Contributions of Conserved Residues at the Gating Interface of Glycine Receptors

Abstract

Glycine receptors (GlyRs) are chloride channels that mediate fast inhibitory neurotransmission and are members of the pentameric ligand-gated ion channel (pLGIC) family. The interface between the ligand binding domain and the transmembrane domain of pLGICs has been proposed to be crucial for channel gating and is lined by a number of charged and aromatic side chains that are highly conserved among different pLGICs. However, little is known about specific interactions between these residues that are likely to be important for gating in ␣1 GlyRs. Here we use the introduction of cysteine pairs and the in vivo nonsense suppression method to incorporate unnatural amino acids to probe the electrostatic and hydrophobic contributions of five highly conserved side chains near the interface, Glu-53, Phe-145, Asp-148, Phe-187, and Arg-218. Our results suggest a salt bridge between Asp-148 in loop 7 and Arg-218 in the pre-M1 domain that is crucial for channel gating. We further propose that Phe-145 and Phe-187 play important roles in stabilizing this interaction by providing a hydrophobic environment. In contrast to the equivalent residues in loop 2 of other pLGICs, the negative charge at Glu-53 ␣1 GlyRs is not crucial for normal channel function. These findings help decipher the GlyR gating pathway and show that distinct residue interaction patterns exist in different pLGICs. Furthermore, a salt bridge between Asp-148 and Arg-218 would provide a possible mechanistic explanation for the pathophysiologically relevant hyperekplexia, or startle disease, mutant Arg-218 3 Gln. The glycine receptor (GlyR)2 chloride channel is a member of the Cys-loop receptor family, a subfamily of the pentameric ligand-gated ion channel (pLGIC) superfamily (1). GlyRs mediate fast inhibitory neurotransmission in the nervous system, and recent studies have provided a wealth of insight into the structure and function of the GlyR and other pLGICs. One of the more studied regions, the N-terminal ligand binding domain (LBD), is composed of a 10-strand ␤-sheet sandwich interconnected by 9 loops and the ligand binding pocket situated at the interface between adjacent subunits (2-8). The transmembrane domain (TMD) contains four ␣-helical segments (M1-M4), including the pore-lining M2 helices.How is the binding of an agonist molecule in the LBD communicated to the channel gate in the TMD, almost 60 Å away? This question has gained considerable attention in the past (9 -11), and previous studies have identified a number of charged residues likely to couple the LBD to the TMD via electrostatic interactions in different pLGICs (12)(13)(14)(15)(16)(17). A number of critical residues have been identified at the interface of LBD and TMD of ␣1 GlyRs (18 -22), but evidence for direct electrostatic interactions is thus far missing. The interface of LBD and TMD in ␣1 GlyRs is of interest not only because of its proposed role in channel gating but also because inherited mutations of side chains near this interface cause hyperekplexia or startle disease (23): Ala-52 ...

Incorporation of Non-Canonical Amino Acids

“…Further, isosteric manipulations that neutralize side-chains have been crucial in determining the roles of putative salt-bridges in pentameric subunit composition and voltage-sensor energetic (Cashin et al 2007; Pless et al 2011b; Pless et al 2011d; Pless et al 2014). Recently a subtle tryptophan derivative that lacks the ability to hydrogen bond (H-bond) at the indole nitrogen (Ind) was used to characterize an intricate H-bond network in the vicinity of the potassium channel selectivity filter that acts as a molecular timing mechanism to control ionic conductance during prolonged exposure to activating voltages (Pless et al 2013).…”

Inhibitory Glycine Receptors: An Update

“…Residues located at the ends or outside of TM2 have been shown to participate in the gating process, such as the GlyR␣1 residues Arg-271, located in the M2-M3 linker (34), and Lys-276, a porefacing residue at the extracellular end of TM2 (50). Presumably, these and other residues that reside at the interface between the ECD and transmembrane domain are essential for coupling the conformational changes occurring in the ECD upon agonist binding to channel opening (4,5,51).…”

Investigation of Congenital Myasthenia Reveals Functional Asymmetry of Invariant Acetylcholine Receptor (AChR) Cys-loop Aspartates

“…In the heteromeric GABA A receptor, analyses of charge reversal or disulfide trapping of residue pairs revealed that the residue equivalent to ␣Asp-138 interacted with cationic or polar residues from the pre-M1 (6) and M2-M3 regions (7). In the homomeric glycine receptor, studies of substituted cysteine pairs suggested that residues equivalent to ␣Asp-138 and ␣Arg-209 form a salt bridge important for receptor function (8). Finally, in the muscle AChR, charge reversal experiments revealed functional interaction between the residue equivalent to ␣Asp-138 and a cationic residue in the C-terminal tail (9).…”