Cation-selective Mutations in the M2 Domain of the Inhibitory Glycine Receptor Channel Reveal Determinants of Ion-Charge Selectivity

Abstract

Ligand-gated ion channel receptors mediate neuronal inhibition or excitation depending on their ion charge selectivity. An investigation into the determinants of ion charge selectivity of the anion-selective α1 homomeric glycine receptor (α1 glycine receptor [GlyR]) was undertaken using point mutations to residues lining the extra- and intracellular ends of the ion channel. Five mutant GlyRs were studied. A single substitution at the intracellular mouth of the channel (A-1′E GlyR) was sufficient to convert the channels to select cations over anions with PCl/PNa = 0.34. This result delimits the selectivity filter and provides evidence that electrostatic interactions between permeating ions and pore residues are a critical factor in ion charge selectivity. The P-2′Δ mutant GlyR retained its anion selectivity (PCl/PNa = 3.81), but it was much reduced compared with the wild-type (WT) GlyR (PCl/PNa = 27.9). When the A-1′E and the P-2′Δ mutations were combined (selectivity double mutant [SDM] GlyR), the relative cation permeability was enhanced (PCl/PNa = 0.13). The SDM GlyR was also Ca2+ permeable (PCa/PNa = 0.29). Neutralizing the extracellular mouth of the SDM GlyR ion channel (SDM+R19′A GlyR) produced a more Ca2+-permeable channel (PCa/PNa = 0.73), without drastically altering monovalent charge selectivity (PCl/PNa = 0.23). The SDM+R19′E GlyR, which introduces a negatively charged ring at the extracellular mouth of the channel, further enhanced Ca2+ permeability (PCa/PNa = 0.92), with little effect on monovalent selectivity (PCl/PNa = 0.19). Estimates of the minimum pore diameter of the A-1′E, SDM, SDM+R19′A, and SDM+R19′E GlyRs revealed that these pores are larger than the α1 GlyR, with the SDM-based GlyRs being comparable in diameter to the cation-selective nicotinic acetylcholine receptors. This result provides evidence that the diameter of the ion channel is also an important factor in ion charge selectivity.