PKC phosphorylates GluA1-Ser831 to enhance AMPA receptor conductance

Abstract

AMPA receptors mediate fast excitatory synaptic transmission in the brain, and are dynamically regulated by phosphorylation of multiple residues within the C-terminal domain. CaMKII phosphorylates Ser831 within the AMPA receptor GluA1 subunit to increase single channel conductance, and biochemical studies show that PKC can also phosphorylate this residue. In light of the discovery of additional PKC phosphorylation sites within the GluA1 C-terminus, it remains unclear whether PKC phosphorylation of Ser831 increases GluA1 conductance in intact receptors. Here, we report that the purified, catalytic subunit of PKC significantly increases the conductance of wild-type GluA1 AMPA receptors expressed in the presence of stargazin in HEK293T cells. Furthermore, the mutation GluA1-S831A blocks the functional effect of PKC. These findings suggest that GluA1 AMPA receptor conductance can be increased by activated CaMKII or PKC, and that phosphorylation at this site provides a mechanism for channel modulation via a variety of protein signaling cascades.

Keywords: AMPA receptor; GluA1; GluR1; PKC; phosphorylation.

Figure 1. PKC increases γ_MEAN of homomeric GluA1-L497Y AMPA receptors. (A) Representative macroscopic current response to 1 mM glutamate in an excised outside-out patch recorded under voltage clamp (V_HOLD = -60 mV). The upper trace shows the response after high pass filtering, illustrating the increase in membrane current noise during the response time course. (B) Current-variance relationship used to determine γ_MEAN for the trace shown in (A). (C) Summary of PKC effects on γ_MEAN of GluA1-LY AMPA receptors expressed in the presence of stargazin in HEK293T cells. *p < 0.001 by Student’s t-test. Data are from 8–14 outside-out patches for each condition.

Figure 2. Scaffolding proteins mediate PKC-dependent vs. CaMKII-dependent GluA1-Ser831 phosphorylation. (A) AKAP79 localizes PKA and calcineurin (PP2B) to the GluA1 C-terminus via SAP97 to allow phosphorylation and dephosphorylation of Ser845 by PKA and PP2B, respectively.^, AKAP79, with SAP97, also promote phosphorylation of GluA1 at Ser831 by PKC. (B) When CaMKII is activated by autophosphorylation, it binds and phosphorylates SAP97, which disrupts phosphorylation of GluA1 by PKC and dephosphorylation of GluA1-Ser845 by PP2B. Phosphorylation at Ser831 is then mediated primarily by CaMKII rather than PKC.^, Our results suggest that phosphorylation of GluA1-Ser831 by either PKC or CaMKII can increase AMPA receptor conductance.