Contribution of changes in the chloride driving force to the fading of IGABA in frog melanotrophs

Frank le Foll, Olivier Soriani, Hubert Vaudry, and Lionel Cazin

European Institute for Peptide Research (Institut Fédératif de Recherches Multidisciplinaires sur les Peptides no. 23), Laboratory of Cellular and Molecular Neuroendocrinology, Institut National de la Santé et de la Recherche Médicale Unité 413, Unité Associée au Centre National de la Recherche Scientifique, University of Rouen, 76821 Mont-Saint-Aignan, France


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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Chloride redistribution during type A gamma -aminobutyric acid (GABAA) currents (IGABA) has been investigated in cultured frog pituitary melanotrophs with imposed intracellular chloride concentration ([Cl-]i) in the whole cell configuration or with unaltered [Cl-]i using the gramicidin-perforated patch approach. Prolonged GABA exposures elicited reproducible decaying currents. The decay of IGABA was associated with both a transient fall of conductance (gGABA) and shift of current reversal potential (EGABA). The shift of EGABA appeared to be time and driving force dependent. In the gramicidin-perforated patch configuration, repeated GABA exposures induced currents that gradually vanished. The fading of IGABA was due to persistent shifts of EGABA as a result of gGABA recovering from one GABA application to another. In cells alternatively clamped at potentials closely flanking resting potential and submitted to a train of brief GABA pulses, a reversal of IGABA was observed after 150 s recording. It is demonstrated that, in intact frog melanotrophs, shifts of EGABA combine with genuine receptor desensitization to depress IGABA. These findings strongly suggest that shifts of EGABA may act as a negative feedback, reducing the bioelectrical and secretory responses induced by an intense release of GABA in vivo.

desensitization; intracellular chloride concentration; gramicidin-perforated patch recording


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INTRODUCTION
MATERIALS AND METHODS
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IN THE MELANOTROPHS of the frog pituitary pars intermedia, gamma -aminobutyric acid (GABA) or GABAA-receptor agonists exert a transient stimulation followed by a prolonged inhibition of alpha -melanocyte-stimulating hormone (alpha -MSH) release in vitro (7). By using the gramicidin-perforated patch technique, which avoids artifactual alterations of the physiological intracellular chloride concentration ([Cl-]i; see Ref. 8), we have recently demonstrated that GABA induces an outward chloride flux from the intracellular compartment containing unusually high [Cl-]i. The depolarization that results from the outward current triggers the activation of voltage-operated sodium and calcium channels, leading to the initial stimulation of alpha -MSH secretion. The delayed inhibitory phase of the secretory response likely originates from the strong shunting effect that accompanies the depolarization induced by high micromolar GABA concentrations and causes cessation of firing (17). Factors affecting the efficacy of the GABAergic transmission are of outstanding importance in the regulation of alpha -MSH secretion because they determine which of the effects, i.e., the depolarization or the resistive shunt, predominantly controls the hormone release. In the frog melanotrophs, the GABAA receptor is subject to extracellular positive or negative modulation by benzodiazepines, barbiturates, and neuroactive steroids (18-20). In addition, in this cell model, the GABAA receptor function is also controlled, at the intracellular level, by the balance between phosphatase and kinase activities (4).

An alternative or concomitant mechanism of regulation of the GABAergic transmission is the fading of the response induced by the agonist itself. In this regard, the time scale of the synaptic events has to be considered. For agonist exposure persisting for minutes to hours, a downregulation of receptors is responsible for a reduction of the number of [3H]muscimol or [3H]flunitrazepam binding sites (23, 28) due to a removal of GABAA receptors from the cell surface (5). During short-term applications of GABA, electrophysiological experiments have demonstrated that the GABAA current (IGABA) declines despite the continuous presence of the agonist (3, 10, 18, 26). This early decay of responsiveness is commonly called desensitization. Accumulating evidence indicates that desensitization may involve distinct processes. A time-dependent decrease of the membrane chloride conductance in the presence of GABA has been observed in neurons and human embryonic kidney (HEK 293) cells expressing recombinant GABAA receptors (9, 26, 34, 35). This phenomenon is a general feature of ligand-gated ion channels that possess nonconducting desensitized states (13, 21) and seems to be functionally equivalent to the inactivation of voltage-sensitive channels. The current decay could also result from a gradual passive redistribution of chloride between both sides of the cell membrane, leading to a shift of the IGABA reversal potential and a reduction of the chloride driving force (EGABA). Such changes of EGABA have been described from experiments using suction pipette (2), sharp microelectrode (32), or whole cell patch-clamp (12) recordings in rat or frog neurons. However, these recording configurations bring some drawbacks because of the rapid chloride exchange between the pipette and cell compartments (17), which likely introduces errors in the evaluation of the magnitude of EGABA displacements. The recently developed gramicidin-perforated patch technique, allowing patch-clamp recordings while preserving intact the physiological [Cl-]i (1, 8, 16), represents a valuable method to investigate the ionic mechanisms responsible for the fading of the IGABA.

