Regulation of GABA A Receptor by Protein Tyrosine Kinases in Frog Pituitary Melanotrophs

The effects of protein tyrosine kinase (PTK) and PTK inhibitors on the GABA A receptor function were studied in cultured frog pituitary melanotrophs by using the patch‐clamp technique. Extracellular application of the PTK inhibitors genistein (10 −9 to 10 −5  M) or lavendustin A (10 −12 to 10 −7  M) provoked a bell‐shaped potentiation of the whole‐cell current induced by GABA (3×10 −6  M). In contrast, at high concentrations, genistein (10 −4  M) and lavendustin A (10 −5  M) reversibly reduced the GABA‐evoked current. Daidzein and lavendustin B, the inactive analogs of genistein and lavendustin A, respectively, did not modify the current induced by GABA. In the inside‐out configuration, bath application of the recombinant PTK pp60 c–src (75 U/ml) inhibited the GABA‐activated chloride current, and the inhibitory effect of pp60 c–src was prevented by genistein (10 −7  M). Immunoblotting revealed that genistein, at doses of 10 −7  M or 10 −4  M, markedly inhibited tyrosine phosphorylation of the β 2/ β 3 subunits of the GABA A receptor. Extracellular application of the PKA activator Bt 2 cAMP (10 −3  M), the PKA/PKC inhibitor H7 (10 −5  M) and the Cam KII inhibitor W7 (10 −5  M) reversibly diminished the whole‐cell GABA‐induced current. Internal application of H7 and W7 (10 −4  M) did not modify the dose‐dependent effects of genistein. Internal application of sodium orthovanadate (10 −4  M), a protein tyrosine phosphatase inhibitor, decreased the GABA‐evoked current and markedly reduced the potentiating effect of genistein. The present study provides the first evidence that, in frog pituitary melanotrophs, the GABA A receptor is phosphorylated at least on its β 2/ β 3 subunits by an endogenous PTK. Our data also demonstrate that tyrosine phosphorylation exerts an inhibitory effect on GABA A receptor function.