Pannexin-1 (Px1) is expressed at postsynaptic sites in pyramidal neurons, suggesting that these hemichannels contribute to dendritic signals associated with synaptic function. We found that, in pyramidal neurons, N -methyl- d -aspartate receptor (NMDAR) activation induced a secondary prolonged current and dye flux that were blocked with a specific inhibitory peptide against Px1 hemichannels; knockdown of Px1 by RNA interference blocked the current in cultured neurons. Enhancing endogenous NMDAR activation in brain slices by removing external magnesium ions (Mg ²⁺ ) triggered epileptiform activity, which had decreased spike amplitude and prolonged interburst interval during application of the Px1 hemichannel blocking peptide. We conclude that Px1 hemichannel opening is triggered by NMDAR stimulation and can contribute to epileptiform seizure activity.

The tyrosine kinase Pyk2 plays a unique role in intracellular signal transduction by linking Ca²⁺influx to tyrosine phosphorylation, but the molecular mechanism of Pyk2 activation is unknown. We report that Pyk2 oligomerization by antibodiesin vitroor overexpression of PSD-95 in PC6-3 cells inducestrans-autophosphorylation of Tyr402, the first step in Pyk2 activation. In neurons, Ca²⁺influx through NMDA-type glutamate receptors causes postsynaptic clustering and autophosphorylation of endogenous Pyk2 via Ca²⁺- and calmodulin-stimulated binding to PSD-95. Accordingly, Ca²⁺influx promotes oligomerization and thereby autoactivation of Pyk2 by stimulating its interaction with PSD-95. We show that this mechanism of Pyk2 activation is critical for long-term potentiation in the hippocampus CA1 region, which is thought to underlie learning and memory.

At CA1 synapses, activation of NMDA receptors (NMDARs) is required for the induction of both long-term potentiation and depression. The basal level of activity of these receptors is controlled by converging cell signals from G-protein-coupled receptors and receptor tyrosine kinases. Pituitary adenylate cyclase activating peptide (PACAP) is implicated in the regulation of synaptic plasticity because it enhances NMDAR responses by stimulating Gα_s-coupled receptors and protein kinase A (Yaka et al., 2003). However, the major hippocampal PACAP1 receptor (PAC₁R) also signals via Gα_qsubunits and protein kinase C (PKC). In CA1 neurons, we showed that PACAP38 (1 nm) enhanced synaptic NMDA, and evoked NMDAR, currents in isolated CA1 neurons via activation of the PAC₁R, Gα_q, and PKC. The signaling was blocked by intracellular applications of the Src inhibitory peptide Src(40-58). Immunoblots confirmed that PACAP38 biochemically activates Src. A Gα_qpathway is responsible for this Src-dependent PACAP enhancement because it was attenuated in mice lacking expression of phospholipase C β1, it was blocked by preventing elevations in intracellular Ca²⁺, and it was eliminated by inhibiting either PKC or cell adhesion kinase β [CAKβ or Pyk2 (proline rich tyrosine kinase 2)]. Peptides that mimic the binding sites for either Fyn or Src on receptor for activated C kinase-1 (RACK1) also enhanced NMDAR in CA1 neurons, but their effects were blocked by Src(40-58), implying that Src is the ultimate regulator of NMDARs. RACK1 serves as a hub for PKC, Fyn, and Src and facilitates the regulation of basal NMDAR activity in CA1 hippocampal neurons.