Herein, we have combined the standard whole cell and the gramicidin-perforated patch approaches to assess the relative contribution of genuine GABAA receptor desensitization and shifts in EGABA to the fading of the IGABA in the cultured frog melanotrophs.


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Animals and cell cultures. Adult male frogs (Rana ridibunda; Couétard, Saint-Hilaire de Riez, France) were used as tissue donors. Frogs were housed in a temperature-controlled room (8°C) under an established 12:12-h light-day photoperiod (lights on from 6:00 AM to 6:00 PM). The animals had free access to running water and were maintained in these conditions for at least 1 wk before use. Animal manipulations were performed according to the recommendations of the French Ethical Committee and under the supervision of authorized investigators. Cell cultures of frog pituitary melanotrophs were prepared as previously described (18). After anesthetization by immersion in 1% MS-222, the animals (body weight 30-40 g) were killed by cervical dislocation and decapitated. Eight neurointermediate lobes were carefully dissected out under sterile conditions. The tissues were rinsed in Leibowitz' 15 mammalian culture medium diluted 1.5× to match amphibian osmolality and were supplemented with 1% (vol/vol) of the kanamycin and the antibiotic-antimycotic solutions. Cells were then enzymatically dissociated (15 min) in the same medium to which 0.15% protease type IX and 0.15% collagenase type IA were added. After mechanical dispersion, cells were centrifuged (60 g) for 5 min and resuspended four times in the culture medium containing 10% FCS and antibiotics. Dispersed cells were then plated at a density of 10,000 cells/35-mm culture dish. Cultured cells were incubated at 22°C in a humidified atmosphere and were used 5-10 days after plating.

Electrophysiological recordings and analysis. Conventional tight-seal whole cell and gramicidin perforated-patch (8, 17) recordings were carried out at room temperature (20-22°C).One-half hour before electrophysiological experiments, the culture medium was replaced by one of the external solutions described in Table 1. During the recording session, the culture dish was superfused constantly with saline (3 ml/min) via a flow pipe aimed at the cell under study. A bath volume of 500 µl was maintained, with the excess bathing solution being continuously aspired via a suction needle. Soft glass patch electrodes were pulled from thin-wall microhematocrit capillaries on a two-step vertical pipette puller (L/M-3P-A; List Medical, Darmstadt, Germany). When filled with one of the pipette solutions (Table 1), the electrodes had 3-5 MOmega resistances. For each combination of external and pipette solutions used, liquid junction potentials were measured and corrected before gaining the whole cell access (25). In the gramicidin-perforated patch configuration, because of the complete lack of chloride permeability, no Donnan equilibrium junction potential was expected to develop (16). Stock solution of gramicidin was prepared by dissolving the antibiotic in methanol (10 mg/ml). Pipette tip filling was accomplished by a brief immersion (1-2 s) in a gramicidin-free pipette solution. The remainder of the pipette was then backfilled with the same solution to which gramicidin (100 µg/ml) was added. The progress of perforation was assayed by monitoring the access resistance deduced from the amplitude of the capacitive transients in response to repetitive 10-mV hyperpolarizing steps. The series resistance, typically 12 MOmega in the whole cell configuration and 14 MOmega after 10 min perforation (17), was partially (60-70%) compensated using the on-board circuitry of the amplifier. GABA was dissolved in the standard extracellular solution and pressure ejected (2-4 psi) through a micropipette (2-4 µm tip diameter). To avoid uncontrolled drug leakage, the ejection pipette was kept away and brought in close proximity (10-20 µm) to the cell soma just before microejection. Currents were recorded using an Axopatch 200A current-to-voltage converter (Axon Instruments, Foster City, CA) interfaced to a Digidata 1200 (Axon instruments) and were directly digitized with pCLAMP 6 software for further off-line analysis. Theoretical equilibrium potentials were computed with the use of the Nernst equation. Current-voltage (I-V) curves were plotted after subtracting the baseline obtained just before the agonist application from the IGABA. EGABA was defined as the x-intercept of the I-V curve or of the instantaneous current in a voltage step or in a voltage-ramp protocol. Slope conductance was determined by linear regression of the less-rectifying more-conducting part of the I-V plot. Nonlinear regressions were performed using the Marquardt-Levenberg algorithm of SigmaPlot 5.0 (Jandel Scientific, Sausalito, CA). Quantitative data are expressed as means ± SE.

