Abstract
Protein kinase A (PKA) enhances synaptic plasticity in the central nervous system by increasing NMDA receptor current amplitude and Ca(2+) flux in an isoform-dependent yet poorly understood manner. PKA phosphorylates multiple residues on GluN1, GluN2A, and GluN2B subunits in vivo, but the functional significance of this multiplicity is unknown. We examined gating and permeation properties of recombinant NMDA receptor isoforms and of receptors with altered C-terminal domain (CTDs) prior to and after pharmacological inhibition of PKA. We found that PKA inhibition decreased GluN1/GluN2B but not GluN1/GluN2A gating; this effect was due to slower rates for receptor activation and resensitization and was mediated exclusively by the GluN2B CTD. In contrast, PKA inhibition reduced NMDA receptor-relative Ca(2+) permeability (PCa/PNa) regardless of the GluN2 isoform and required the GluN1 CTD; this effect was due primarily to decreased unitary Ca(2+) conductance, because neither Na(+) conductance nor Ca(2+)-dependent block was altered substantially. Finally, we show that both the gating and permeation effects can be reproduced by changing the phosphorylation state of a single residue: GluN2B Ser-1166 and GluN1 Ser-897, respectively. We conclude that PKA effects on NMDA receptor gating and Ca(2+) permeability rely on distinct phosphorylation sites located on the CTD of GluN2B and GluN1 subunits. This separate control of NMDA receptor properties by PKA may account for the specific effects of PKA on plasticity during synaptic development and may lead to drugs targeted to alter NMDA receptor gating or Ca(2+) permeability.
Highlights
Protein kinase A (PKA) increases NMDA receptor responses and phosphorylates multiple residues on C-terminal domains (CTD)
We asked whether bath-applied myristoylated Protein kinase A inhibitor (PKI)(14 –22) (myr-PKI) produced effects that were similar to those produced by intracellularly applied myr-PKI and whether this modulation occurred in the modified conditions usually employed for NMDA receptor gating measurements, which require low concentrations of protons and divalent cations (Ca2ϩ and Mg2ϩ) [35]
We observed that peak current amplitudes recorded immediately after break-in were stable when recorded with regular intracellular solutions but declined within minutes when the intracellular solution contained myr-PKI or non-myristoylated PKI(6 –22) (Fig. 1B)
Summary
PKA increases NMDA receptor responses and phosphorylates multiple residues on C-terminal domains (CTD). We conclude that PKA effects on NMDA receptor gating and Ca2؉ permeability rely on distinct phosphorylation sites located on the CTD of GluN2B and GluN1 subunits. PKA inhibition has specific effects on N2A- and N2B-containing receptors; it decreases both Ca2ϩ influx and macroscopic current amplitude in N2B-containing receptors, but it reduces Ca2ϩ flux without changing the macroscopic traces of N2A-containing receptors [13] Consistent with these observations, PKA phosphorylation of NMDA receptors can alter the size and threshold of synaptic plasticity (13, 18, 20 –22). We found that PKA activity influenced gating and Ca2ϩ permeability of NMDA receptors by separate mechanisms, and these effects were mediated by sites located on N2B and N1 subunits, respectively
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