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¹ Department of Physiology and Pharmacology, University of New South Wales, Sydney 2052, Australia
² The Garvan Institute of Medical Research, Darlinghurst, Sydney 2010, Australia

Address correspondence to Peter H. Barry, Department of Physiology and Pharmacology, University of New South Wales, Sydney 2052, Australia. Tel.: (61) 2-9385-1101; Fax: (61) 2-9385-1099; E-mail: p.barry{at}unsw.edu.au

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 1 homomeric glycine receptor (1 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 P_Cl/P_Na = 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' mutant GlyR retained its anion selectivity (P_Cl/P_Na = 3.81), but it was much reduced compared with the wild-type (WT) GlyR (P_Cl/P_Na = 27.9). When the A-1'E and the P-2' mutations were combined (selectivity double mutant [SDM] GlyR), the relative cation permeability was enhanced (P_Cl/P_Na = 0.13). The SDM GlyR was also Ca²⁺ permeable (P_Ca/P_Na = 0.29). Neutralizing the extracellular mouth of the SDM GlyR ion channel (SDM+R19'A GlyR) produced a more Ca²⁺-permeable channel (P_Ca/P_Na = 0.73), without drastically altering monovalent charge selectivity (P_Cl/P_Na = 0.23). The SDM+R19'E GlyR, which introduces a negatively charged ring at the extracellular mouth of the channel, further enhanced Ca²⁺ permeability (P_Ca/P_Na = 0.92), with little effect on monovalent selectivity (P_Cl/P_Na = 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 1 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.

Key Words: ligand-gated ion channels • electrostatics • pore diameter • permeability • selectivity filter

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