Figure 1. Hydrophobic determinants of Zn²⁺ inhibition.

(a) Partial protein alignment of Cys–loop receptor ECDs showing: GlyR α1 hydrophobic residues that are examined (red); where hydrophobicity is retained in other Cys–loop receptors (red); conserved residues (boxed); and the GlyR Zn²⁺ inhibition binding site residues (underlined). Hydrophobic motifs are labelled and colour–coded in accordance with (b, c), the GlyR α1 ECD homology model based on TornAChRα₁ ¹. Clustered hydrophobic residues (orange) connect the Zn²⁺ inhibition site at the inner–subunit interface (black and light blue motifs – note, black motif is not a β–strand in the model but referred to as such in main text for clarity ²), to glycine binding loops A (green), D (dark blue) and E (mauve) of outer–subunit interface. Zn²⁺ inhibition site residues, His¹⁰⁷, His¹⁰⁹, Thr¹¹² and Thr¹³³, are in grey (nitrogen atoms are blue and oxygens are red). Dotted line depicts the interface with (+) and (–) subunit sides ²⁸. Glycine, displayed in CPK, spacefill, was fitted manually in accordance with ³⁵; Zn²⁺, fitted manually, in grey, spacefill. Exact side chain orientations presented here are speculative, based on best alignment and modelling estimations. Inset, blue box – pentamer plan view showing viewing angle (arrow) for main picture, and revealing the separation between the Zn²⁺ binding site (grey circle) and glycine binding site (mauve circle); enlarged plan view of interface shown in black box. (d, e) Zn²⁺ concentration response curves for modulation of EC₅₀ glycine currents on wild–type (WT; dashed line) and alanine–substituted receptors. α1^I120A did not traffic to cell surface so no recordings were made (Supplementary Figure 2). Curves fitted with Hill equation. All points are means ± s.e.m. (n = 3–6).