Cations But Not Anions Regulate the Responsiveness of Kainate Receptors

Abstract

Kainate-selective ionotropic glutamate receptors are unique among ligand-gated ion channels in their obligate requirement of external anions and cations for activation. Although it is established that the degree of kainate receptor (KAR) activation is shaped by the chemical nature of the agonist molecule, the possible complementary role of external ions has yet to be examined. Here we show that external cations but not anions regulate the responsiveness to a range of full and partial agonists acting on rat GluK2 receptors. This observation is unexpected as previous work has assumed anions and cations affect KARs in an identical manner through functionally coupled binding sites. However, our data demonstrate that anion-and cation-binding pockets behave discretely. We suggest cations uniquely regulate a pregating or flipping step that impacts the closed-cleft stability of the agonist-binding domain (ABD). This model departs from a previous proposal that KAR agonist efficacy is governed by the degree of closure elicited in the ABD by ligand binding. Our findings are, however, in line with recent studies on Cys-loop ligand-gated ion channels suggesting that the "flipping" mechanism has been conserved by structurally diverse ligand-gated ion channel families as a common means of regulating neurotransmitter behavior.

Transmembrane AMPA receptor regulatory protein regulation of competitive antagonism: a problem of interpretation

“…All experiments described in this study were performed on outside‐out patches excised from transfected tsA201 cells as described previously (MacLean et al 2011). Briefly, cDNAs encoding enhanced green fluorescent protein (eGFP), the GluA1 AMPAR subunit and/or stargazin were transiently transfected using the calcium phosphate method at ratios of (eGFP:GluA1:stargazin) 1:10:15 or 1:10:20 for 10–14 h. We included 10 μ m NBQX in the media to inhibit cell death.…”

Retour aux sources: defining the structural basis of glutamate receptor activation

“…Importantly, the authors were later able to more directly examine agonist efficacy by studying AMPARs at the single-channel level, taking into account sub-conductance levels. These studies suggested a model whereby more efficacious agonists can better stabilize the closed conformation of a dynamic ligand binding cleft (Zhang et al 2008), an idea that has also been used to explain agonist behaviour at KARs (Maclean et al 2011). In agreement, spectroscopic measurements of the willardiine-bound GluA2 S1S2 constructs pinpointed specific residues in the cleft that undergo dynamic motions correlated with agonist potency (Fenwick & Oswald, 2008).…”

“…Accordingly, studies of the LBD dimer interface have often examined how individual residues affect the kinetics of desensitization (Horning & Mayer, 2004), with less attention paid to their role in agonist efficacy. However, other studies show that mutations along the KAR interface affect the relative efficacies of L-Glu and KA (Fleck et al 2003;Zhang et al 2006;Maclean et al 2011), in addition to the time course of desensitization.…”

“…), an idea that has also been used to explain agonist behaviour at KARs (Maclean et al . ). In agreement, spectroscopic measurements of the willardiine‐bound GluA2 S1S2 constructs pinpointed specific residues in the cleft that undergo dynamic motions correlated with agonist potency (Fenwick & Oswald, ).…”

“…; Maclean et al . ), in addition to the time course of desensitization. Comparisons of agonist behaviour at GluA2 AMPARs in conditions with desensitization present or attenuated (in the presence of cyclothiazide or atop the L–Y mutation) even suggested that desensitization generally inverts the rank order of efficacy (Jin et al .…”