SMALL-CONDUCTANCE CL- CHANNELS IN RABBIT PARIETAL-CELLS ACTIVATED BY PROSTAGLANDIN-E2 AND INHIBITED BY GTP-GAMMA-S

1. Small-conductance, sub-picosiemens (sub-pS) Cl- channels in the basolateral membrane of non-stimulated parietal cells in isolated rabbit gastric glands were studied by whole-cell patch-clamp and noise analysis techniques. 2. Voltage-independent whole-cell Cl- currents were recorded from parietal cells equilibrated with Cl--containing solutions. Intracellular application of guanosine-5-'O-(3-thiotriphosphate) (GTPgammaS, 5-200 mum) slowly decreased the whole-cell Cl- current, and its steady-state effect was observed about 6 min after the start of the dialysis. The half-maximal inhibitory concentration of GTPgammaS was about 60 mum. 3. The single Cl- channel conductance was estimated to be 0.37 pS from the variance noise analysis during the GTPgammaS-induced inhibitory process of the whole-cell Cl- current. It is in agreement with the value obtained by a method of power spectrum analysis (0.47 ps). 4. The whole-cell Cl- current was increased by prostaglandin E2 (10 mum). The increased Cl- current was reduced by the subsequent application of GTPgammaS (50 mum), whereas the GTPgammaS (50 muM)-induced inhibition of the Cl- current was not reversed by the subsequent application of prostaglandin E2 (10 muM). 5. The combined intracellular application of GTPgammaS (50 mum) and GDPbetaS (500 mum) markedly reduced the inhibitory effect of GTPgammaS, indicating that a GTP-binding protein is involved in the regulation of the Cl- channel. 6. Treatment of parietal cells with pertussis toxin (PTX, 500 ng/ml) for 90-140 min did not affect the GTPgammaS-induced inhibition of the whole-cell Cl- current. 7. Intracellular application of cyclic AMP-dependent protein kinase inhibitor peptide (100 mug/ml) or 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) (5 mm, pCa 8) did not affect the GTPgammaS-induced inhibition of the whole-cell Cl- current. 8. The present study has suggested that the opening of the sub-pS Cl- channel is modulated negatively by a PTX-insensitive GTP-binding protein and positively by prostaglandin E2.