration was lowered but was unchanged when the sodium concentration was decreased. These results indicate that harmaline does not compete with sodium for a binding site on the carrier as has been suggested for other sodium-coupled transport systems, and that instead, chloride may be required for harmaline binding to the beta-alanine transporter.     

gamma-Aminobutyric acid transport in reconstituted preparations from rat brain: coupled sodium and chloride fluxes

Transport of gamma-aminobutyric acid (GABA) is electrogenic and completely depends on the presence of both sodium and chloride ions. These ions appear to be cotransported with gamma-aminobutyric acid through its transporter [reviewed in Kanner, B. I. (1983) Biochim. Biophys. Acta 726, 293-316]. Using proteoliposomes into which a partially purified gamma-aminobutyric acid transporter preparation was reconstituted, we have been able--for the first time--to provide direct evidence for sodium- and chloride-coupled gamma-aminobutyric acid transport. This has been done by measuring the fluxes of 22Na+, 36Cl-, and [3H]GABA. These fluxes have the following characteristics: There are components of the net fluxes of sodium and chloride that are gamma-aminobutyric acid dependent. The sodium flux is chloride dependent; i.e., when Cl- is replaced by inorganic phosphate or by SO4(2-), gamma-aminobutyric acid dependent sodium fluxes are abolished. The chloride flux is sodium dependent; i.e., when Na+ is replaced by Tris+ or by Li+, gamma-aminobutyric acid dependent chloride fluxes are abolished. Thus, the gamma-aminobutyric acid dependent sodium and chloride fluxes appear to be catalyzed by the transporter. Using these fluxes we have attempted to determine the stoichiometry of the process. We measured the initial rate of sodium-dependent gamma-aminobutyric acid fluxes and that of gamma-aminobutyric acid dependent sodium fluxes. This yields the stoichiometry between sodium and gamma-aminobutyric acid (2.58 +/- 0.99). Similarly, we measured the stoichiometry between chloride and gamma-aminobutyric acid, which is found to be 1.27 +/- 0.12.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of cations on taurine,hypotaurine, and GABA uptake in mouse brain slices

The effects of cations on taurine, hypotaurine and GABA uptake were studied in mouse brain slices under identical experimental conditions. The uptakes were all strictly sodium-dependent. The omission or excess of K⁺ inhibited similarly taurine, hypotaurine and GABA uptake. The effects of omission of Ca²⁺ or Mg²⁺ were less pronounced. In both normal-sodium and low-sodium media all uptakes were saturable, consisting of both low-and high-affinity transport components. TheK _m constants for both low-and high-affinity transport components of hypotaurine and GABA increased in low-sodium medium, suggesting that sodium ions are necessary for their attachment to possible carrier sites in plasma membranes. In the case of taurine, however, the translation rate rather than the affinity of carrier sites was affected in Na⁺-free media. More than two sodium ions may be involved in the transport of one hypotaurine and one GABA molecule, whereas the coupling ratio between sodium and taurine was at least three. In its cation dependence hypotaurine uptake thus resembled more GABA uptake than taurine uptake.     

Rapid substrate-induced charge movements of the GABA transporter GAT1

The GABA transporter GAT1 removes the neurotransmitter GABA from the synaptic cleft by coupling of GABA uptake to the co-transport of two sodium ions and one chloride ion. The aim of this work was to investigate the individual reaction steps of GAT1 after a GABA concentration jump. GAT1 was transiently expressed in HEK293 cells and its pre-steady-state kinetics were studied by combining the patch-clamp technique with the laser-pulse photolysis of caged GABA, which allowed us to generate GABA concentration jumps within <100 micros. Recordings of transport currents generated by GAT1, both in forward and exchange transport modes, showed multiple charge movements that can be separated along the time axis. The individual reactions associated with these charge movements differ from the well-characterized electrogenic "sodium-occlusion" reaction by GAT1. One of the observed electrogenic reactions is shown to be associated with the GABA-translocating half-cycle of the transporter, in contradiction to previous studies that showed no charge movements associated with these reactions. Interestingly, reactions of the GABA-bound transporter were not affected by the absence of extracellular chloride, suggesting that Cl- may not be co-translocated with GABA. Based on the results, a new alternating access sequential-binding model is proposed for GAT1's transport cycle that describes the results presented here and those by others.     

