Modulation of NMDA receptors by pituitary adenylate cyclase activating peptide in CA1 neurons requires G alpha q, protein kinase C, and activation of Src.

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 Galphas-coupled receptors and protein kinase A (Yaka et al., 2003). However, the major hippocampal PACAP1 receptor (PAC1R) also signals via Galphaq subunits 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, Galphaq, 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 Galphaq pathway is responsible for this Src-dependent PACAP enhancement because it was attenuated in mice lacking expression of phospholipase C beta1, it was blocked by preventing elevations in intracellular Ca2+, and it was eliminated by inhibiting either PKC or cell adhesion kinase beta [CAKbeta 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.

State-dependent barium block of wild-type and inactivation-deficient HERG channels in Xenopus oocytes.

The effects of Ba2+ on current resulting from the heterologous expression of the human ether-à-go-go related gene (HERG) (IHERG) was studied with two-electrode voltage clamp techniques in Xenopus oocytes. Ba2+ produced time- and voltage-dependent block of IHERG. Significant inhibition was seen at concentrations as low as 1 microM. Inhibition was greatest at step potentials between -40 and 0 mV; at more positive potentials, inhibition decreased in association with time-dependent unblocking of channels. An inactivation-attenuated mutant of HERG (S631A) was prepared and expressed in Xenopus oocytes. Ba2+ block of S631A differed from that of HERG in that extensive unblocking was no longer seen at positive potentials and the voltage dependence of step current block was greatly attenuated. A mathematical model was applied to analyse quantitatively the inhibitory effects of Ba2+ on IHERG. The model suggested similar voltage-dependent affinity of Ba2+ for the open and closed states, along with absence of binding to the inactivated state, and accounted well for Ba2+ effects on both wild-type and S631A channels. We conclude that Ba2+ potently inhibits IHERG in a characteristic state-dependent fashion, with strong unblocking at positive potentials related to the presence of an intact C-type inactivation mechanism.