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Programme
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The ligand-gated glycine receptor ion channel (GlyR) mediates fast inhibitory synaptic transmission in the central nervous system. The wild-type (WT) homomeric α1 GlyR, with a minimum pore diameter of 5.3 Å, is predominantly permeant to Cl- anions, while a proline deletion (P-2'Δ) creates a larger (6.9 Å) minimum pore diameter and a smaller anion-cation permeability (e.g., Keramidas et al., 2004).
In a series of whole cell dilution potential experiments to investigate the mechanism of counter-ion permeation in the above two channels expressed in HEK 293 cells, we have already measured relative permeabilities of counter-ion cations to anions and shown that the hydrated counter-ion size determines the anion-cation permeability (Sugiharto et al., 2006). The effective hydrated diameters of Cl-, NO3-, Cs+, Na+ and Li+ ions of 5.0, 5.2, 4.9, 6.5 and 7.4 Å, respectively, were calculated de novo, or re-calculated, from published data (Robinson & Stokes, 1965). We used the GHK (Goldman-Hodgkin-Katz) equation with activity corrections and PCl/Pcation values were determined in LiCl, NaCl and CsCl solutions from reversal potential shifts (corrected for liquid junction potentials) with ∼50% and ∼25% external salt dilutions. We showed that in the smaller WT GlyR channels, PCl/PCs = 5.1 ± 0.5, PCl/PNa = 12.4 ± 0.4 and PCl/PLi = 32 ± 5. However, in the larger mutant P-2'Δ GlyR channels, PCl/PCs = 2.0 ± 0.1, PCl/PNa = 3.5 ± 0.2 and PCl/PLi = 6.6 ± 0.5. For both channels, as the hydrated counter-ion cation size increased so did PCl/Pcation. In addition, the smaller channel displayed the greater range of relative permeabilities (Sugiharto et al., 2006). In anion-cation permeability measurements in a neuronal chloride channel, Franciolini & Nonner (1994) had previously shown that their counter-ion/co-ion permeability ratio was approximately constant for different anions.
We used whole-cell patch-clamp measurements to explore the counter-ion/co-ion permeability ratio for different anions to see if the same relationship held true for the WT GlyR channel, and if it could elicit further information about the mechanism of counter-ion permeation in that channel. We chose the nitrate anion, NO3-, which is similar in size to Cl- but has a different bi-ionic permeability (Lee et al., 2003). The solutions contained either 145 mM NaCl or NaNO3, together with 10 mM HEPES titrated to a pH of 7.4 with NaOH. In addition, each internal solution contained 2 mM CaCl2 and 5 mM EGTA, and the internal NaNO3 solution had 5 mM of its NaNO3 replaced by 5 mM NaCl to maintain a well-defined Ag/AgCl electrode potential. The diluted external solutions had the diluted Na-salt replaced by an osmotically equal concentration of sucrose. We initially measured the reversal potential in bi-ionic NaNO3 : NaCl solutions and determined a relative PNO3/PCl of 1.6 ± 0.1. We then did dilution potential measurements in NaNO3 solutions, determined their reversal potentials and, fitting the data to the GHK equation, showed that PNO3/PNa = 12.8 ± 0.6. This was very similar to the PCl/PNa value of 12.4 ± 0.4 and indicates that this counter-ion/co-ion permeability is not primarily determined by the anion permeability. It is supportive of counter-ion permeation being via neutral anion-cation pairs with PCl/Pcation determined by the larger hydrated size of the cation counter-ions. It can readily be explained if (1) the ions permeate as neutral ion pairs, (2) the rate at which the ion pairs are formed is proportional to the rate at which the anions permeate, and (3) the hydrated sizes of the anions are smaller than those of the cations.
Franciolini F & Nonner W. (1994) Journal of General Physiology, 104: 711-23.
Keramidas A, Moorhouse AJ, Schofield & Barry PH. (2004) Progress in Biophysics and Molecular Biology, 86: 161-204.
Lee D-J.S, Keramidas A, Moorhouse AJ, Schofield PR & Barry PH. (2003) Neuroscience Letters, 351: 96-200.
Robinson RA & Stokes RH (1965) Electrolyte Solutions, 2nd Ed. Rev., Butterworths, London.
Sugiharto S, Moorhouse AJ, Lewis TM, Schofield PR & Barry PH. (2006) Proceedings of the Australian Society for Biochemistry and Molecular Biology, 38: 106 (Oral-01-06).