The action of Avermectin (MK 936) on identified central neurones from Helix and its interaction with acetylcholine and gamma-aminobutyric acid (GABA) responses

Intracellular recordings were made from identified neurones in the suboesophageal ganglionic mass of the snail, Helix aspersa. Avermectin, MK 936, 0.01-1.0 microM, induced an outward current in certain neurones. The size of this current varied from one cell type to another. This direct effect of Avermectin occurred irrespective of whether the neurones were sensitive to GABA or not and was generally irreversible. Avermectin, 0.1 microM, reduced the chloride mediated inhibitory GABA response and potentiated the largely sodium mediated excitatory GABA response. Avermectin, 0.1 microM, reduced the chloride mediated acetylcholine inhibitory response and potentiated the sodium mediated excitatory acetylcholine response. In neurones which showed a biphasic response to acetylcholine, Avermectin enhanced the excitatory and depressed the inhibitory component. It is concluded that Avermectin can interact with chloride ionophores to induce an outward current and can reduce chloride mediated responses associated with acetylcholine and GABA.     

The electrical potential profile of gallbladder epithelium.

In this study the relative ionic permeabilities of the cell membranes of Necturus gallbladder epithelium have been determined by means of simultaneous measurement of transmural and transmucosal membrane potential differences (PD) and by ionic substitution experiments with sodium, potassium and chloride ions. It is shown that the mucosal membrane is permeable to sodium and to potassium ions. The baso-lateral membrane PD is only sensitive to potassium ions. In both membranes chloride conductance is negligible or absent. The ratio of the resistances of the mucosal and baso-lateral membranes, RM/RS, increases upon reducing the sodium concentration in the mucosal solution. The same ratio decreases when sodium is replaced by potassium which implies a greater potassium than sodium conductance in the mucosal membrane. The relative permeability of the shunt for potassium, sodium and chloride ions is: PK/PNa/PCl=1.81:1.00:0.32. From the results obtained in this study a value for the PK/PNa ratio of the mucosal membrane could be evaluated. This ratio is 2.7. From the same data the magnitude of the electromotive forces generated across the cell membranes could be calculated. The EMF's are -15mV across the mucosal membrane and -81mV across the baso-lateral one. Due to the presence of the low resistance shunt the transmucosal membrane PD is -53.2mV (cell inside negative) and the transmural PD is +2.6mV (serosal side positive). The change in potential profile brought about by the low resistance shunt favors passive entry of Na ions into the cell across the mucosal membrane. Calculations show that this passive Na influx is maximally 64% of the net Na flux estimated from fluid transport measurements. The C-1 conductive of the baso-lateral membrane is too small to allow electrogenic coupling of C1 with Na transport across this membrane. Experiments with rabbit gallbladder epithelium indicate that the membrane properties in this tissue are qualitatively similar to those of Necturus gallbladder epithelium.     

Ionic events following GABA receptor activation in an identified insect motor neuron

The ionic events underlying gamma-aminobutyric acid (GABA) receptor activation on the cell body of a cockroach identified motor neuron were investigated by using current-clamp and voltage-clamp techniques. The reversal potential for GABA-induced hyperpolarization was -77.0 +/- 2.4 mV (mean +/- s.e.m.; n = 22). The reversal potential for GABA was highly sensitive to changes in external chloride, only weakly affected by changes in external potassium, and independent of changes in either sodium or calcium ion concentration. Intracellular ion-sensitive microelectrodes confirmed that an influx of chloride ions mediated the GABA response. Intracellular injection of acetate, citrate, sulphate, fluoride or ammonium caused no change in the reversal potential for GABA. Intracellular injection of chloride, bromide, chlorate, bromate, or methyl sulphate shifted the reversal potential for GABA to values more positive than resting membrane potential. Evidence for chloride accumulating and for extrusion mechanisms was examined by using putative inhibitors. However, internal application of ammonium ions, and external application of 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid (SITS), 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS), acetazolamide, furosemide, ammonium, zinc and copper ions, were all without effect on the reversal potential for GABA.     

