Identification and characterization of heptapeptide modulators of the glycine receptor

The glycine receptor is a member of the Cys-loop receptor superfamily of ligand-gated ion channels and is implicated as a possible therapeutic target for the treatment of diseases such as alcoholism and inflammatory pain. In humans, four glycine receptor subtypes (alpha 1, alpha 2, alpha 3, and beta) co-assemble to form pentameric channel proteins as either alpha homomers or alpha beta heteromers. To date, few agents have been identified that can selectively modulate the glycine receptor, especially those possessing subtype specificity. We used a cell-based method of phage display panning, coupled with two-electrode voltage clamp electrophysiology in Xenopus laevis oocytes, to identify novel heptapeptide modulators of the alpha 1 beta glycine receptor. This involved a panning procedure in which the phage library initially underwent subtractive panning against Human Embryonic Kidney (HEK) 293 cells expressing alternative glycine receptor subtypes before panning the remaining library over HEK 293 cells expressing the target, the alpha 1 beta glycine receptor. Peptides were identified that act with selectivity on alpha 1 beta and alpha 3 beta, compared to alpha 2 beta, glycine receptors. In addition, peptide activity at the glycine receptor decreased when zinc was chelated by tricine, similar to previous observations of a decrease in ethanol's enhancing actions at the receptor in the absence of zinc. Comparisons of the amino acid sequences of heptapeptides capable of potentiating glycine receptor function revealed several consensus sequences that may be predictive of a peptide's enhancing ability. (C) 2016 Elsevier B.V. All rights reserved.