Abstract
To learn about the mechanism of ion charge selectivity by invertebrate glutamate-gated chloride (GluCl) channels, we swapped segments between the GluClbeta receptor of Caenorhabditis elegans and the vertebrate cationic alpha7-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 GluClbeta 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 GluClbeta 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 GluClbeta receptor's mutants retained high capacity to select Cl(-) over Na(+). These results indicate that, unlike the proposed case of anionic Gly- and gamma-aminobutyric acid-gated ion channels, positively charged residues do not play the key role in the selection of ionic charge by the GluClbeta receptor. Taken together with measurements of the effective open pore diameter and with structural modeling, the study presented here collectively indicates that in the most constricted part of the open GluClbeta receptor's channel, Cl(-) interacts with backbone amides, where it undergoes partial dehydration necessary for traversing the pore.
Highlights
The invertebrate GluCl2 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) [1,2,3,4,5,6,7,8]
Unlike all other homomeric anionic Cys-loop receptors studied far, the  subunit of the GluCl receptor does not have a proline residue at position Ϫ2Ј, a feature that minimizes the likelihood of causing drastic local conformational changes when mutating its M1-M2 loop
Chimeric Design and Current Amplitudes of the Various Mutants— As a first step, we generated a chimeric subunit where the extracellular segment of the AChR ␣7 subunit was fused to the GluCl subunit segment that folds in the membrane and cytoplasm
Summary
Chimeras and Mutants—The ␣7-GluCl chimeric subunit was prepared as performed previously with the ␣7-5HT3AR [19], by fusing the N-terminal half of the chick ␣7 subunit (Fig. 1A, red segment) (SwissProt accession number P22770) to the C-terminal half of the  subunit of the GluClR (Fig. 1A, non-red segments) (Swiss-Prot accession number Q17328). Note that in one exceptional case (chimera 4), the composition of the diluted solutions was slightly different, giving external NaCl concentrations of (in mM): B1Ј ϭ 100, B2Ј ϭ 40, B3Ј ϭ 20, B4Ј ϭ 10, B5 ϭ 0. After modeling the M1-M2 loop, it was tilted together with the M1 and M2 segments to an intermediate position between the closed and open states previously elaborated by Paas et al [20] in the case of another Cys-loop chimeric receptor. HϪ5Ј of the GluClR’s model is not in contact with the permeating ions This modeling observation is in line with the incapacity of Zn2ϩ to block the chimeric ␣7-GluClR (data not shown), unlike the case of a chimera having the transmembrane segments of the 5HT3AR with the sequence PPDLHSTAG between M1 and M2 [20]. The root mean square difference between the backbone atoms of the M1-M2 loops (plus their flanking residues, IϪ10Ј and R0Ј) of these structures is 0.08 Å
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