Modulation of GABA(A) receptor function by tyrosine phosphorylation of β subunits

Abstract

Protein tyrosine phosphorylation is a key event in diverse intracellular signaling pathways and has been implicated in modification of neuronal functioning. We investigated the role of tyrosine phosphorylation in regulating type A GABA (GABA(A)) receptors in cultured CNS neurons. Extracellular application of genistein (50 μM), a membrane-permeable inhibitor of protein tyrosine kinases (PTKs), produced a reversible reduction in the amplitude of GABA(A) receptor-mediated whole-cell currents, and this effect was not reproduced by daidzein (50 μM), an inactive analog of genistein. In contrast, intracellular application of the PTK pp60(c-src) (30 U/ml) resulted in a progressive increase in current amplitude, and this potentiation was prevented by pretreatment of the neurons with genistein. Immunoprecipitation and immunoblotting of cultured neuronal homogenates indicated that the β2/β3 subunit(s) of the GABA(A) receptor are tyrosine phosphorylated in situ. Moreover, genistein (50 μM) was found to be capable of decreasing GABA(A) currents in human embryonic kidney 293 cells transiently expressing functional GABA(A) receptors containing the β2 subunit. Thus, the present work provides the first evidence that native GABA(A) receptors are phosphorylated and modulated in situ by endogenous PTKs in cultured CNS neurons and that phosphorylation of the β subunits may be sufficient to support such a modulation. Given the prominent role of GABA(A) receptors in mediating many brain functions and dysfunctions, modulation of these receptors by PTKs may be important in a wide range of physiological and pathological processes in the CNS.