                              
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Table 1.   Composition of recording solutions

Reagents. GABA, MS-222, protease type IX, collagenase type IA, Leibovitz L-15 medium, gramicidin D, HEPES, EGTA, ATP potassium salt, TTX, and tetraethylammonium were purchased from Sigma (St. Louis, MO). Kanamycin, the antibiotic-antimycotic solutions, and FCS were obtained from Boehringer Mannheim (Meylan, France). Tissue culture dishes were supplied by Costar (Cambridge, MA).


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INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Whole cell IGABA were recorded from cultured frog melanotrophs by using either the tight-seal ruptured-patch or the gramicidin-perforated patch techniques in the voltage-clamp mode. All of the melanotrophs examined (n = 109) responded to GABA.

Decay of the IGABA in melanotrophs with imposed [Cl-]i. In experiments carried out in the whole cell configuration, a prolonged ejection of GABA (10 µM, 15 s) elicited a current that rapidly peaked and then gradually decayed to 26.2 ± 1.9% (n = 25) of the maximum amplitude within 15 s (Fig. 1A). The late amplitude was defined as the residual current at the end of the GABA application. Repeated 15-s GABA ejections at 2-min intervals generated currents with reproducible peak and late amplitudes (Fig. 1B). Successive I-V relationships established at 2-min intervals from a single melanotroph displayed similar EGABA. Moreover, the slope conductances remained unchanged (Fig. 1, C and D). To monitor the rate at which IGABA recovered from the fading provoked by a prolonged (15-s) GABA application, brief test pulses (600 ms) of GABA were delivered at incremental intervals (5-s steps, Fig. 2A). These test pulses were supposed to induce no additional current decrease. The exponential curve fitted to the data yielded a time constant (tau ) = 12.9 s for recovery in the present experimental conditions (Fig. 2B).


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Fig. 1.   Currents evoked by repeated administrations of gamma -aminobutyric acid (GABA) in the whole cell configuration. Recordings were carried out using a low-chloride solution in the patch pipette [External 1 (E1)/Whole Cell 1 (WC1), chloride equilibrium potential (ECl) = -75 mV]. A: currents evoked in a single melanotroph by 5 successive ejections of GABA (10 µM, 15 s; horizontal bars) at 2-min intervals. Holding potential was 0 mV. B: time course of the peak () and late () currents shown in A. C: three families of currents (1, 2, and 3) evoked by GABA (10 µM, 5 s) at 2-min intervals from another cell. In each family, membrane potential was set for 2 min at values ranging from -95 to +65 mV by 20-mV steps. D: superimposed current-voltage (I-V) curves corresponding to C. Peak current amplitude was plotted against the membrane potential. Reversal potentials for families of currents 1, 2, and were -73 (), -82 (triangle ), and -76 mV (X), respectively.



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Fig. 2.   Fading and recovery from fading of the GABAA current (IGABA). Currents were recorded in the whole cell configuration with low internal chloride (E1/WC1, ECl = -75 mV). Holding potential was 0 mV. A: superimposed IGABA traces recorded from a single cell. Series of three successive GABA (10 µM) ejections were repeated 10 times at 2-min intervals. In each series, a brief pulse of GABA (600 ms, a) eliciting a control response was followed within 10 s by a prolonged GABA application (15 s, b), provoking a current decay. Recovery was monitored by a brief test pulse of GABA (600 ms, c) applied at incremental intervals (5-s steps) after the end of the 15-s GABA ejection. B: time course of recovery. Relative amplitude of the current evoked by pulse c was expressed as a fraction of the control current induced by pulse a and was plotted against the interpulse interval between b and c (n = 6-11). Data were fitted to a monoexponential function, yielding a time constant of 12.9 s for recovery of the control current.

