Modulation of GABA_AReceptor 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 M), 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 M), 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 ␤2/␤3 subunit(s) of the GABA A receptor are tyrosine phosphorylated in situ. Moreover, genistein (50 M) was found to be capable of decreasing GABA A currents in human embryonic kidney 293 cells transiently expressing functional GABA A receptors containing the ␤2 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 ␤ 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.

Phosphatase inhibitors remove the run-down of γ-aminobutyric acid type A receptors in the human epileptic brain

“…Such process may be positively modulated by inhibition of protein phosphatases. For instance, okadaic acid inhibition of protein phosphatases 2A results in hyperphosphorylation of the ␤3 subunit, yielding an enhanced activity of the GABA A -receptor (26,27), and orthovanadate inhibition of tyrosine phosphatase reduces GABA-current run-down acting at tyrosine kinasedependent sites in ␤ subunits (24,26,34).…”

Ischemic Insults Direct Glutamate Receptor Subunit 2-Lacking AMPA Receptors to Synaptic Sites

“…Coimmunoprecipitation and immunoblotting assays were performed as described previously (Wan et al, 1997a;. Homogenates from control and OGD-treated hippocampal cultures were incubated with anti-adaptin ␤ 2 antibody (Sigma) or GluR2 (Chemicon) in 500 l of 50 mM Tris-HCl, 150 mM NaCl, and 0.1% Triton X-100 for 4 h at 4°C.…”

Evidence for a Functional Interaction between the ClC-2 Chloride Channel and the Retrograde Motor Dynein Complex

“…Co-immunoprecipitation-Hippocampal slices (300-m thickness) were prepared from adult male rats (Sprague-Dawley, 150 -200 g; Charles River) as described previously (13). A crude membrane preparation was obtained from tissue homogenate (in PBS with protease inhibitors by centrifugation at 100,000 ϫ g for 1 h following a 10-min low speed spin at 2,000 ϫ g to pellet nuclei and unlysed cells.…”