The roles of aromatic residues in the glycine receptor transmembrane domain

Bijun Tang,Sarah C R Lummis

doi:10.1186/s12868-018-0454-8

Abstract

BackgroundCys-loop receptors play important roles in fast neuronal signal transmission. Functional receptors are pentamers, with each subunit having an extracellular, transmembrane (TM) and intracellular domain. Each TM domain contains 4 α-helices (M1–M4) joined by loops of varying lengths. Many of the amino acid residues that constitute these α-helices are hydrophobic, and there has been particular interest in aromatic residues, especially those in M4, which have the potential to contribute to the assembly and function of the receptor via a range of interactions with nearby residues.ResultsHere we show that many aromatic residues in the M1, M3 and M4 α-helices of the glycine receptor are involved in the function of the receptor. The residues were explored by creating a range of mutant receptors, characterising them using two electrode voltage clamp in Xenopus oocytes, and interpreting changes in receptor parameters using currently available structural information on the open and closed states of the receptor. For 7 residues function was ablated with an Ala substitution: 3 Tyr residues at the extracellular end of M1, 2 Trp residues located towards the centers of M1 and M3, and a Phe and a Tyr residue in M4. For many of these an alternative aromatic residue restored wild-type-like function indicating the importance of the π ring. EC50s were increased with Ala substitution of 8 other aromatic residues, with those in M1 and M4 also having reduced currents, indicating a role in receptor assembly. The structure shows many potential interactions with nearby residues, especially between those that form the M1/M3/M4 interface, and we identify those that are supported by the functional data.ConclusionThe data reveal the importance and interactions of aromatic residues in the GlyR M1, M3 and M4 α-helices, many of which are essential for receptor function.

Highlights

Cys-loop receptors play important roles in fast neuronal signal transmission
To probe the roles of the aromatic residues in the transmembrane α-helices of the Gly receptors (GlyR), we mutated each to Ala, and determined changes in functional characteristics following expression in Xenopus oocytes
Mutation of seven of the nineteen aromatic residues to Ala resulted in nonfunctional receptors (Table 1), and, apart from one (F295A), all others resulted in increased E C50s and/or lower maximal currents (Fig. 3), indicating these residues are important for GlyR expression and/or function

Summary

Introduction

Cys-loop receptors play important roles in fast neuronal signal transmission. Functional receptors are pentamers, with each subunit having an extracellular, transmembrane (TM) and intracellular domain. Cys-loop receptors, which include nicotinic acetylcholine (nACh), 5-HT3, GABAA, and glycine (Gly) receptors, are pentameric ligand-gated ion channels (pLGIC) responsible for fast excitatory and inhibitory synaptic neurotransmission in the central and peripheral nervous systems [1,2,3,4]. Members of this family are pentameric, with each of the subunits having an extracellular domain (ECD), a transmembrane domain (TMD), and an intracellular domain.

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