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.

Correspondence should be addressed to Y. T.W. (y.t.wang@utoronto.ca) Melatonin, a hormone principally produced and released by the pineal gland, has been shown to regulate a variety of biological functions including circadian rhythms, sleep-wake cycles and reproduction 1 , presumably through activating high-affinity G-protein-coupled receptors 2-5 . We report here that these subtypes can differentially modulate the function of type-A g-aminobutyric acid (GABA A ) receptor, the principal neurotransmitter receptor mediating synaptic inhibition in the CNS 6,7 . This work demonstrates that melatonin, through activation of different receptor subtypes, can exert opposite effects on the same substrate, suggesting that receptor subtype is the primary molecular basis for the diversity of melatonin effects.Two mammalian melatonin receptor subtypes, Mel 1a and Mel 1b , have been cloned 2-4 , but, because of a lack of subtype-specific agonists and antagonists, it has proven difficult to assign specific roles to each subtype 2,5 . Probably the best-characterized actions of melatonin in

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.