External divalent cations increase anion–cation permeability ratio in glycine receptor channels

The functional role of ligand-gated ion channels in the central nervous system depends on their relative anion–cation permeability. Using standard whole-cell patch clamp measurements and NaCl dilution potential measurements, we explored the effect of external divalent ions on anion–cation selectivity in α1-homomeric wild-type glycine receptor channels. We show that increasing external Ca 2+ from 0 to 4 mM resulted in a sigmoidal increase in anion–cation permeability by 37%, reaching a maximum above about 2 mM. Our accurate quantification of this effect required rigorous correction for liquid junction potentials (LJPs) using ion activities, and allowing for an initial offset potential. Failure to do this results in a considerable overestimation of the Ca 2+ -induced increase in anion–cation permeability by almost three-fold at 4 mM external Ca 2+ . Calculations of LJPs (using activities)_ were validated by precise agreement with direct experimental measurements. External SO 4 2− was found to decrease anion–cation permeability. Single-channel conductance measurements indicated that external Ca 2+ both decreased Na + permeability and increased Cl − permeability. There was no evidence of Ca 2+ changing channel pore diameter. Theoretical modeling indicates that the effect is not surface charge related. Rather, we propose that, under dilution conditions, the presence of an impermeant Ca 2+ ion in the channel pore region just external to the selectivity filter tends to electrostatically retard outward movement of Na + ions and to enhance movement of Cl − ions down their energy gradients.