In another set of experiments, a protocol was developed to assess the relative contribution of the decrease of the GABA-dependent membrane conductance (gGABA) and the shift of EGABA to the decay of IGABA. Both parameters were followed throughout a prolonged (25-s) GABA exposure, by applying repeated voltage ramps at 3.5-s intervals. A very fast sweep rate (800 mV/s) was employed to minimize the influence of the ramp on the global current decay. The resulting I-V relationships were used to determine EGABA and the slope gGABA (Fig. 3, A and B). In these experiments, the intrapipette chloride concentration (33 mM) was close to the physiological [Cl-]i (27 mM) previously measured in the frog melanotrophs (17). To verify that the voltage ramps did not influence the values of EGABA and gGABA, control experiments were performed on single cells. In all cells studied (n = 8), the values of EGABA and gGABA obtained using voltage ramps did differ from that measured with voltage steps (data not shown). In the presence of GABA, a time-dependent decrease of gGABA occurred. Concomitantly, a gradual shift of EGABA toward 0 mV was observed (Fig. 3, B and C). Although IGABA fell down to 33 ± 6% of the peak current, gGABA dropped to 44 ± 6% of the control, and EGABA markedly drifted from -31.5 ± 0.9 to -20.0 ± 3.2 mV (n = 8, Fig. 3, D-F).


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Fig. 3.   Redistribution of chloride ions during the response to GABA in the whole cell configuration. Melanotrophs were held at 0 mV with a physiological internal chloride concentration (E2/WC3, ECl = -31 mV). A: current evoked by a prolonged GABA application (10 µM, 25 s; horizontal bar). High-speed depolarizing triangular voltage ramps (800 mV/s, from 0 to +75 mV and from +75 to 0 mV) followed by mirror-image hyperpolarizing ramps were applied before (a) and during (b-h) the GABA application. B: superimposed instantaneous I-V relationships corresponding to A. The leak current (a) has been subtracted from the currents recorded during GABA exposure (b-h). C: enlarged detail of the I-V curves shown in B. Arrows indicate the zero-current potential for each I-V curve. D-F: changes in IGABA (D), GABA reversal potential (EGABA; E), and GABA conductance (gGABA; F) occurring during the prolonged GABA administration. Relative current amplitude and slope conductance in c-h were expressed as a fraction of their respective values in b. Each point corresponds to the mean ± SE of data obtained as in A from 8 cells.

The role of the chloride flux in the decay of IGABA was further studied for various chloride driving forces. Therefore, the above-described repeated-ramp protocol was used at three different holding potentials (Fig. 4, A-C). At -80 mV, the decay phase of the GABA-evoked inward current was accompanied by a decrease of gGABA, which was associated with a slight shift of EGABA toward more negative potentials. At -35 mV, a value close to the chloride equilibrium potential (ECl) in the present recording conditions, GABA failed to elicit any current or noticeable change in EGABA. Nevertheless, a time-dependent decrease of gGABA was still detected. At +30 mV, GABA generated a robust outward current characterized by a high initial conductance compared with that measured at negative potentials. Thereafter, gGABA markedly decayed during GABA exposure. In addition, EGABA sharply drifted toward 0 mV (Fig. 4, D and E).


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Fig. 4.   Dependence of the changes in EGABA and gGABA on the holding potential (Vh) in a single melanotroph. A-C: recordings were performed in the whole cell configuration with a physiological internal chloride concentration (E2/WC3, ECl = -31 mV). Currents were evoked by prolonged GABA applications (10 µM, 25 s; horizontal bars) at holding potentials of -80 (A), -35 (B), or +30 (C) mV. High-speed triangular voltage ramps were applied before and during GABA exposure, as described in Fig. 3. Voltage ramp pulses ran from -5 to -155 mV (A), +35 to -115 mV (B), and +105 to -45 mV (C). Resulting instantaneous I-V curves are presented below the current traces. D-E: changes in ggaba (D) and EGABA (E) accompanying the IGABA, at holding potentials of -80 (), -35 (), or +30 (black-triangle) mV. Relative slope conductance measured at the different times was expressed as a fraction of the initial conductance at -80 mV.

