The organization and regulation of excitatory synapses in the mammalian CNS entails complex molecular and cellular processes. In the postsynaptic membrane, scaffolding proteins bring together glutamate receptors with multiple regulatory proteins involved in signal transduction. This gives rise to an elaborate postsynaptic structure known as the postsynaptic density (PSD). This protein network plays a critical role in the regulation of glutamate receptor function and thus in synaptic plasticity. To study this regulation, we have developed a system in which ionotropic glutamate receptors (iGluRs) can be recorded, in the steady state, by the patch clamp technique in isolated PSDs incorporated into giant liposomes. In this preparation, ionotropic glutamate receptors maintain their characteristic physiological and pharmacological properties. The recordings reflect the presence of channel clusters, as multiple conductance and subconductance states are observed. Each of the receptor subtypes is activated by a specific set of kinases that are activated differentially by Ca(2+): the "kainate receptor kinases" are active even in the presence of EGTA, i.e. they are not calcium-dependent; the "N-methyl-D-aspartate receptor (NMDAR) channel kinases" are active in the presence of submicromolar calcium concentrations, whereas the "alpha-amino-3- hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor kinases" need microM calcium for activation. The NMDA receptor showed its characteristic voltage-dependent Mg(2+) blockade, and activation by phosphorylation was in part a consequence of a relief of Mg(2+) blockade. These results allow us to propose a model in which phosphorylation of NMDA receptors can contribute to a long-lasting and self-maintained change in synaptic function. The experimental approach we present will allow us to test the functional consequence of activation of the multiple signal transduction pathways thought to regulate excitatory neurotransmission in the adult CNS.