Reduction of tyrosine kinase activity and protein tyrosine dephosphorylation by anoxic stimulation in vitro.

Tyrosine-specific protein phosphorylation has been recently implicated in mediating pathological changes associated with cerebral ischemia. In the present study, acute hypoxia/ischemia (anoxia) was simulated in vitro by incubating rat hippocampal slices in glucose-free artificial cerebrospinal fluid saturated with 95% N2/5% CO2. A marked decrease in the level of tyrosine phosphorylation of many protein bands compared with the control was observed. Immunoprecipitation and western blot confirmed that the NR2A/2B subunits of the N-methyl-D-aspartate receptors are among the dephosphorylated proteins. Maximal dephosphorylation of bands corresponding to NR2A/2B was reached after 10 min, and no recovery was observed even after 1 h in normal, oxygenated artificial cerebrospinal fluid. The effect was partially blocked by dephostatin, a membrane-permeable inhibitor of protein tyrosine phosphatases, but was not affected by the presence of glutamate receptor antagonists, or by removing extracellular Ca2+ or chelating intracellular Ca2+. Enzyme assay showed that anoxic stimulation resulted in a selective reduction in protein tyrosine kinase activity without affecting protein tyrosine phosphatase activity. Thus the present work suggests that anoxic stimulation produces a selective inhibition of protein tyrosine kinase activity leading to tyrosine-dephosphorylation of several proteins including the N-methyl-D-aspartate receptors. The underlying mechanism may involve a novel signal transduction pathway, which may protect neurons from degeneration during ischemic stress.

Recruitment of functional GABA(A) receptors to postsynaptic domains by insulin.

Modification of synaptic strength in the mammalian central nervous system (CNS) occurs at both pre- and postsynaptic sites. However, because postsynaptic receptors are likely to be saturated by released transmitter, an increase in the number of active postsynaptic receptors may be a more efficient way of strengthening synaptic efficacy. But there has been no evidence for a rapid recruitment of neurotransmitter receptors to the postsynaptic membrane in the CNS. Here we report that insulin causes the type A gamma-aminobutyric acid (GABA[A]) receptor, the principal receptor that mediates synaptic inhibition in the CNS, to translocate rapidly from the intracellular compartment to the plasma membrane in transfected HEK 293 cells, and that this relocation requires the beta2 subunit of the GABA(A) receptor. In CNS neurons, insulin increases the expression of GABA(A) receptors on the postsynaptic and dendritic membranes. We found that insulin increases the number of functional postsynaptic GABA(A) receptors, thereby increasing the amplitude of the GABA(A)-receptor-mediated miniature inhibitory postsynaptic currents (mIPSCs) without altering their time course. These results provide evidence for a rapid recruitment of functional receptors to the postsynaptic plasma membrane, suggesting a fundamental mechanism for the generation of synaptic plasticity.

Modulation of GABAA receptor function by tyrosine phosphorylation of beta subunits.

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 (GABAA) receptors in cultured CNS neurons. Extracellular application of genistein (50 microM), a membrane-permeable inhibitor of protein tyrosine kinases (PTKs), produced a reversible reduction in the amplitude of GABAA receptor-mediated whole-cell currents, and this effect was not reproduced by daidzein (50 microM), 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 beta2/beta3 subunit(s) of the GABAA receptor are tyrosine phosphorylated in situ. Moreover, genistein (50 microM) was found to be capable of decreasing GABAA currents in human embryonic kidney 293 cells transiently expressing functional GABAA receptors containing the beta2 subunit. Thus, the present work provides the first evidence that native GABAA receptors are phosphorylated and modulated in situ by endogenous PTKs in cultured CNS neurons and that phosphorylation of the beta subunits may be sufficient to support such a modulation. Given the prominent role of GABAA 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.