Gramicidin-perforated patch revealed depolarizing effect of GABA in cultured frog melanotrophs

Abstract

1. In frog pituitary melanotrophs, GABA induces a transient stimulation followed by prolonged inhibition of hormone secretion. This biphasic effect is inconsistent with the elevation of cytosolic calcium and the inhibition of electrical activity also provoked by GABA in single melanotrophs. In the present study, standard patch-clamp configurations and gramicidin-perforated patches were used to investigate the physiological GABAA receptor-mediated response and intracellular chloride concentration ([Cl-]i) in cultured frog melanotrophs. 2. In the gramicidin-perforated patch configuration, 1 microM GABA caused a depolarization associated with an action potential discharge and a slight fall of membrane resistance. In contrast, at a higher concentration (10 microM) GABA elicited a depolarization accompanied by a transient volley of action potentials, followed by a sustained inhibitory plateau and a marked fall of membrane resistance. Isoguvacine mimicked the GABA-evoked responses, indicating a mediation by GABAA receptors. 3. In gramicidin-perforated cells, the depolarizing excitatory effect of 1 microM GABA was converted into a depolarizing inhibitory action when 0.4 microM allopregnanolone was added to the bath solution. 4. After gaining the whole-cell configuration, the amplitude and/or direction of the GABA-evoked current (IGABA) rapidly changed before stabilizing. After stabilization, the reversal potential of IGABA followed the values predicted by the Nernst equation for chloride ions when [Cl-]i was varied. 5. In gramicidin-perforated cells, the steady-state I-V relationships of 10 microM GABA- or isoguvacine-evoked currents yielded reversal potentials of -37.5 +/- 1.6 (n = 17) and -38.6 +/- 2.0 mV (n = 8), respectively. These values were close to those obtained by using a voltage-ramp protocol in the presence of Na+, K+ and Ca2+ channel blockers. The current evoked by 1 microM GABA also reversed at these potentials. 6. We conclude that, in frog pituitary melanotrophs, chloride is the exclusive charge carrier of IGABA. In intact cells, the reversal potential of IGABA is positive to the resting potential because of a relatively high [Cl-]i (26.5 mM). Under these conditions, GABA induces a chloride efflux responsible for a depolarization triggering action potentials. However, GABA at a high concentration or in the presence of the potentiating steroid allopregnanolone exerts a concomitant shunting effect leading to a rapid inhibition of the spontaneous firing.

Figure 1. Relationships between GABA-evoked voltage responses and membrane potential in cultured frog melanotrophs

A, voltage responses to GABA (10 μM) recorded in the whole-cell configuration from a single cell held at different membrane potentials as indicated beside the traces. Chloride concentration in the patch pipette solution was 6 mM (E_Cl= -75 mV). Applications of GABA (10 μM, 5 s duration, filled bar) hyperpolarized the cell at potentials more positive than -70 mV. At membrane potentials allowing spontaneous firing, GABA concomitantly inhibited the action potentials. B, amplitude of the GABA-evoked voltage responses shown in A, plotted against membrane potential. The deduced reversal potential (-72 mV) corresponded to E_Cl. C, voltage responses to GABA (10 μM) recorded in the gramicidin-perforated patch configuration from another melanotroph. Chloride concentration in the patch pipette solution was 115 mM (E_Cl= -0.6 mV). GABA was pressure-ejected at various membrane potentials as in A. GABA depolarized the cell at potentials more negative than -30 mV. At potentials where activity was infrequent or absent (-70 and -80 mV), GABA produced first a volley of action potentials followed by a sustained inhibitory plateau. D, V-V curve corresponding to the traces shown in C, displaying a reversal potential of -30 mV.

