Disruption of an intersubunit electrostatic bond is a critical step in glycine receptor activation

Abstract

Proper regulation of neurotransmission requires that ligandactivated ion channels remain closed until agonist binds. How channels then open remains poorly understood. Glycine receptor (GlyR) gating is initiated by agonist binding at interfaces between adjacent subunits in the extracellular domain. Aspartate-97, located at the α1 GlyR interface, is a conserved residue in the cys-loop receptor superfamily. The mutation of D97 to arginine (D97R) causes spontaneous channel opening, with open and closed dwell times similar to those of maximally activated WT GlyR. Using a model of the N-terminal domain of the α1 GlyR, we hypothesized that an arginine-119 residue was forming intersubunit electrostatic bonds with D97. The D97R/R119E charge reversal restored this interaction, stabilizing channels in their closed states. Cysteine substitution shows that this link occurs between adjacent subunits. This intersubunit electrostatic interaction among GlyR subunits thus contributes to the stabilization of the closed channel state, and its disruption represents a critical step in GlyR activation.cysteine substitution | electrophysiology | mutagenesis | Xenopus oocytes G lycine receptors (GlyR) are pentameric anion-conducting members of the cys-loop receptor superfamily, with their subunits arranged around a central ion pore. Each subunit consists of a large extracellular N-terminal ligand binding domain and four transmembrane segments (TM1-TM4); TM2 of each subunit lines the pore (1). When glycine binds to initiate channel opening it interacts with specific amino acids located at intersubunit interfaces, and spontaneous openings do not occur in the absence of neurotransmitter (2). Six loops of amino acids located on adjacent subunits constitute the known glycine binding site. On the plus (+) side of the interface on one subunit are loops A-C, whereas loops D-F are located on the minus (−) side of an adjacent subunit (3). In the related nicotinic acetylcholine receptor (nAChR), signal transduction after agonist binding is described as a "Brownian conformational wave" that travels down the interface between subunits (4). Auerbach and colleagues used ϕ analysis to show that the binding pocket region of the N-terminal domain is the first to move after ligand binds. Loops 2 and 7 (the cys-loop) of the Nterminal domain interact with the extracellular end of TM1 and the TM2-3 linker region to transmit binding signals to the channel gate (5-7). In the α1 GlyR subunit, D148 in loop 7 forms an electrostatic bridge with K276 in the TM2-3 linker (8).Our previous work demonstrated that mutation of D97 in loop A results in spontaneous channel opening (9). This D97 residue is conserved within the cys-loop receptor superfamily (Fig. 1), suggesting its critical role in channel function. In this article we report on an electrostatic interaction between specific charged amino acid residues at the interfaces of adjacent subunits that contribute to the stabilization of the closed channel state in the absence of neurotransmitter. Disruption of these ...

Disruption of a putative intersubunit electrostatic bond enhances agonist efficacy at the human α1 glycine receptor

“…In the absence of ligand and contaminating Zn 2+ , the D97R receptor displayed spontaneous channel openings grouped into clusters of activity separated by sojourns into what are likely desensitized states. These clusters had a P open of 0.949 ± 0.004 and exhibited the same behavior as first described by Todorovic et al (2010). When saturating concentrations of taurine or glycine were applied, the P open of the clusters increased slightly to 0.990 ± 0.002 and 0.990 ± 0.001, respectively (Figs.…”

“…This amino acid is conserved across the entire cys-loop subunit superfamily, residing at a subunit interface near residues previously implicated in ligand binding (Brejc, 2001). Our previous work strongly suggested that D97 forms an intersubunit electrostatic bond with R119 (Todorovic et al, 2010), a residue earlier shown to play a role in determining glycine affinity (Grudzinska et al, 2005). In our previous study we engineered cysteine mutations at the D97 and R119 positions, and used those to show that receptor function could be markedly affected by addition of oxidizing, cross-linking or reducing agents (Todorovic et al, 2010).…”

“…Previously we showed that mutation of the aspartate-97 residue to arginine (D97R) in loop A in the LBD of the α1 subunit favors spontaneous transitions of channels to the open state (Beckstead et al, 2002). Subsequently we found that this spontaneous activity was due to the disruption of a putative intersubunit electrostatic bond involving D97 and at least R119 of an adjacent subunit (Todorovic et al, 2010). Single channel recordings showed that clusters of spontaneous D97R GlyR openings had a P open of 0.91, very similar to that observed when wildtype α1 GlyR are exposed to saturating concentrations of glycine.…”

Synaptic Neurotransmitter-Gated Receptors

“…It is therefore not surprising that destabilizing the interface by mutation can lead to spontaneous channel activation (Miller et al 2008;Todorovic et al 2010).…”

A State-Dependent Salt-Bridge Interaction Exists across the β/α Intersubunit Interface of the GABAAReceptor

“…Laha and Wagner at ASPET Journals on May 12, 2018 molpharm.aspetjournals.org dependent intersubunit electrostatic interaction (Todorovic et al, 2010). This interaction is proposed to exist when the glycine receptor is in the unbound state and is broken upon the binding of agonist.…”