Fading of the IGABA in melanotrophs with unaltered physiological [Cl-]i. In cells studied in the whole cell configuration, chloride exchange between pipette and cell compartments could distort the time course of the current. In this respect, the gramicidin-perforated patch technique provides an alternative approach that allows recordings of IGABA without imposing [Cl-]i (1, 16, 17). In recordings performed in the gramicidin-perforated patch configuration, a prolonged GABA ejection (10 µM) µM, 15 s) elicited a decaying current. Repeated 15-s GABA ejections at 2-min intervals generated currents that gradually vanished (Fig. 5A). Peak and late amplitudes of IGABA declined with similar time courses (Fig 5B). To establish successive I-V relationships from a single melanotroph, families of IGABA at increasing membrane potentials were recorded at 2-min intervals (Fig 5C). Interestingly, from one family of currents to another, the I-V curves exhibited an irreversible drift of EGABA from -25 to 0 mV, without any marked modification in the slope conductances (Fig. 5D).


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Fig. 5.   Currents evoked by repeated administrations of GABA in the gramicidin-perforated patch configuration. Recordings were carried out using a KCl solution in the patch pipette (E1/patch pipette 1 (PP1), ECl = -0.6 mV), to which gramicidin D (100 µg/ml) was added. GABA was applied after a complete cell membrane perforation, indicated by stabilization of the capacitive transients generated by 10-mV hyperpolarizing pulses. A: currents evoked from a single melanotroph by successive ejections of GABA (10 µM, 15 s; horizontal bars) at 2-min intervals. Holding potential was -80 mV. B: time course of the peak () and late () currents shown in A. C: three families of currents (1, 2, and 3) evoked by GABA (10 µM, 5 s) at 2-min intervals from another cell. In each family, membrane potential was set for 2 min at values ranging from -80 to +80 mV by 20-mV steps. D: superimposed I-V curves corresponding to C. Peak current amplitude was plotted against the membrane potential. Reversal potentials for families of currents 1, 2 and 3 were -31 (), -9 (triangle ), and 0 (X) mV, respectively.

To evaluate the relative contribution of changes in gGABA and EGABA to the decay of IGABA recorded in the gramicidin-perforated patch configuration, the repeated-ramp protocol described in Fig. 3 was used. Under these conditions, the late current amplitude decreased to 28 ± 3% of the peak during a 25-s GABA (10 µM) ejection. Concomitantly, gGABA diminished to 45 ± 2% of its control value, and EGABA shifted from -34.8 ± 2.2 to -20.9 ± 2.3 mV (n = 8, Fig. 6, A-C). In addition, in all melanotrophs (n = 5) submitted to three consecutive repeated-ramp protocols at 2-min intervals, long-lasting and additive shifts of EGABA were observed. As shown in the representative example of Fig. 6, D and E, the EGABA drifted from -39.6 to -29.3 mV, -31.9 to -22.7 mV, and -24.4 to -17.2 mV throughout the first, the second, and the third GABA ejection, respectively.


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Fig. 6.   Redistribution of chloride ions during the response to GABA in the gramicidin-perforated patch configuration. Currents were recorded using a CsCl and gramicidin D solution in the patch pipette (E2/PP2, ECl = -0.6 mV). Holding potential was 0 mV. High-speed triangular voltage ramps were applied before and during 25-s GABA (10 µM) exposures, as described in Fig. 3. A-C, with changes in IGABA (A), EGABA (B), and gGABA (C) occurring during the prolonged GABA administrations. Relative current amplitude and slope conductance were expressed as a fraction of their initial values. Each point indicates the mean ± SE of data obtained from 8 cells. Only one GABA pulse was delivered per cell under study. D: three currents (1, 2, and 3) were evoked by prolonged GABA applications at 2-min intervals in a single melanotroph. The corresponding instantaneous I-V curves focus on the current reversal potentials. E: changes in EGABA observed during successive IGABA 1 (), 2(), and 3 (black-triangle) shown in D.

The persistent drift of EGABA toward more depolarized potentials observed in intact melanotrophs could be accounted for by a continuous accumulation of chloride in the intracellular compartment, through specific transport systems and/or chloride channels in addition to the GABAA receptor. To address this issue, a protocol was designed and applied to single cells (n = 5, Fig. 7). First, three successive families of IGABA were recorded at 2-min intervals. EGABA, deduced from the corresponding I-V relationships, shifted from its native value (-38 mV) to a more depolarized and stable level (-11 mV). Thereafter, to lower [Cl-]i, the cell was submitted to a series of five GABA ejections at -80 mV. Subsequently, EGABA measured from a first family of IGABA was -35 mV, a value close to that obtained initially. In a second determination, EGABA shifted to -10 mV, as already observed at the beginning of the recording session. Finally, after another series of five GABA ejections at -80 mV, again EGABA (-36 mV) recovered (Fig. 7, A and C).


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Fig. 7.   Reversibility of the shifts of EGABA in the gramicidin-perforated patch configuration. A: recordings were carried out using a KCl and gramicidin D solution in the patch pipette (E1/PP1, ECl = -0.6 mV). A single melanotroph was submitted to the voltage-clamp protocol indicated above the current traces. In this representation of the voltage protocol, the time scale is not constant. Repeated GABA (10 µM, 5 s) ejections were performed at 2-min intervals, as indicated by the vertical arrows. The stimulation procedure included 6 I-V protocols (noted 1 to 6) and two series of 5 successive GABA ejections at -80 mV. B: superimposed I-V curves obtained from the I-V protocols shown in A. , triangle , open circle , , black-triangle, and  correspond to the families of currents 1, 2, 3, 4, 5, and 6, respectively. C: changes in EGABA during the stimulation procedure followed in A. The values of EGABA were deduced from the I-V curves in B.

The possible influence of EGABA changes on the postsynaptic response of melanotrophs to GABA was investigated by using an experimental procedure closer to physiological conditions. The cell was alternatively clamped at potentials (-50 and -40 mV) closely flanking the mean resting potential of the melanotrophs (-46.3 ± 1.5 mV, n = 29). A train of brief GABA pulses (10 µM, 100 ms, 1.1 Hz) was delivered. At -50 mV, the GABA-evoked inward current was initially large and gradually diminished over the course of the experiment. In contrast, at -40 mV, the inward current was markedly weaker and reversed after 150 s recording (Fig. 8).


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Fig. 8.   Inversion of IGABA elicited by a train of GABA pulses in a cell alternatively clamped at 2 membrane potentials in the gramicidin-perforated patch configuration. Current recordings were performed using a KCl and gramicidin D solution in the patch pipette (E1/PP1, ECl = -0.6 mV). A single melanotroph was alternatively held at -50 or -40 mV at 15-s intervals (top). IGABA (bottom) were evoked by a train of brief GABA pulses (10 µM, 100 ms) applied at the frequency of 1.1 Hz (middle). Extended current traces shown at bottom display inversion of IGABA at -40 mV in the second part of the recording. Horizontal dashed lines superimposed to the current traces indicate the zero-current levels at -40 and -50 mV.


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In the frog melanotrophs bathed in HEPES-buffered media, chloride is the exclusive charge carrier of the IGABA (17, 20). By using the gramicidin-perforated patch technique, we have recently found that, in this cell model, EGABA is ~10 mV positive to the resting potential. As a consequence, GABA provokes a depolarization, triggering action potentials. The voltage change due to GABA is underlaid by a dramatic increase of the membrane conductance, which shunts the voltage-activated currents. Thus the initial burst of activity is followed by a shunt-induced abolition of firing, with GABA clamping membrane potential at EGABA (17). We now demonstrate that, in frog melanotrophs with unaltered [Cl-]i, EGABA is unexpectedly a labile parameter. It appears that long-lasting shifts of EGABA can account for a GABAA receptor use-dependent fading of the response to GABA.

