Transmembrane AMPA Receptor Regulatory Proteins and Cornichon-2 Allosterically Regulate AMPA Receptor Antagonists and Potentiators

Douglas A Schober,Martin B Gill,Hong Yu,Douglas L Gernert,Matthew W Jeffries,Paul L Ornstein,Akihiko S Kato,Christian C Felder,David S Bredt

doi:10.1074/jbc.m110.212522

Abstract

AMPA receptors mediate fast excitatory transmission in the brain. Neuronal AMPA receptors comprise GluA pore-forming principal subunits and can associate with multiple modulatory components, including transmembrane AMPA receptor regulatory proteins (TARPs) and CNIHs (cornichons). AMPA receptor potentiators and non-competitive antagonists represent potential targets for a variety of neuropsychiatric disorders. Previous studies showed that the AMPA receptor antagonist GYKI-53655 displaces binding of a potentiator from brain receptors but not from recombinant GluA subunits. Here, we asked whether AMPA receptor modulatory subunits might resolve this discrepancy. We find that the cerebellar TARP, stargazin (γ-2), enhances the binding affinity of the AMPA receptor potentiator [(3)H]-LY450295 and confers sensitivity to displacement by non-competitive antagonists. In cerebellar membranes from stargazer mice, [(3)H]-LY450295 binding is reduced and relatively resistant to displacement by non-competitive antagonists. Coexpression of AMPA receptors with CNIH-2, which is expressed in the hippocampus and at low levels in the cerebellar Purkinje neurons, confers partial sensitivity of [(3)H]-LY450295 potentiator binding to displacement by non-competitive antagonists. Autoradiography of [(3)H]-LY450295 binding to stargazer and γ-8-deficient mouse brain sections, demonstrates that TARPs regulate the pharmacology of allosteric AMPA potentiators and antagonists in the cerebellum and hippocampus, respectively. These studies demonstrate that accessory proteins define AMPA receptor pharmacology by functionally linking allosteric AMPA receptor potentiator and antagonist sites.

Highlights

Ficking [6] and modulate channel gating [7,8,9,10]
Using GluA2 because of its predominant incorporation into cerebellar neuronal AMPA receptors [37], we observed that GYKI-53655 readily displaced the AMPA potentiator, [3H]-LY450295, from cerebellar membranes but did not displace [3H]-LY450295 binding from transfected GluA2 membranes (Fig. 1A)
The principal finding of this study is that transmembrane AMPA receptor regulatory proteins (TARPs) subunits functionally link potentiator and antagonist binding sites within AMPA receptors

Summary

To whom correspondence should be addressed

Ficking [6] and modulate channel gating [7,8,9,10]. Neuronal AMPA receptors may associate with other transmembrane proteins, including CNIH (cornichon) proteins and CKAMP44, which can further modulate receptor trafficking and channel function [11,12,13,14]. Multiple classes of AMPA receptor antagonists have been identified Antagonists, such as 6-cyano7-nitroquinoxaline-2,3-dione, compete with glutamate at the agonist binding site and occlude channel opening [29]. GYKI-53655 potently blocks binding of [3H]-LY395153 to AMPA receptors in brain membranes but does not affect binding to recombinant receptors [35] We addressed this discrepancy and discovered an unexpected role for AMPA receptor accessory proteins in functionally linking AMPA receptor potentiator and non-competitive antagonist binding sites. These results provide valuable insights for understanding and refining the neuropharmacology of AMPA receptors

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION