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

Abstract

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 alpha1-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.

Further analysis of counterion permeation through anion-selective glycine receptor channels

“…less, only P Cl /P Li is minimally increased by the initial offset potential correction. In order to clarify whether the changes in relative permeability with different counterions were reflecting changes in counterion movement (Case 1), rather than the counterion affecting anion movement, with the counterion conductance being independent of the nature of the cation (Case 2), conductance measurements were made in a biionic situation where the cell pipette solution contained (4) where g Cs , g Li and g Cl are the individual Cs + , Li + and Clcomponents of conductance respectively, which since concentrations of cations and anions in both solutions are the same would be expected to be proportional to permeabilities (Suppl. Material).…”

“…It should be noted that a similarly increased contribution of the initial offset potential correction was observed in the presence of external calcium, which if ignored further increased P Cl /P Na for the WT GlyR channel. 4 The updated relative counterion permeability values for the two channels can be compared with the sizes of the two GlyR channels and the ionic and hydrated sizes of the different ions in Figure 2, where the hydrated size of the Li + ion with its large equivalent hydrated size now has a much higher relative permeability in the smaller WT GlyR channel compared to Na + and Cs + (Fig. 6 of ref.…”

“…the theoretical shifts in LJPs (∆V LJ ) were calculated using concentrations (Col. 3) and activities (Col. 4) and compared with values measured "experimentally" (∆V LJ m ; Col. 5) at 22 o C. n represents the number of bracketed measurements used for each pair of solutions (e.g., 1.0 LiCl:0.5 LiCl). it should be noted that the experimental and calculated ∆V LJ values for naCl dilutions in this table are the same as those given in the Supplementary Material of Sugiharto et al 4 relationship between γ and √m (where m = molal salt concentration) using SigmaPlot 9 (Systat Software, Point Richmond, CA). For simplicity, m was approximated by the molar (M) concentrations, the errors generally being less than 0.2% and the relative errors in final activities being much less than this.…”

“…While using the shifts in reversal potentials automatically eliminates any pipette tip potential, it also eliminates any small offset potential across the cell membrane. Another article, in press, 4 on the effects of divalent cations on P Cl /P Na in these GlyR channels indicates that although correcting for such offset potentials only has a small effect on its value in the absence of external calcium, in the presence of 4 mM CaCl 2 , which enhances P Cl /P Na , the effect of the offset potential is much more marked (Sugiharto et al 4 ).…”

“…1 analyses, all the relative permeabilities were still averaged from values determined from individual dilution potential experiments, and are given as the mean ± Se, with the number of observations shown in parenthesis. it should be noted that the naCl data set and new P Cl /P na values for the Wt Glyr only are the same as the control naCl values in Sugiharto et al4…”