Figure 2. Effects of low GABA concentrations on the bioelectrical activity of frog melanotrophs with intact internal chloride concentration

GABA was pressure-ejected in the vicinity of the cells as indicated by the filled bars above the traces. A, recordings obtained from two distinct melanotrophs in the cell-attached configuration. Left, in a cell which did not exhibit spontaneous firing, repeated applications of 0.1 μM GABA elicited action current discharges. Right, in a spontaneously spiking cell, 0.1 μM GABA increased the action current frequency. Note the decrease of the action current amplitude during GABA exposure. B-D, effects of 1 μM GABA in three melanotrophs recorded in the gramicidin-perforated patch configuration. B, membrane potential was adjusted from the resting value (-49 mV, top trace) to -55 mV (middle trace) and -65 mV (bottom trace) in order to modify the firing rate of the cell. For each distinct discharge pattern, an increase in the spike frequency persisted throughout the application of GABA. C, fast sweep exhibiting a GABA (1 μM)-induced depolarization associated with a burst of action potentials. To hamper spontaneous spiking activity, the cell was hyperpolarized from resting potential (-47 mV) to -60 mV. D, the cell input resistance was monitored by hyperpolarizing pulses (-20 pA, 400 ms, 0.2 Hz) superimposed to the holding current. The GABA (1 μM)-evoked depolarization was accompanied by a slight fall in the membrane resistance.

Figure 3. Effects of high GABA concentrations on the bioelectrical activity of frog melanotrophs with intact internal chloride concentration

A, cell-attached recording obtained from a spontaneously spiking cell. GABA (10 μM, filled bar) provoked a reversible arrest of action current discharges. B-D, gramicidin-perforated patch recordings performed in three other melanotrophs. B, resting potential was -44 mV. At a concentration of 5 μM, GABA induced a marked depolarization associated with an arrest of firing. The fast sweep shows the initial depolarization-induced spike (arrow) preceding a sustained inhibitory plateau. C, resting potential was -49 mV. The cell input resistance was monitored by hyperpolarizing pulses (-20 pA, 400 ms, 0.2 Hz) superimposed to the holding current. Note that the depolarization induced by 5 μM GABA was accompanied by a dramatic drop in the membrane resistance. D, voltage and input resistance responses to 10 μM GABA at various membrane potentials in the presence of voltage-gated ion channel blockers. The bath solution contained (mM): 92 NaCl, 20 TEA-Cl, 15 Hepes, 2 CoCl₂ and 0.001 TTX. The pipette was filled with a solution containing (mM): 100 CsCl and 15 Hepes. During GABA applications the membrane resistance, monitored as in C, fell markedly to reach a level that did not appear to depend on the membrane potential.

Figure 4. Isoguvacine-evoked membrane potential and input resistance changes recorded from gramicidin-perforated patches in melanotrophs

Isoguvacine (1 or 10 μM) was administrated as indicated by the filled bars above the traces. A, the cell was hyperpolarized from resting potential (-54 mV) to -65 mV. Isoguvacine (1 μM) induced slight depolarizations accompanied by action potential discharges. B, the cell input resistance was monitored by hyperpolarizing pulses (-20 pA, 400 ms, 0.2 Hz) superimposed to the holding current. In the presence of isoguvacine (1 μM), spike discharges were evoked by the hyperpolarizing pulses without any apparent modification of the membrane resistance. C, recording obtained from another melanotroph. Resting potential was -41 mV. At a higher concentration (10 μM), isoguvacine induced a depolarization accompanied by an arrest of firing. D, the cell input resistance was monitored as in B. The depolarization induced by 10 μM isoguvacine was associated with a pronounced reduction in the membrane resistance.

Figure 5. Effect of allopregnanolone on the GABA-evoked voltage responses recorded in the gramicidin-perforated patch configuration in melanotrophs

GABA (1 μM) was pressure-ejected as indicated by the filled bars above the traces. A, resting potential was -54 mV. GABA provoked a transient increase in the action potential frequency (left). During bath perfusion with 0.4 μM allopregnanolone (open bar), GABA induced a depolarization associated with an early volley of action potentials, followed by an inhibitory plateau (middle). Recovery of the control response was obtained after washout (right). B, the cell was hyperpolarized from resting potential (-51 mV) to -63 mV, to hamper spontaneous action potentials. Hyperpolarizing pulses (-6 pA, 400 ms, 0.2 Hz) were superimposed to the holding current to monitor the cell input resistance. A fast sweep is presented below each trace in order to detail both the evoked action potentials and the voltage jumps (arrows) caused by the hyperpolarizing current pulses. GABA induced a clear-cut depolarization accompanied by a burst of action potentials (left). The membrane resistance fell from 4.5 GΩ in control to 2.1 GΩ during GABA administration. In the presence of allopregnanolone (0.4 μM, open bar), the GABA-evoked depolarization triggered a brief flurry of action potentials followed by a complete arrest of firing (middle). Membrane resistance throughout depolarization was 0.6 GΩ. After washout, recovery of the control response was obtained (right).

