AMPA Receptor Binding Cleft Mutations That Alter Affinity, Efficacy, and Recovery from Desensitization

Abstract

Glutamate binds to AMPA receptors within a deep cleft between two globular protein domains (domains 1 and 2). Once glutamate binds, the cleft closes, and agonist-bound structures of the isolated ligand binding core suggest that closure of the binding cleft is sufficiently complete that it essentially prevents ligand dissociation. There is also considerable evidence supporting the view that cleft closure is the initial conformational change that triggers receptor activation and desensitization, and it has been clearly demonstrated that there is a correlation between the degree of cleft closure and agonist efficacy. It is unknown, however, whether the stability of binding cleft closure also influences receptor-channel properties. The crystallographic structures indicate that closed-cleft conformations are stabilized by the formation of hydrogen bonds that involve amino acid side chains of residues in domains 1 and 2. We show here that mutations that disrupt one such cross-cleft hydrogen bond (in the AMPA receptor subunit GluR2) decrease both agonist affinity and efficacy. The same mutations also hasten recovery from desensitization. We conclude that the stability of binding cleft closure has a significant impact on AMPA receptor function and is a major determinant of the apparent affinity of agonists. The results suggest that the stability of cleft closure has been tuned so that glutamate dissociates as rapidly as possible yet remains a full agonist.

Structural and Single-Channel Results Indicate That the Rates of Ligand Binding Domain Closing and Opening Directly Impact AMPA Receptor Gating

“…Compared with glutamate-evoked currents through wildtype channels or currents evoked by quisqualate, the rate of desensitization was also substantially slower for the T686A mutants and steady-state desensitization was reduced (Fig. 1d), results consistent with our previous observations (Robert et al, 2005).…”

“…Although there is no direct evidence that LBD closure necessarily precedes channel gating, these and other results strongly suggest that closing of the LBD is the initial large-scale conformational change that triggers the subsequent gating rearrangements that result in channel opening and desensitization (Sun et al, 2002;Robert et al, 2005;Valentine and Palmer, 2005;Armstrong et al, 2006;Mayer, 2006;Ahmed et al, 2007;Hansen et al, 2007). The available evidence therefore indicates that, for glutamate to be an effective and fast neurotransmitter, the kinetics of LBD closing and opening must be such that two apparently opposing requirements are met: the LBD must be closed for effective gating but open for glutamate to dissociate.…”

“…tsA201 cells were maintained and transfected as described previously (Robert et al, 2005). Recordings from outside-out patches held at Ϫ100 mV were performed 12-24 h after transfection at room temperature with an EPC 9 amplifier (HEKA Elektronik, Lambrecht/Pfalz, Germany).…”

“…When glutamate-and quisqualateevoked currents were compared after reducing desensitization of GluR2 channels with CTZ, mutation of threonine 686 to serine or alanine altered the relative efficacy of the two agonists, reducing the maximal glutamate-evoked currents to ϳ75 and 45%, respectively, of those seen with quisqualate (Robert et al, 2005). To verify that these results still held when desensitization was intact, we first compared peak currents evoked by rapid application of the two agonists in patches containing wildtype GluR2 channels (GluR2-wt) or GluR2 channels carrying the T686A mutation (both GluR2 constructs were flip versions and had a glutamine at the Q/R editing site).…”

“…Disruption of one such cross-cleft interaction (between threonine 686 and glutamate 402) significantly increases EC 50 values for glutamate and quisqualate (Quis) and speeds deactivation (Robert et al, 2005), results consistent with subsequent work on kainate and AMPA receptors and the idea that the rate at which the LBD opens is a major determinant of agonist affinity (Weston et al, 2006). Serine and alanine mutations at T686 also selectively reduced the efficacy of glutamate and slowed the rate of receptor desensitization, raising the possibility that subsequent gating transitions were also altered, perhaps because the LBD closed differently (Robert et al, 2005).…”