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

Frank Le Foll, Hélène Castel, Olivier Soriani, Hubert Vaudry and Lionel Cazin

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 GABA_A receptor-mediated response and intracellular chloride concentration ([Cl^-]_i) in cultured frog melanotrophs.

2. In the gramicidin-perforated patch configuration, 1 µM GABA caused a depolarization associated with an action potential discharge and a slight fall of membrane resistance. In contrast, at a higher concentration (10 µM) 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 GABA_A receptors.

3. In gramicidin-perforated cells, the depolarizing excitatory effect of 1 µM GABA was converted into a depolarizing inhibitory action when 0·4 µM allopregnanolone was added to the bath solution.

4. After gaining the whole-cell configuration, the amplitude and/or direction of the GABA-evoked current (I_GABA) rapidly changed before stabilizing. After stabilization, the reversal potential of I_GABA 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 µM 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 µM GABA also reversed at these potentials.

6. We conclude that, in frog pituitary melanotrophs, chloride is the exclusive charge carrier of I_GABA. In intact cells, the reversal potential of I_GABA 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.

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