Cation-selective mutations in the M2 domain of the inhibitory glycine receptor channel reveal determinants of ion-charge selectivity.
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 alpha1 homomeric glycine receptor (alpha1 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'Delta 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'Delta 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 Ca(2+) permeable (P(Ca)/P(Na) = 0.29). Neutralizing the extracellular mouth of the SDM GlyR ion channel (SDM+R19'A GlyR) produced a more Ca(2+)-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 Ca(2+) 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 alpha1 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.
Keramidas
Angelo
A
Department of Physiology and Pharmacology, University of New South Wales, Sydney 2052, Australia.
Moorhouse
Andrew J
AJ
Pierce
Kerrie D
KD
Schofield
Peter R
PR
Barry
Peter H
PH
D000595:Amino Acid Sequence; D002118:Calcium / Q000378:metabolism; D002412:Cations / Q000378:metabolism; D002460:Cell Line; D002463:Cell Membrane Permeability / Q000235:genetics; D006801:Humans; D015640:Ion Channel Gating / Q000235:genetics*; D008024:Ligands; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009433:Neural Inhibition / Q000235:genetics*; D017354:Point Mutation / Q000502:physiology*; D018009:Receptors, Glycine / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D002412:Cations; D008024:Ligands; D018009:Receptors, Glycine; D002118:Calcium