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 gamma-aminobutyric acid type A receptors. This approach can readily be adapted for use with other channels that currently lack specific allosteric modulators.
Highlights
The effects of volatile anesthetics [1]
We used a commercially available phage display library, expressing more than two billion unique sequences, to isolate peptides that bind to glycine receptor (GlyR) ␣1 expressed in Human Embryonic Kidney (HEK) 293 cells
Because ethanol clearly affects multiple biochemical targets in addition to the GlyR, it has proved difficult to determine conclusively the roles that individual putative targets play in the various behavioral effects of this agent
Summary
Phage Display—On panning day 1, a plate of control HEK cells was washed three times with 0.01 M phosphate-buffered saline (PBS) containing 8.2 mM NaPO4, 1.5 mM KH2PO4, 137 mM NaCl, and 2.7 mM KCl with 1.5% bovine serum albumin (BSA) and 0.1% Tween (PBS/BSAϩT). Specific Peptide Modulators of the Glycine Receptor negative selection procedure were removed from the plate with a pipette, applied to the plate of GlyR-expressing cells, and rocked gently at room temperature for 60 min. The pellet was resuspended in 1 ml of Tris-buffered saline (50 mM Tris-HCl (pH 7.5) and 150 mM NaCl), transferred to a 1.7-ml microcentrifuge tube, and spun at 10,000 rpm for 5 min at 4 °C. The pellet was resuspended in Tris-buffered saline and spun again for 1 min, and the supernatant was transferred to a fresh tube and stored at 4 °C. The percent enhancement or inhibition values obtained in the presence of peptide were compared by either one- or two-way analysis of variance, as indicated, to determine statistical significance, with a criterion of p Ͻ 0.05 required
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