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

  • Silas Sugiharto
  • Jane E. Carland
  • Trevor M. Lewis
  • Andrew J. Moorhouse
  • Peter H. BarryEmail author
Ion Channels, Receptors and Transporters

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 α1-homomeric wild-type glycine receptor channels. We show that increasing external Ca2+ 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 Ca2+-induced increase in anion–cation permeability by almost three-fold at 4 mM external Ca2+. 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 Ca2+ both decreased Na+ permeability and increased Cl permeability. There was no evidence of Ca2+ 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 Ca2+ 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.

Keywords

Cl channels Glycine receptors Ion selectivity Calcium Patch clamp 

Notes

Acknowledgments

This work was supported by the National Health and Medical Research Council of Australia and by a Goldstar Award from the University of New South Wales. J.E.C. was supported by a UNSW Vice-Chancellor’s Postdoctoral Research Fellowship.

Supplementary material

424_2010_792_MOESM1_ESM.pdf (323 kb)
ESM 1 (PDF 323 kb)

References

  1. 1.
    Barry PH (1994) JPCalc—a software package for calculating liquid junction potential corrections in patch-clamp, intracellular, epithelial and bilayer measurements and for correcting junction potential measurements. J Neurosci Methods 51:107–116CrossRefPubMedGoogle Scholar
  2. 2.
    Barry PH (2006) The reliability of relative anion–cation permeabilities deduced from reversal (dilution) potential measurements in ion channel studies. Cell Biochem Biophys 46:143–154CrossRefPubMedGoogle Scholar
  3. 3.
    Barry PH, Diamond JM (1970) Junction potentials, electrode potentials, and other problems in interpreting electrical properties of membranes. J Membrane Biol 3:93–122CrossRefGoogle Scholar
  4. 4.
    Barry PH, Gage PW (1984) Ion selectivity of channels at the end-plate. Curr Top Membr Transp (Academic Press) 21:1–51Google Scholar
  5. 5.
    Carland JE, Cooper MA, Sugiharto S, Jeong H-J, Lewis TM, Barry PH, Peters JA, Lambert JJ, Moorhouse AJ (2009) Characterization of the effects of charged residues in the intracellular loop on ion permeation in α1 glycine receptor channels. J Biol Chem 284:2023–2030CrossRefPubMedGoogle Scholar
  6. 6.
    Corringer J-P, Bertrand S, Galzi J-L, Devillers-Thiery A, Changeux J-P, Bertrand D (1999) Mutational analysis of the charge selectivity filter of the α7 nicotinic acetylcholine receptor. Neuron 22:831–843CrossRefPubMedGoogle Scholar
  7. 7.
    D’Arrigo J (1978) Screening of membrane surface charges by divalent cations: an atomic representation. Am J Physiol 235:C109–C117PubMedGoogle Scholar
  8. 8.
    Dweck D, Reyes-Alfonso A Jr, Potter JD (2005) Expanding the range of free calcium regulation in biological solutions. Anal Chem 347:303–315Google Scholar
  9. 9.
    Franciolini F, Nonner W (1994) A multi-ion permeation mechanism in neuronal background chloride channels. J Gen Physiol 104:725–746CrossRefPubMedGoogle Scholar
  10. 10.
    Grahame DC (1947) The electric double layer and the theory of electrocapillarity. Chem Rev 41:441–501CrossRefPubMedGoogle Scholar
  11. 11.
    Gunthorpe MJ, Lummis SCR (2001) Conversion of the ion selectivity of the 5-HT3A receptor from cationic to anionic reveals a conserved feature of the ligand-gated ion channel superfamily. J Biol Chem 276:10977–10983CrossRefGoogle Scholar
