Phosphatidylinositol-4,5-bisphosphate (PIP2) has been shown to regulate many ion channels, transporters, and other signaling proteins, but it is not known whether it also regulates neurotransmitter-gated channels. The NMDA receptors (NMDARs) are gated by glutamate and serve as a critical control point in synaptic function. Here we demonstrate that PIP2 supports NMDAR activity. In Xenopus oocytes, overexpression of phospholipase Cgamma (PLCgamma) or preincubation with 10 microm wortmannin markedly reduced NMDA currents. Stimulation of the epidermal growth factor receptor (EGFR) promoted the formation of an immunocomplex between PLCgamma and NMDAR subunits. Stimulation of EGFR or the PLCbeta-coupled M1 acetylcholine receptor produced a robust transient inhibition of NMDA currents. Wortmannin application blocked the recovery of NMDA currents from the inhibition. Using mutagenesis, we identified the structural elements on NMDAR intracellular tails that transduce the receptor-mediated inhibition, which pinpoint to the binding site for the cytoskeletal protein alpha-actinin. Mutation of the PIP2-binding residues of alpha-actinin dramatically reduced NMDA currents and occluded the effect of EGF. Interestingly, EGF or wortmannin affected the interaction between NMDAR subunits and alpha-actinin, suggesting that this protein mediates the effect of PIP2 on NMDARs. In mature hippocampal neurons, expression of the mutant alpha-actinin reduced NMDA currents and accelerated inactivation. We propose a model in which alpha-actinin supports NMDAR activity via tethering their intracellular tails to plasma membrane PIP2. Thus, our results extend the influence of PIP2 to the NMDA ionotropic glutamate receptors and introduce a novel mechanism of "indirect" regulation of transmembrane protein activity by PIP2.