The monensin-mediated transport of Na+ and K+ through phospholipid bilayers studied by 23Na- and 39K-NMR

Addition of monesin to preparations of large unilamellar vesicles made from egg yolk phosphatidylcholine (EPC) in sodium or potassium chloride solution and from dioleoylphosphatidylcholine (DOPC) in sodium chloride solutions gives rise to dynamic 23Na- and 39K-NMR spectra. The dynamic spectra arise from the monensin-mediated transport of the metal ions through the membrane. The kinetics of the transport are followed as a function of monensin and metal ion concentrations and are compatible with a model in which one monensin molecule transports one metal ion. Rate constants for the association and dissociation of the monensin-metal complex in the membrane/water interface are extracted and the stability constants for complex formation are evaluated. The rate constants in DOPC are similar to those in EPC, confirming that diffusion is not rate-limiting in the transport process and that dissociation of the complex is the rate-limiting step. Although potassium on its own is transported more rapidly, sodium forms the more stable complex and is therefore transported preferentially in competition with potassium.     

Beta-alanine transport in synaptic plasma membrane vesicles from rat brain. Efflux, exchange and stoichiometry

The efflux and exchange of beta-alanine were studied in synaptic plasma membrane vesicles from rat brain. The mechanism of beta-alanine translocation has been probed by comparing the ion dependence of net efflux to that of exchange. Dilution-induced efflux requires the simultaneous presence of internal sodium and chloride ions while influx is dependent on the presence of these two ions on the outside [Zafra, F., Aragón, M. C., Valdivieso, F. and Giménez, C. (1984) Neurochem Res. 9, 695-707]. These data show that the release of beta-alanine occurs via the carrier system and that it is cotransported with sodium and chloride ions. beta-Alanine efflux from the membrane vesicles is stimulated by external beta-alanine. This exchange does not require external sodium and chloride but it is dependent on the external concentration of beta-alanine. Half-maximal stimulation is obtained at a beta-alanine concentration similar to the Km for beta-alanine influx. Results of the direct measurements of the coupling of sodium and chloride to the transport of beta-alanine by using a kinetic approach allow us to propose a stoichiometry for the translocation cycle catalyzed by the beta-alanine transporter of three sodium ions and one chloride ion per beta-alanine zwitterion. To account for all the observed effects of external ions, beta-alanine concentrations and membrane potential on beta-alanine influx and efflux, a kinetic model of the Na+/Cl-/beta-alanine cotransport system is discussed.     

Identification of a lithium interaction site in the gamma-aminobutyric acid (GABA) transporter GAT-1

The sodium- and chloride-dependent electrogenic gamma-aminobutyric acid (GABA) transporter GAT-1, which transports two sodium ions together with GABA, is essential for synaptic transmission by this neurotransmitter. Although lithium by itself does not support GABA transport, it has been proposed that lithium can replace sodium at one of the binding sites but not at the other. To identify putative lithium selectivity determinants, we have mutated the five GAT-1 residues corresponding to those whose side chains participate in the sodium binding sites Na1 and Na2 of the bacterial leucine-transporting homologue LeuT(Aa). In GAT-1 and in most other neurotransmitter transporter family members, four of these residues are conserved, but aspartate 395 replaces the Na2 residue threonine 354. At varying extracellular sodium, lithium stimulated sodium-dependent transport currents as well as [3H]GABA uptake in wild type GAT-1. The extent of this stimulation was dependent on the GABA concentration. In mutants in which aspartate 395 was replaced by threonine or serine, the stimulation of transport by lithium was abolished. Moreover, these mutants were unable to mediate the lithium leak currents. This phenotype was not observed in mutants at the four other positions, although their transport properties were severely impacted. Thus at saturating GABA, the site corresponding to Na2 behaves as a low affinity sodium binding site where lithium can replace sodium. We propose that GABA participates in the other sodium binding site, just like leucine does in the Na1 site, and that at limiting GABA, this site determines the apparent sodium affinity of GABA transport.     

The transport of Na+ and K+ ions through phospholipid bilayers mediated by the antibiotics salinomycin and narasin studied by 23Na- and 39K-NMR spectroscopy

Addition of the ionophoric antibiotics salinomycin or narasin to preparations of large unilamellar vesicles made from egg yolk phosphatidylcholine in sodium or potassium chloride solutions gives rise to dynamic effects in the 23Na- and 39K-NMR spectra. The dynamic spectra arise from the ionophore-mediated transport of the metal ions through the membrane. The kinetics of the transport are followed as a function of the concentrations of ionophore and the metal ion and are compatible in all cases with a model in which one ionophore molecule transports one metal ion. For both ionophores the transport of potassium ions is appreciably faster than that of sodium and in both cases the rate-limiting step for sodium transport is dissociation of the ionophore-metal complex. Assuming dissociation to be rate limiting in all four cases it is shown that the transport rate differences between the pairs of complexes of each metal arise solely from differences in the rates of formation. The stability constants for ionophore-metal complex formation in the membrane/water interface are evaluated.     

L-serine and GABA uptake by synaptosomes during postnatal development of rat

Postnatal development changes in mechanisms of synaptosomal amino acid transport have been studied in rat cerebral cortex. Specific uptake of radiolabeled L-serine was examined and compared with that of radiolabeled GABA using synaptosomes-enriched fractions freshly prepared from cerebral cortex at different postnatal days from the birth to young adulthood. The preparations were incubated with 10 nM of [3H]L-serine and 10 nM of [3H]-GABA in either the presence or absence of NaCl, KCl or choline chloride, at 2 and 30 degrees C, for different periods up to 30 min. The uptake of [3H]l-serine was temperature dependent in synaptosomal fractions prepared from cerebral cortex of rats in postnatal days 5, 7, 13 and 21, but stronger dependence was observed in adult brain, irrespective of the presence of Na+, K+ or choline ions. At all postnatal ages studied, [3H]-GABA uptake showed a high activity in the presence of Na+ ions and at 30 degrees C. The values of Km were 90-489 microM in L-serine uptake. However, in the uptake of GABA the values of Km were 80-150 microM. The highest values of Vmax were obtained at 5 and 21 postnatal days for both transport systems. These results indicate that the uptake of l-serine and GABA are regulated differentially during postnatal development.     

A glutamine residue conserved in the neurotransmitter:sodium:symporters is essential for the interaction of chloride with the GABA transporter GAT-1

Neurotransmitter:sodium symporters are crucial for efficient synaptic transmission. The transporter GAT-1 mediates electrogenic cotransport of GABA, sodium, and chloride. The presence of chloride enables the transporter to couple the transport of the neurotransmitter to multiple sodium ions, thereby enabling its accumulation against steep concentration gradients. Here we study the functional impact of mutations of the putative chloride-binding residues on transport by GAT-1, with the emphasis on a conserved glutamine residue. In contrast to another putative chloride coordinating residue, Ser-331, where mutation to glutamate led to chloride-independent GABA transport, the Q291E mutant was devoid of any transport activity, despite substantial expression at the plasma membrane. Low but significant transport activity was observed with substitution mutants with small side chains such as Q291S/A/G. Remarkably, when these mutations were combined with the S331E mutation, transport was increased significantly, even though the activity of the S331E single mutant was only ～25% of that of wild type GAT-1. Transport by these double mutants was largely chloride-independent. Like mutants of other putative chloride coordinating residues, the apparent affinity of the active Gln-291 single mutants for chloride was markedly reduced along with a change their anion selectivity. In addition to the interaction of the transporter with chloride, Gln-291 is also required at an additional step during transport. Electrophysiological analysis of the Q291N and Q291S mutants, expressed in Xenopus laevis oocytes, is consistent with the idea that this additional step is associated with the gating of the transporter.     