  12. 12.
    Hille B (2001) Ion channels of excitable membranes, 3rd edn. Sinauer Associates, SunderlandGoogle Scholar
  13. 13.
    Hille B, Woodhull AM, Shapiro BI (1975) Negative surface charge near sodium channels of nerve: divalent ions, monovalent ions, and pH. Phil Trans R Soc Lond B 270:301–318CrossRefGoogle Scholar
  14. 14.
    Jensen ML, Schousboe A, Ahring PK (2005) Review. Charge selectivity of the Cys-loop family of ligand-gated ion channels. J Neurochem 92:217–225CrossRefPubMedGoogle Scholar
  15. 15.
    Jensen ML, Timmermann DB, Johansen TH, Schousbe A, Varming T, Ahring PK (2002) The β subunit determines the ion selectivity of the GABAA receptor. J Biol Chem 277:41438–41447CrossRefPubMedGoogle Scholar
  16. 16.
    Jensen ML, Pedersen LN, Timmermann DB, Schousboe A, Ahring PK (2005) Mutational studies using a cation-conducting GABAA receptor reveal the selectivity determinants of the Cys-loop family of ligand-gated ion channels. J Neurochem 92:962–972CrossRefPubMedGoogle Scholar
  17. 17.
    Keramidas A, Moorhouse AJ, French CR, Schofield PR, Barry PH (2000) M2 pore mutations convert the glycine receptor channel from being anion- to cation-selective. Biophys J 78:247–259CrossRefGoogle Scholar
  18. 18.
    Keramidas A, Moorhouse AJ, Pierce KD, Schofield PR, Barry PH (2002) Cation-selective mutations of the inhibitory glycine receptor channel reveal determinants of ion-charge selectivity. J Gen Physiol 119:393–410CrossRefPubMedGoogle Scholar
  19. 19.
    Keramidas A, Moorhouse AJ, Schofield PR, Barry PH (2004) Ligand gated ion channels: mechanisms underlying ion selectivity. Prog Biophys Molec Biol 86:61–204CrossRefGoogle Scholar
  20. 20.
    Lee DJ-S, Keramidas A, Moorhouse AJ, Schofield PR, Barry PH (2003) The contribution of proline 250 (P-2′) to pore diameter and ion selectivity in the human glycine receptor channel. Neurosci Letts 351:96–200Google Scholar
  21. 21.
    MacInnes DA (1961) The principles of electrochemistry. Dover, New YorkGoogle Scholar
  22. 22.
    Moorhouse AJ, Keramidas A, Zaykin A, Schofield PR, Barry PH (2002) Single channel analysis of conductance and rectification in cation-selective, mutant glycine receptor channels. J Gen Physiol 119:411–425CrossRefPubMedGoogle Scholar
  23. 23.
    Robinson RA, Stokes RH (1965) Electrolyte Solutions, Revised 2nd edn. Butterworths, LondonGoogle Scholar
  24. 24.
    Sugiharto S, Lewis TM, Moorhouse AJ, Schofield PR, Barry PH (2008) Anion–cation permeability correlates with hydrated counterion size in glycine receptor channels. Biophys J 95:4698–4715CrossRefPubMedGoogle Scholar
  25. 25.
    Weast RC (ed) (1980) CRC handbook of chemistry and physics, 1980–1981, 61st edn. CRC, Boca RatonGoogle Scholar
  26. 26.
    Wotring VE, Miller TS, Weiss DS (2003) Mutations at the GABA receptor selectivity filter: a possible role for effective charges. J Physiol (Lond) 548:527–540CrossRefGoogle Scholar
  27. 27.
    Wotring VE, Weiss DS (2008) Charge scan reveals an extended region at the intracellular end of the GABA receptor pore that can influence ion selectivity. J Gen Physiol 131:87–97CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Silas Sugiharto
    • 1
  • Jane E. Carland
    • 1
  • Trevor M. Lewis
    • 1
  • Andrew J. Moorhouse
    • 1
  • Peter H. Barry
    • 1
    Email author
  1. 1.Department of Physiology, School of Medical SciencesUniversity of New South WalesSydneyAustralia

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