Structural and Functional Analysis of Two New Positive Allosteric Modulators of GluA2 Desensitization and Deactivation

Abstract

At the dimer interface of the extracellular ligand-binding domain of ␣-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors a hydrophilic pocket is formed that is known to interact with two classes of positive allosteric modulators, represented by cyclothiazide and the ampakine 2H,3H,6aH-pyrrolidino(2,1-3Ј,2Ј)1,3-oxazino(6Ј,5Ј-5,4)benzo(e)1,4-dioxan-10-one (CX614). Here, we present structural and functional data on two new positive allosteric modulators of AMPA receptors, phenyl-1,4-bisalkylsulfonamide (CMPDA) and phenyl-1,4-bis-carboxythiophene (CMPDB). Crystallographic data show that these compounds bind within the modulator-binding pocket and that substituents of each compound overlap with distinct moieties of cyclothiazide and CX614. The goals of the present study were to determine 1) the degree of modulation by CMPDA and CMPDB of AMPA receptor deactivation and desensitization; 2) whether these compounds are splice isoform-selective; and 3) whether predictions of mechanism of action could be inferred by comparing molecular interactions between the ligand-binding domain and each compound with those of cyclothiazide and CX614. CMPDB was found to be more isoform-selective than would be predicted from initial binding assays. It is noteworthy that these new compounds are both more potent and more effective and may be more clinically relevant than the AMPA receptor modulators described previously.

A Charge-inverting Mutation in the “Linker” Region of α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptors Alters Agonist Binding and Gating Kinetics Independently of Allosteric Modulators

“…To address whether the addition of the fluorophore impacted our results, we compared the kinetics of the fusion protein with that of untagged recombinant receptors and concluded that the addition of the C-terminal fluorophore to GluA2 did not alter receptor kinetics. Specifically, the time constants for deactivation and desensitization of the WT receptor, 0.79 and 1.64 ms, respectively, are very consistent with time constants reported in other published reports (our own and others) using untagged receptors with deactivation kinetics in those reports ranging from 0.71 to 0.92 ms and desensitization kinetics ranging from 1.18 to 1.87 ms (3,11,41,42). Thus, although there is a possibility that the unique functional properties of R628E may have been influenced by the C-terminal reporter protein, this is a less likely interpretation of our results as both the WT and mutant proteins contained the enhanced GFP sequences.…”

“…In this study, wild type constructs were modified to include the R607Q mutation to enhance currents for patch clamp electrophysiology as described previously (11). Thus, the term wild type ("WT") used in this study refers to GluA2 R607Q.…”

“…where R is the universal gas constant, T is the temperature (293 K), k B is the Boltzmann constant, h is the Planck constant, and k is the rate constant of interest (11,18). Data Analysis and Statistics-Digitized current recordings were imported to IGOR Pro 6.22 software using custom macros written by M. Benveniste.…”

“…Allosteric Modulation of R628E Is Preserved-The ability of the R628E mutation to perturb both receptor deactivation and desensitization is reminiscent of the actions of positive allosteric modulators that bind at the dimer interface of the ligandbinding core (11,16,(25)(26)(27)(28) and is also similar to the allosteric modulation of receptor gating conferred by transmembrane accessory receptor proteins such as stargazin (29,30). Therefore, we hypothesized that assessing the efficacy of positive allosteric modulators on gating of the R628E mutation might provide insight into the molecular mechanism through which the mutation perturbs channel gating compared with wild type receptors.…”

Kinetic Contributions to Gating by Interactions Unique to N-methyl-d-aspartate (NMDA) Receptors

“…Site II Side-chain Truncations Impart Sensitivity to AMPA Receptor Positive Modulators-In AMPA receptors, the equivalent location of site II residues defines the binding pocket for small allosteric modulators, which potentiate currents by slowing macroscopic deactivation (11,30,31,(43)(44)(45)(46). The "floor" of this binding pocket is lined by serine residues absent in NMDA receptors (Fig.…”