GABA efflux from synaptosomes: Effects of membrane potential, and external GABA and cations

Nelson, Mark T.; Blaustein, Mordecai P.

doi:10.1007/bf01870400

Cited by 63 publications

(32 citation statements)

References 31 publications

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“…We propose that, in addition to the coupled mode, GAT1 also has a channel mode. Ionic pathways through cotransporters have serotonin cotransporters (for review see Lester, Mager, Quick & Corey, 1994), noradrenaline cotransporters (Galli, DeFelice, Duke, Moore & Blakely, 1995) (Martin, 1973;Blaustein & King, 1976; Nelson & Blaustein, 1982). Such release would be in addition to vesicle fusion-mediated efflux (Atwell et al 1993).…”

Section: Discussionmentioning

confidence: 99%

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Sodium‐dependent GABA‐induced currents in GAT1‐transfected HeLa cells.

Risso

DeFelice

Blakely

1996

The Journal of Physiology

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1. HeLa cells were infected with recombinant vaccinia virus containing the T7 RNA polymerase gene and transfected with the cDNA for a rat GABA transporter, GAT1, cloned downstream of a T7 RNA polymerase promoter. Six to sixteen hours after transfection, whole-cell recording with a voltage ramp in the range -90 to 50 mV revealed GABAinduced currents (approximately -100 pA at -60 mV in 100 uM GABA, 16 h after transfection at room temperature). No GABA-induced currents were observed in parental HeLa cells or in mock-transfected cells. 2. GABA-induced currents were suppressed by extracellular perfusion with GABA-free solutions or addition of GATI inhibitors SKF89976-A or SKF100330-A. At fixed voltage the GABA dependence of the inward current fitted the Michaelis-Menten equation with aHill coefficient, n, near unity and an equilibrium constant, Km, near 3 /tM. The Na+ dependence of the inward currents fitted the Michaelis-Menten equation with n t 2 and Km ; 10 mM. The constants n and Km for GABA and Na+ were independent of voltage in the range -90 to -30 mV. 3. GABA-induced currents reverse direction in the range 5-10 mV. The implication of this result is that GAT1 can mediate electrogenic (electrophoretic) influx or efflux of GABA depending on the membrane voltage. The presence of an outward current in our experiments is consistent with radioactive-labelled flux data from resealed vesicle studies. However, it is inconsistent with frog oocyte expression experiments using the same clone. In oocytes, GAT1 generates no outward current in a similar voltage range. Smaller intracellular volume or higher turnover rates in the mammalian expression system may explain the outward currents. 4. External GABA induces inward current, and internal GABA induces outward current.However, in cells initially devoid of internal GABA, external GABA can also facilitate an outward current. This GATi -mediated outward current occurs only after applying negative potentials to the cell. These data are consistent with the concept that negative potentials drive GABA and Nae into the cell, which then leads to electrogenic efflux through GATI at positive voltages. 5. Assuming coupled transport, we estimate the number of transporters, N, times the turnover rate, r, to be Nr t 109 s-' under nominal conditions ( V = -60 mV, 30 /M GABA, 130 mm Nae and room temperature). This indicates either very high levels of expression (0104 um-2), assuming published turnover rates (-1 0 s-1), or turnover rates that are significantly greater than previously reported. As an alternative, a channel may exist in the GAT1 protein that is gated by GABA and Nae and blocked by GAT1 antagonists. The channel mode of conduction would exist in addition to the coupled, fixed-stoichiometry transporter mode of conduction.

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Section: Discussionmentioning

confidence: 99%

“…It had been suggested, partly on the basis of tracer flux experiments, that membrane voltage regulates the efflux of neurotransmitters by this mechanism (Martin, 1973;Blaustein & King, 1976;Nelson & Blaustein, 1982). However, tracer flux experiments provide no direct information on the mechanism of voltagedependent uptake or voltage-dependent release.…”

mentioning

confidence: 99%

Sodium‐dependent GABA‐induced currents in GAT1‐transfected HeLa cells.

Risso

DeFelice

Blakely

1996

The Journal of Physiology

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“…By assuming that both ions and 5_HT need to bind to the transport system prior to membrane translocation, each model predicts a unique sequence of molecular events for the generalized case of transport binding by an ion and 5_HT. The respective Michaelis-Menten derivations for these models are analogous to those used previously for analysing Na¤ dependence in GABA transport (Wheeler & Hollingsworth, 1979;Nelson & Blaustein, 1982). These models and their corresponding predictions are as follows.…”

Section: Kinetic Modellingmentioning

confidence: 93%

Mechanistic analyses of ion dependences in a high‐affinity human serotonin transport system in transfected murine fibroblast cells

Chang

Lam

1998

The Journal of Physiology

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A clonal cell line, L‐S1, has been identified from transfection of human genomic DNA into cultured mouse L‐M fibroblasts. Because this transfectant cell line stably expresses a high‐affinity serotonin (5‐HT) transport mechanism with kinetic and pharmacological properties comparable to those of other serotonin uptake systems, it was used to investigate the mechanistic involvement of Na+ and Cl− ions in the ligand binding and kinetic uptake processes of this system. Intact transfectant cells, when incubated at low temperature (4 °C), enabled quantitative assessment of imipramine‐displaceable 5‐[3H]HT binding to the 5‐HT transport system. This binding activity is insensitive to the presence of various ligands specific for 5‐HT receptor subtypes. Imipramine‐displaceable 5‐[3H]HT binding to intact L‐S1 cells was shown to be a Cl−‐dependent but Na+‐independent process. Chloride ions lack binding co‐operativity in facilitating ligand binding. Changes in external Cl− concentration altered the Kd but not the Bmax of binding. The overall transport activity was observed to be highly dependent on both external Na+ and Cl− concentrations, characterized by a 5‐HT:Na+:Cl− coupling ratio of 1:1:1 per transport cycle. Alterations in the external concentrations of both Na+ and Cl− ions altered only the Km and not the Vmax of transport. Both binding and kinetic results are consistent with kinetic modelling predictions of the Cl− ion in facilitating 5‐HT binding to the transport system, and of the Na+ ion in enabling translocation of bound 5‐HT across the plasma membrane. Thus, Na+ and Cl− ions facilitate mechanistically distinct and discernible functions in the transport cycle.

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“…The consequent depolarization of the nerve terminals is accompanied by an influx of Ca2+ (Blaustein, 1975;Akerman and Nicholls, 1981;Adam-Vizi and Ligeti, 1986), paralleled by an increase in transmitter release (Blaustein, 1975;Adam-Vizi and Ashley, 1987). However, in the absence of external Ca2+, veratridine is also effective in enhancing the release of GABA (Benjamin and Quastel, 1972;Haycock et al, 1978;Sandoval, 1980;Nelson and Blaustein, 1982;Rhoads et al, 1983;Carvalho et al, 1986), noradrenaline (Schoffelmeer and Mulder, 1983), ACh (Meyer and Cooper, 1981;Vyas and Marchbanks, 198 1 ;Adam-Vizi and Ligeti, 1984;Carroll, 1984;Carroll and Benishin, 1984), dopamine (Raiteri et al, 1979), glutamate (Levi et al, 1980;Rhoads et al, 1983;McMahon et al, 1990), glycine and B-alanine (Aragon et al, 1988). These effects of veratndine, in both the presence and the absence of Ca2+, were specific with respect to being sensitive to the sodium channel blocker tetrodotoxin (TTX).…”

Section: Raised Internal Na+ Concentrationmentioning

confidence: 99%