Mechanism of Cl- Selection by a Glutamate-gated Chloride (GluCl) Receptor Revealed through Mutations in the Selectivity Filter

Abstract

To learn about the mechanism of ion charge selectivity by invertebrate glutamate-gated chloride (GluCl) channels, we swapped segments between the GluCl␤ receptor of Caenorhabditis elegans and the vertebrate cationic ␣7-acetylcholine receptor and monitored anionic/cationic permeability ratios. Complete conversion of the ion charge selectivity in a set of receptor microchimeras indicates that the selectivity filter of the GluCl␤ receptor is created by a sequence connecting the first with the second transmembrane segments. A single substitution of a negatively charged residue within this sequence converted the selectivity of the GluCl␤ receptor's pore from anionic to cationic. Unexpectedly, elimination of the charge of each basic residue of the selectivity filter, one at a time or concomitantly, moderately reduced the P Cl /P Na ratios, but the GluCl␤ receptor's mutants retained high capacity to select Cl The invertebrate GluCl 2 receptor channels are pentameric transmembrane receptors belonging to a wide superfamily of Cys-loop receptors activated by various neurotransmitters such as acetylcholine (ACh), serotonin (5-hydroxtryptamine, 5HT), ␥-aminobutyric acid (GABA), Gly, Glu, or histamine (Fig. 1A) (1-8). This superfamily consists of cationic channels permeable to Na ϩ , K ϩ , and, in many subunit combinations, to Ca 2ϩ ions, as well as of anionic channels selective to Cl Ϫ ions (reviewed by Keramidas et al. (9)). Structural similarities shared by Cys-loop receptors enabled swapping of pore sequences between cationic and anionic channels so as to assess the involvement of specific amino acids in ion charge selectivity. It was previously shown that concomitant replacement of the residues at positions Ϫ2Ј, Ϫ1Ј, and 13Ј ( Fig. 1, B and C) of cationic receptors by the residues found at the homologous positions of anionic receptors, and vice versa, lead to conversion of ion charge selectivity (10 -13).Further mutagenesis studies led to the recognition that the different capacities of cationic versus anionic Cys-loop receptors to distinguish between the charge of ions rely on the differences in the amino acid composition at positions Ϫ1Ј and Ϫ2Ј (Fig. 1C) (13-16). The conserved pore-facing Glu residue at position Ϫ1Ј of cationic Cys-loop receptors was further inferred to form, around the axis of ion conduction, a negatively charged ring that plays the key role in cationic selectivity by interacting with cations and repulsing anions (12,13,15,16). Conversely, a conserved arginine at position 0Ј of anionic Cys-loop receptors was inferred to interact with anions and repulse cations. A basic residue at position 0Ј is also typical of all cationic Cys-loop receptors (Fig. 1C and the ligand-gated ion channels data base), but it was suggested that local conformational differences in the M1-M2 connecting segment (M1-M2 loop) orient this basic residue to the pore lumen only in anionic Cys-loop receptors (9, 15). These local conformational differences have been attributed to a proline residue, which is present exclusively at pos...

The structural basis of function in Cys-loop receptors

“…The structural importance of the Pro was further illustrated by changes in functional properties of the mutant receptors, including differences in activation, desensitization, EC 50 , Hill co-efficient and spontaneous channel openings (Corringer et al 1999a ;Wotring et al 2003). At the invertebrate GluCl receptor, charge selectivity remains unaltered in similar Pro mutants and a structural role was also concluded (Sunesen et al 2006). Further complications were presented in a study by Wotring & Weiss (2008), who also showed that the introduction of Glu residues within an eight amino-acid stretch (x2k to 5k) of GABA r1 produces varied permeability ratios depending upon the location of the substitution.…”

Channel Replacement Therapy for Cystic Fibrosis

“…Class 1, the cys-loop ligand-gated ion channel superfamily of neurotransmitter receptors, includes the anion-selective inhibitory post-synaptic glycine (GlyR) and aminobutyric acid type A (GABAAR) receptors, as well as cation selective nicotinic acetylcholine (nAChR), and 5-hydroxytryptamine type 3 (5-HT3R) receptors Sine and Engel, 2006) and the invertebrate post-synaptic glutamate receptor (Sunesen et al, 2006). These related structures all have a simple central pore defined by the parallel association of the second TM or 'M2' segments contributed by each of the five assembled subunits.…”

Identifying the elusive link between amino acid sequence and charge selectivity in pentameric ligand-gated ion channels

“…2A and SI Materials and Methods). Means and SEs of these values are indicated in Tables S1-S4 for the wild-type and mutant constructs of the muscle AChR, the homomeric serotonin type 3A receptor (5-HT 3A R), the heteromeric serotonin type 3A-3B receptor (5-HT 3A-3B R), and the α1 glycine receptor (GlyR) from mammals, respectively; in Table S5 for a chimeric chick-Caenorhabditis elegans α7-AChR-β-GluCl receptor channel (16); and in Table S6 for the bacterial Erwinia chrysanthemi ligand-gated ion channel (ELIC). To establish a "baseline," we started by studying the wild-type constructs (including the chimera) and found all six of them to be highly selective for either cations or anions (P K + /P Cl -= 97 for the muscle AChR; 31 for the 5-HT 3A R; 42 for the 5-HT 3A-3B R; and 44 for ELIC; P Cl -/P K + = 119 for the α1 GlyR; and 410 for the α7-AChR-β-GluCl chimera).…”

“…2 B-D and F). Furthermore, we estimated the charge selectivity of a α7-AChR-β-GluCl chimeric construct consisting of the extracellular domain of the (cation-selective) α7 AChR and the transmembrane and intracellular domains of the (anion-selective) β GluCl channel (16). As such, this channel carries an aspartate at the aligned extracellular position (Asp-96)-and also a glutamate at the adjacent position 97-that could compromise the anion selectivity of its β GluCl pore.…”

“…For example, whereas some authors have suggested that the charge selectivity of pLGICs is governed solely by interactions between the passing ions and backbone atoms (2,4,16), others have favored the notion that only pore-facing charged side chains underlie this process in both the cation-selective and the anion-selective pLGICs (5). Notably, thus far, no unifying mechanism has been proposed that can account for the results of mutational studies obtained with different pLGICs and that explains the molecular basis of charge selectivity for the entire superfamily.…”