Abstract
Neurotransmitter binding to Cys-loop receptors promotes a prodigious transmembrane flux of several million ions/s, but to date, structural determinants of ion flux have been identified flanking the membrane-spanning region. Using x-ray crystallography, sequence analysis, and single-channel recording, we identified a novel determinant of ion conductance near the point of entry of permeant ions. Co-crystallization of acetylcholine-binding protein with sulfate anions revealed coordination of SO4(2-) with a ring of lysines at a position equivalent to 24 A above the lipid membrane in homologous Cys-loop receptors. Analysis of multiple sequence alignments revealed that residues equivalent to the ring of lysines are negatively charged in cation-selective receptors but are positively charged in anion-selective receptors. Charge reversal of side chains at homologous positions in the nicotinic receptor from the motor end plate decreases unitary conductance up to 80%. Selectivity filters stemming from transmembrane alpha-helices have similar pore diameters and compositions of amino acids. These findings establish that when the channel opens under a physiological electrochemical gradient, permeant ions are initially stabilized within the extracellular vestibule of Cys-loop receptors, and this stabilization is a major determinant of ion conductance.
Highlights
Ion selectivity defines two major classes of Cys-loop receptors
Using x-ray crystallography, sequence analysis, and single-channel recording, we identified a novel determinant of ion conductance near the point of entry of permeant ions
Mutations of residues in a channel cytoplasmic region altered conductance in 5-HT3A receptors [5], suggesting that other domains form vestibules leading into the channel that may influence ion conductance and selectivity
Summary
Ion selectivity defines two major classes of Cys-loop receptors. Receptors that selectively translocate cations are excitatory and include vertebrate nAChRs3 and 5-HT3 receptors, whereas receptors that selectively translocate anions are inhibitory and include ␥-aminobutyric acid and glycine receptors. Co-crystallization of acetylcholine-binding protein with sulfate anions revealed coordination of SO42؊ with a ring of lysines at a position equivalent to 24 Aabove the lipid membrane in homologous Cys-loop receptors.
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