Mechanisms of Modal Activation of GluA3 Receptors

Abstract

AMPA receptors are the major excitatory neurotransmitter receptors in the central nervous system and are involved in numerous neurological disorders. An agonist-binding site is present in each of four subunits that form a functional channel. Binding consists of three steps: docking of agonist to the bilobed ligand binding domain (LBD), closure of the LBD, and increased stability of the closed-lobe conformation through interlobe hydrogen bonding. We describe GluA3 single channel currents activated by nitrowillardiine (NO 2 W) and chlorowillardiine (ClW) in the presence of cyclothiazide, in conjunction with crystal structures of GluA2 and GluA3 LBDs bound to fluorowillardiine (FW), ClW, and NO 2 W. When bound to NO 2 W or ClW, the GluA3 channel opens to three conductance levels with comparable open probabilities and displays modal behavior similar to that obtained with glutamate and FW as agonists (Poon et al., 2010). At lower concentrations, ClW evoked an alternate kinetic behavior, consisting of high open probability in lower conductance states. The structure of ClW bound to GluA3 LBD exhibits a unique partially open hydrogen bonding structure that may be associated with these alternative kinetics. NO 2 W evoked longer open times than seen for other agonists in high and very high modes. The structure ofGluA2 LBD bound to NO 2 W exhibits fully closed lobes with additional interlobe interactions mediated by the nitro group. Beyond differences in efficacy between full and partial agonists, the complexities of the single channel behavior of AMPA receptors may also be associated with small interactions that modify the stability of various degrees of closure.

Role of Conformational Dynamics in α-Amino-3-hydroxy-5-methylisoxazole-4-propionic Acid (AMPA) Receptor Partial Agonism

“…1. The data shown in the bar graph are consistent with the numerous previous more detailed investigations and show that the willardiines are partial agonists with iodowillardiine have a lower response relative to chloro-and nitro-willardiines (31,32,39). A two-tailed repeated measures t test was performed for statistical analysis of differences in the measured currents, and a p value of Յ0.05 was considered significant in our experiments.…”

“…Nitrowillardiine and cholorwillardiine have similar electrophysiological responses; however, the average smFRET distances indicate that the nitrowillardiine-bound state is on average slightly more closed relative to chlorowillardiine bound state. A similar breakdown was observed in the crystal structures where no significant changes were observed in the distances at the positions labeled for the FRET measurements between these willardiines (shown in Table 1) (39).…”

“…The willardiines were initially thought to be classical examples for showing a graded cleft closure consistent with their activation (50). However, further studies using crystal structures (39) and NMR showed that the correlation is not as straightforward as first thought (31,49). NMR studies investigating the exchange at the side chain methyl groups showed differences among the various willardiines and indicated that the dynamics must also play a role in the activation.…”

“…4) and add to these studies by showing the whole range of conformations probed. Additionally, the crystal structures of GluA2 and GluA3 agonist-binding domain in complex with chlorowillardiine show that despite the small size of the chloro substituent, the chlorowillardiine-bound form is on average more open than the glutamate-bound form (39). The nitrowillardiinebound form, despite nitro being a larger substituent, is more closed in six of the nine structures studied relative to the fluorowillardiine-bound form.…”

Mechanism of AMPA Receptor Activation by Partial Agonists

“…Single channel recording experiments (12) have demonstrated that full and partial agonists can activate AMPA receptor channels to the same three or four distinct conductance levels. At saturating agonist concentrations in the absence of desensitization, partial agonists exhibit lower currents than full agonists because the lower conductance levels are preferentially populated relative to that seen with full agonists (12,14,42). The different conductance levels have been assumed to arise from the activation of individual gates on each of four subunits (43).…”