The main purpose of our study was to decipher, in the frog melanotrophs, the mechanisms involved in the fading of IGABA occurring in the presence of the agonist. In the whole cell configuration, GABA generated a decaying current that was reproducible over repeated agonist applications at 2-min intervals. This indicates that the parameters (EGABA and gGABA) controlling the current flow were modified throughout the agonist exposure and recovered within the delay between the GABA pulses. The kinetics of recovery (tau  = 12.9 s) were found to be similar to that described in neurons (3, 13, 15). Recovery may involve both true resensitization, i.e., removal of desensitized states (13), and restoration of the initial transmembrane chloride gradient by ion diffusion between the patch pipette and the cell interior (17). The observation that successive I-V curves were superimposed confirmed that neither the chloride driving force nor the conductance varied from one GABA application to another. In contrast, by using the repeated-ramp protocol to follow EGABA throughout a prolonged GABA exposure, it appeared that EGABA gradually and transiently drifted during the chloride flow. The shift of EGABA toward potentials corresponding to lower driving forces reflects a passive redistribution of chloride, which leads, together with the decrease of gGABA, to the fading of IGABA in the continued presence of the agonist. This observation is in good agreement with previous findings in rat and frog neurons (2, 12, 32). To verify the role of the chloride flux in the current decay, the variations of EGABA and gGABA have been monitored at various holding potentials. The data reveal that the displacement of EGABA was more pronounced at increasing chloride driving forces and was inverted when IGABA reversed. In these experiments, the initial increase or decrease of gGABA observed at holding potentials of +30 or -80 mV, respectively, was attributed to the voltage sensitivity of the GABAA receptor channel. Altogether, the present results demonstrate that, in frog melanotrophs, the GABA-evoked chloride flux induces transient time- and driving force-dependent shifts of EGABA.

Because of the relatively rapid ion equilibration between the pipette and cell compartments, the whole cell recordings may lead to an underestimation of the magnitude of EGABA changes during the response to GABA. To avoid such a limitation, the fading of IGABA has been examined using the gramicidin-perforated patch approach (16, 17). In the gramicidin-perforated patch recordings, the decay of IGABA observed during a single 25-s GABA exposure was underlaid by a fall of gGABA and a shift of EGABA, which were characterized by magnitudes and time courses very similar to those obtained from whole cell recordings. Thus we conclude that, in the whole cell configuration, the chemical access to the intracellular compartment did not alter the transient changes of EGABA caused by the chloride flux itself. This probably originates from the fact that chloride exchanges between pipette and cell compartments are much less intense than chloride fluxes evoked by GABA through membrane channels. However, when GABA was repeatedly ejected while the physiological [Cl-]i remained unaltered, IGABA gradually vanished from one GABA application to the next. This response pattern markedly differed from that obtained in the whole cell recordings in which repeated IGABA were reproducible. Interestingly, in the gramicidin-perforated patch configuration, successive GABA ejections resulted in increased shifts of EGABA that persisted during washout while gGABA recovered. Moreover, the present work demonstrates that the long-lasting redistributions of chloride ions were highly reproducible and mostly depended on the direction of the evoked chloride flux. Hence the possible contribution of a resting chloride permeability to the regulation of [Cl-]i is likely negligible. Taken together, the above-described observations indicate that, in intact frog melanotrophs, [Cl-]i is severely altered by the passive chloride flux through the GABAA receptor channels and seems not to be subject to any other short-term regulation.

To influence the response to GABA in vivo, the shifts of EGABA must be driven by deviations of membrane potential from the native ECl to maintain a significant chloride flux throughout the presence of GABA in the synaptic cleft. This implies that GABA released in synapses formed between hypothalamic nerve endings and melanotrophs activates only a few channels so that the resistive shunt still remains negligible and does not result in a voltage clamp of the membrane at ECl. Accumulating evidence now supports the occurrence of such a situation in vivo (6, 17, 24, 27, 29). Moreover, it has recently been proposed that, during an inhibitory postsynaptic current, a substantial number of postsynaptic receptors must be exposed to subsaturating concentrations of GABA because of the time course of GABA release, the action of transmitter clearance mechanisms, the cleft configuration, and the "spillover" of GABA, which leads to activation of extrasynaptic GABAA receptors (11). As a result, GABA must actually open only a part of the postsynaptic GABAA receptor channels for relatively long periods, contrary to that often obtained by rapid jumps of saturating GABA concentrations (1 mM, 0.5-3 ms) in patch experiments. In addition, modification of the membrane potential is a very common mechanism shared by a panoply of neurotransmitters and neuropeptides in frog melanotrophs (for review, see Ref. 33). It has also been shown that inhibitory postsynaptic potentials are often associated with depolarizations in neurons (22, 30, 31). These voltage fluctuations likely contribute to move the membrane potential beyond ECl. Herein, we demonstrate that variations of membrane potential in a restricted range (10 mV) spanning the resting potential, imposed during a train of 100-ms GABA pulses, were sufficient to induce the reversal of IGABA. This indicates that crucial changes of EGABA may occur after small variations of membrane potential and in response to GABA pulses, which are both relevant to physiological conditions.

