Identification of Novel Specific Allosteric Modulators of the Glycine Receptor Using Phage Display

Abstract

The glycine receptor (GlyR) is a member of the Cys-loop superfamily of ligand-gated ion channels and the major mediator of inhibitory neurotransmission in the spinal cord and brainstem. Many allosteric modulators affect the functioning of members of this superfamily, with some such as benzodiazepines showing great specificity and others such as zinc, alcohols, and volatile anesthetics acting on multiple members. To date, no potent and efficacious allosteric modulator acting specifically at the GlyR has been identified, hindering both experimental characterization of the receptor and development of GlyR-related therapeutics. We used phage display to identify novel peptides that specifically modulate GlyR function. Peptide D12-116 markedly enhanced GlyR currents at low micromolar concentrations but had no effects on the closely related ␥-aminobutyric acid type A receptors. This approach can readily be adapted for use with other channels that currently lack specific allosteric modulators.The glycine receptor (GlyR) 2 is a member of the Cys-loop superfamily of ligand-gated ion channels, including also the ␥-aminobutyric acid type A (GABA A ) and serotonin-3 receptors. They share a number of structural features, including ligand-binding sites in the extracellular N-terminal domain and a transmembrane domain consisting of four segments, with a large intracellular loop connecting segments 3 and 4. Individual channels consist of five subunits co-localized with the transmembrane domain 2 segment of each subunit lining the anionconducting pore (1, 2). Two classes of GlyR subunits have been identified: the ␣ subunits, of which there are four subtypes, and a single ␤ subunit (3). Most native GlyRs in adult animals consist of heteromeric ␣1␤ subunits, although homomeric ␣2 receptors are the predominant form found prenatally (4). GlyRs constitute the major inhibitory neurotransmitter receptor system in the brainstem and spinal cord (5), where they are thought to play a role in the modulation of pain signals and in the effects of volatile anesthetics (1). Some GlyR mutations result in the startle disorder hyperekplexia. GlyRs are also found throughout the brain, including the thalamus, hippocampus, and nucleus accumbens, where they were recently shown to be involved in the reinforcing properties of ethanol (6). The GlyR is only one of multiple ion channels and receptors thought to play a role in pain perception and alcohol and volatile anesthetic effects and in determining the state of neuronal excitability. The isolation of the role of the GlyR is hindered by the fact that, to date, no potent and efficacious allosteric modulator acting specifically at the GlyR has been identified. Phage display involves the expression of a random library of peptides on the coat proteins of bacteriophage. This method has long been used to identify peptides that can bind with high affinity to selected targets and to aid in identifying binding motifs (7). We combined phage display technology with standard electrophysiological testing to identi...

Contaminating levels of zinc found in commonly-used labware and buffers affect glycine receptor currents

“…Responsible for the majority of fast inhibitory neurotransmission in the brainstem and spinal, GlyR are also found in many higher brain regions including the hippocampus, nucleus accumbens, and prefrontal cortex (Baer et al, 2009; Jonsson et al, 2012, 2009; Lynch, 2004). GlyR function is modulated by inhaled anesthetic, alcohols and inhalants, making it a possible target for the development of therapeutics for the treatment of alcoholism (Beckstead et al, 2000; Mascia et al, 1996; Tipps et al, 2010). …”

Physiological concentrations of zinc reduce taurine-activated GlyR responses to drugs of abuse

“…Thus, the GlyR has emerged as a logical target for the possible development of pharmacological agents to treat substance abuse (Tipps et al, 2010). Allosteric modulators exert their greatest enhancing effects on low concentrations of glycine that are unlikely to be seen at GlyR in vivo except at the initiation or tail-end of synaptic events or perhaps at extrasynaptic receptors (Scimemi and Beato, 2009).…”

Identification and characterization of heptapeptide modulators of the glycine receptor

“…While αl- and α2-containing receptors seem to be the most likely in vivo targets of alcohol (Blednov et al, 2015), there would be considerable utility in the development of glycine receptor modulators with subunit selectivity, to definitively determine the relative contributions of each particular subtype to alcohol’s pharmacological effects. Our previous work validated the use of phage display for the discovery of novel peptide modulators of the glycine receptor (Tipps et al, 2010). In the present report, we expand on these studies by identifying and characterizing the action of peptides with selectivity for α1β and α3β over α2β-containing glycine receptors and identify several possible amino acid consensus sequences within the heptapeptides.…”

“…The Ph.D.-7 phage library was purchased from New England Biolabs (Ipswich, MA) and the panning procedures followed were the same as described in Tipps et al (2010), except as noted below. The phage display procedure consisted of two separate panning series, D7.1 and D7.2.…”