Figure 6. Currents evoked by GABA_A receptor agonists in the whole-cell and gramicidin-perforated patch configurations

A, time course of the GABA-evoked current amplitude within the first seconds following whole-cell access. Chloride concentration in the patch pipette solution was 6 mM (E_Cl= -75 mV). Left, the arrow indicates the onset of whole-cell access. Holding potential was -40 mV. Repeated 3 s pulses of GABA (10 μM, filled bar) were delivered 1, 6, 11, 16 and 21 s after membrane rupture. Note that the first pulse of GABA induced an inward current while the following pulses generated outward currents. Right, time course of the GABA-evoked current amplitude in three cells clamped at holding potentials of -80 (○), -40 (□) or -35 mV (▵). The data were fitted to monoexponential functions yielding time constants of 5.2, 4.6 and 4.3 s, respectively. Note that the initial GABA-evoked current was inwardly directed at -80 and -40 mV, whilst it was outwardly directed at -35 mV. B, chloride concentration in the patch pipette solution was 115 mM (E_Cl= -0.6 mV). Isoguvacine (10 μM) was pressure-ejected (5 s, filled bar) in the vicinity of a melanotroph clamped at -20 (top traces) or -50 mV (bottom traces). The perforated patch recording (left) was subsequently converted into a whole-cell recording (right) by a suction pulse-induced rupture of the underlying plasma membrane. Isoguvacine-induced currents were studied within the first minute after gaining whole-cell access to avoid gramicidin perforation of the whole-cell membrane. In the gramicidin-perforated patch recording, the currents evoked by isoguvacine at -20 and -50 mV were opposite, whilst in the whole-cell configuration they both remained inwardly directed.

Figure 7. Steady-state I-V relationships of currents evoked by GABA_A receptor agonists in melanotrophs

A, I-V relationships of GABA-evoked currents recorded in the whole-cell configuration. Chloride concentration in the patch pipette was 6 mM (E_Cl= -75 mV). The amplitude of the currents evoked by GABA (10 μM) at potentials varying from -80 to +80 mV by 20 mV steps (inset) was measured at the peak and plotted against the membrane potential. The curve displayed an outward rectification. Reversal potential (-71 mV) was very close to E_Cl. B, chloride dependence of the reversal potential of the current evoked by GABA. Intracellular chloride was varied by partial replacement of KCl with potassium glutamate. All reversal potentials were determined by interpolation from I-V relationships of the type illustrated in A. Data points are means ±s.e.m. and values indicated in parentheses are numbers of melanotrophs examined. The straight line, fitted to the data by linear regression, has a slope of 56.3 mV. The dotted line corresponds to E_Cl values calculated from the Nernst equation. C, I-V relationship of the GABA-evoked current recorded in the gramicidin-perforated patch configuration. Left, family of currents evoked by GABA as in A. Right, the corresponding I-V curve yielded a reversal potential of -39 mV. D, same as in C, GABA being replaced by isoguvacine (10 μM). The deduced reversal potential was -42 mV.

Figure 8. Instantaneous I-V relationships of currents evoked by GABA recorded in the gramicidin-perforated patch configuration in melanotrophs

The bath solution contained (mM): 92 NaCl, 20 TEA-Cl, 15 Hepes, 2 CoCl₂ and 0.001 TTX. The pipette was filled with a solution containing (mM): 100 CsCl and 15 Hepes. The cells were submitted to depolarizing voltage ramps (150 mV s⁻¹) from -80 to +80 mV. The instantaneous current recorded in the absence of the agonist was subtracted from the current obtained in the presence of 1 (A) or 10 μM (B) GABA. Reversal potentials were measured as the x-intercept value of the resulting current. The deduced reversal potentials were -32 (A) and -35 mV (B) for 1 and 10 μM GABA, respectively.