GABA reverse transport by the neuronal cotransporter GAT1: influence of internal chloride depletion

The role of intracellular ions on the reverse GABA transport by the neuronal transporter GAT1 was studied using voltage-clamp and [(3)H]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 during early neuronal development, similarly to the functional alterations seen in GABA receptors caused by KCC2 activity.     

Molecular heterogeneity of the gamma-aminobutyric acid (GABA) transport system. Cloning of two novel high affinity GABA transporters from rat brain.

cDNA clones encoding two novel gamma-aminobutyric acid (GABA) transporters (designated GAT-2 and GAT-3) have been isolated from rat brain, and their functional properties have been examined in mammalian cells. The transporters display high affinity for GABA (Km approximately 10 microM) and exhibit pharmacological properties distinct from the previously cloned neuronal GABA transporter (GAT-1). Both transporters require sodium and chloride for transport activity. The nucleotide sequences of GAT-2 and GAT-3 predict proteins of 602 and 627 amino acids, respectively, which can be modeled with 12 transmembrane domains, similar to the topology proposed for other cloned neurotransmitter transporters. Localization studies indicate that both transporters are present in brain and retina, while GAT-2 is also present in peripheral tissues. The cloning of these transporter genes from rat brain reveals previously undescribed heterogeneity in GABA transporters.     

Low-affinity gamma-aminobutyric acid transport in rat brain

The low-affinity (Km = 100-200 microM) gamma-aminobutyric acid (GABA) transporter from membrane vesicles from rat brain has been characterized and found to be in many aspects similar to the well-known sodium- and chloride-coupled high-affinity gamma-aminobutyric acid transporter (Km = 2-4 microM). Influx by this system is sodium and chloride dependent and stimulated by an interior negative membrane potential. Steady-state levels obtained by both systems are lowered by the sodium channel openers veratridine and aconitine. However, while the channel blocker tetrodotoxin fully reverses this inhibition with the high-affinity system, this is not the case for its low-affinity counterpart. Furthermore, the toxin from the scorpion Androctonus australis Hector inhibited high-affinity transport only. Efflux of gamma-aminobutyric acid taken up by the high-affinity system displayed a Km of about 100 microM. Exchange catalyzed by the low-affinity system was observed in the absence of external sodium and chloride. Furthermore, both activities copurified in the fractionation procedure developed to purify the high-affinity transporter. All these observations are consistent with the idea that both activities are manifestations of only one gamma-aminobutyric acid transporter. The high-affinity binding site represents the extracellular and the low-affinity site the cytosolic aspect of the transporter. In addition, it was found that right-side-out synaptosomes also contain a low-affinity GABA transporter. This apparently represents a different transport protein.     

Cation Dependence of Hypotaurine Uptake in Mouse Brain Slices

Abstract: Mouse brain slices take up hypotaurine (2-aminoethanesulphinic acid) from medium by means of two concentrative low- and high-affinity transport systems. [³⁵S]Hypotaurine uptake by the slices was significantly reduced in the absence of external potassium, calcium, or magnesium ions. An excess of potassium ions also inhibited hypotaurine uptake by one-half. Uptake was almost completely abolished on removal of sodium ions. The K _m constants for both low- and high-affinity transport components increased in a low-sodium medium, suggesting that sodium ions are required when hypotaurine is attached to its possible carrier sites in plasma membranes. Sodium ions also mimicked allosteric effectors of hypotaurine transport, showing positive cooperativity. More than two sodium ions may be involved in the transport of one hypotaurine molecule across the cell membrane. The calculated activation energies of transport were fairly similar in normal and sodium-deficient media and thus sodium ions may not participate in the activation mechanisms of the transport. With respect to cation dependence, hypotaurine transport in brain slices exhibits features characteristic of neurotransmitter amino acids.