Principles of agonist recognition in Cys-loop receptors

Abstract

Cys-loop receptors are ligand-gated ion channels that are activated by a structurally diverse array of neurotransmitters, including acetylcholine, serotonin, glycine, and GABA. After the term "chemoreceptor" emerged over 100 years ago, there was some wait until affinity labeling, molecular cloning, functional studies, and X-ray crystallography experiments identified the extracellular interface of adjacent subunits as the principal site of agonist binding. The question of how subtle differences at and around agonist-binding sites of different Cys-loop receptors can accommodate transmitters as chemically diverse as glycine and serotonin has been subject to intense research over the last three decades. This review outlines the functional diversity and current structural understanding of agonist-binding sites, including those of invertebrate Cys-loop receptors. Together, this provides a framework to understand the atomic determinants involved in how these valuable therapeutic targets recognize and bind their ligands.

Keywords: Cys-loop receptors; GABA-A receptors; GluCl; glycine receptors; ion channels; ligand recognition; nicotinic acetylcholine receptors; serotonin receptors.

Figure 1

Fundamental Cys-loop receptor architecture. (A) Pentameric Cys-loop receptor, viewed from within the membrane (gray) plane. Each of the five subunits is indicated by a different color and contains an extracellular N-terminal extracellular domain (ECD) consisting of two β-sheets and a membrane-spanning domain comprised of four α-helices. (B) Magnified view of the interface of adjacent ECDs: one subunit in green, one in blue. The outer β-sheet of the green and the inner β-sheet of the blue form the principal and complementary faces, respectively, of the agonist-binding site. (C) Magnified view of the agonist-binding site, showing agonist-binding loops A–C of the principal face and D–G of the complementary face. All images are based on the glutamate-bound Caenorhabditis elegans α GluCl crystal structure, Protein DataBase reference 3RIF (Hibbs and Gouaux, 2011).

Figure 2

Conventional Cys-loop receptor agonists. Glycine, GABA, and glutamate are neurotransmitters that bind to inhibitory receptors, while serotonin and acetylcholine bind to excitatory receptors. Note the different electrical state of these agonists under physiological conditions: glycine and GABA are zwitterions, while acetylcholine and serotonin carry a single positive charge and glutamate a net negative charge.

Figure 3

Amino acid sequence alignment. Only side chains in agonist-binding loops are shown, plus small segments abutting Loop A and Loop B. Hum, human; Lym, Lymnaea stagnalis; Erw, Erwinia chrysanthemi; Glo, Gloeobacter violaceus; Cae, Caenorhabditis elegans; Dro, Drosophila melanogaster. ^*Refers to nAChR isoform 1, which is up-regulated in muscle but less abundant in humans than isoform 2, which contains a 25 amino acid insert in Loop D (Beeson et al., ; Talib et al., 1993). While ELIC gates in response to primary amines, GLIC is a proton-gated cation channel. HisCl 1 (Zheng et al., 2002), ACC-1 (Putrenko et al., 2005), MOD-1 (Ranganathan et al., 2000), and LGC-55 (Ringstad et al., 2009) are histamine, acetylcholine, serotonin, and tyromine-gated chloride channels, respectively. Gray boxes indicate conserved aromatic side chains A, B, C1 and C2. Arrows indicate the position of functionally important side chains described in the main text, left-to-right: pre-Loop B threonine in 5-HT₃Rs; post-Loop B side chain in nAChRs; Loop C threonine in GlyRs, GABA_ARs, and GluCls; Loop D aromatic side chain in nAChRs; Loop D arginine/glutamine side chain in GluCls/AChBP; and Loop G arginine in GluCls. Performed in ClustalW2 (Larkin et al., 2007).

Figure 4

Relative abundance of amino acids in Cys-loop receptor agonist-binding loops. (A) Relative frequency (y-axis) at which each amino acid (x-axis) appears in a set of 520 proteins (dark columns; Brooks et al., 2002) and in the 25 Cys-loop receptor agonist-binding loops as shown in Figure 3 (light columns). (B) Fold increase (positive numbers) or decrease in frequency at which amino acids appear in Cys-loop receptor agonist-binding loops compared to the general protein set.

Figure 5

X-ray crystallographic structures of agonist-bound receptors. (A) L. stagnalis AChBP in complex with carbamylcholine (PDB entry 1UV6). (B) C. elegans α GluCl (GLC-1) in complex with glutamate (and ivermectin in the transmembrane domain; PDB entry 3RIF). In each illustration, numerous segments have been removed for clarity, including Loop C, of which only selected side chains are visible.

Figure 6

X-ray crystallographic structures of other notable agonists. (A) L. stagnalis AChBP in complex with nicotine (PDB entry 1UW6). (B) L. stagnalis AChBP in complex with imidacloprid (PDB entry 2ZJU). A red sphere illustrates the oxygen atom of a water molecule that bridges agonist pyridines to backbone Leu102 carbonyl and Met114 amide groups. In each illustration, numerous segments have been removed for clarity, including Loop C, of which only selected side chains are visible.