The findings of the present work are schematically illustrated in Fig. 9. In the whole cell configuration, the transient shift of EGABA induced by the chloride flow combines with the gradual decline of gGABA to entail the decay of IGABA. The patch pipette solution does not instantaneously clamp but rather slowly buffers the [Cl-]i. In the gramicidin-perforated patch configuration, the physiological [Cl-]i remains unaltered, and the redistribution of chloride persists over the successive GABA ejections. As a result, the shift of EGABA leads to a tachyphylaxis phenomenon.


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Fig. 9.   Schematic interpretation of the contribution of shifts in EGABA to the fading of the IGABA. Decrease of IGABA in the presence of GABA is ascribed to a "true" desensitization, i.e., a fall of gGABA, associated with a shift of EGABA. Because IGABA = gGABA (membrane potential - EGABA) and EGABA = ECl, local variations of intracellular chloride concentration ([Cl-]i) due to the chloride flux are responsible for a transient reduction of the chloride driving force, leading to a fading of the current. In whole cell recordings, the chemical pipette/cell exchange results in a fast, but not instantaneous, buffering of [Cl-]i (shaded bands) so that repeated GABA applications elicit reproducible responses. In contrast, in gramicidin-perforated patch recordings, redistribution of chloride between intra- and extracellular compartments persists and can account for a tachyphylaxis phenomenon. This latter observation suggests that, in melanotrophs, [Cl-]i is not subject to a short-term regulation. ICl, chloride current.

Among these lines, it must be mentioned that, so far, no information is available concerning the regulation of [Cl-]i by pumps, channels, or any metabolically dependent active transport systems in melanotrophs. Nevertheless, because [Cl-]i is less than extracellular chloride concentration, mechanisms responsible for a net chloride extrusion may exist in these cells. In addition, in the present work, we have intentionally employed HEPES-buffered saline without CO2 equilibration precisely to eliminate any involvement of HCO-3 in the dynamics of the responses. Thus the shifts of EGABA can exclusively be attributed to chloride movements. However, in other studies using different cell types, GABAA anion channels were shown to display a permeability ratio of 0.2 to 0.3 for HCO-3 vs. chloride. In addition, it has also been reported that HCO-3 is involved in various transmembrane exchange systems (for review, see Ref. 14). Then, to study more accurately the physiological role of EGABA changes, further investigations need to be carried out using a preparation closer to the in vivo situation, i.e., in the presence of HCO-3 in the extracellular solution. A detailed comparison between HCO-3- buffered gramicidin-perforated patch recordings and the above-reported results would provide interesting information concerning the role of chloride, HCO-3, internal pH, and ion exchange systems in the homeostasis of the response to GABA in melanotrophs.

The regulations of IGABA reported herein have some functional significance. It is speculated that, in physiological conditions, the melanotrophs can be switched between excitatory and inhibitory modes of GABA signaling. For instance, the shift of EGABA could be considered as a negative feedback of the GABAergic inputs, regulating the amplitude of the response as a function of the intensity and duration of previous chloride fluxes.


    ACKNOWLEDGEMENTS

We thank Catherine Buquet for excellent technical assistance.


    FOOTNOTES

This research was supported by grants from Institut National de la Santé et de la Recherche Médicale (U 413), the European Union (Human Capital and Mobility Program; ERBCHRXCT920017), and the Conseil Régional de Haute-Normandie. F. Le Foll was a recipient of a doctoral fellowship from La Direction Générale de la Recherche et de la Technologie.

Present addresses: F. Le Foll, Laboratory of Ecotoxicology, University of Le Havre, 25 rue Philippe Lebon, 76058 Le Havre Cedex, France; O. Soriani: CNRS ERS 1253, Université de Nice Sophia-Antipolis, Laboratoire Jean Maetz, La Darse BP68, 06238 Villefranche-sur-Mer Cedex 1, France.

Present address and address for reprint requests and other correspondence: L. Cazin, Laboratoire de Microbiologie du Froid, UPRESS 2123, University of Rouen, 76821 Mont-Saint-Aignan, Cedex, France (E-mail: lionel.cazin{at}univ-rouen.fr).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Received 8 June 1999; accepted in final form 27 September 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Endocrinol Metab 278(3):E430-E443
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