Skip to main content
NCBI home page
Biophysical Journal logoLink to Biophysical Journal
. 2000 Mar;78(3):1335–1348. doi: 10.1016/s0006-3495(00)76688-4

Protonation of lysine residues inverts cation/anion selectivity in a model channel.

V Borisenko 1, M S Sansom 1, G A Woolley 1
PMCID: PMC1300733  PMID: 10692320

Abstract

A dimeric alamethicin analog with lysine at position 18 in the sequence (alm-K18) was previously shown to form stable anion-selective channels in membranes at pH 7.0 [Starostin, A. V., R. Butan, V. Borisenko, D. A. James, H. Wenschuh, M. S. Sansom, and G. A. Woolley. 1999. Biochemistry. 38:6144-6150]. To probe the charge state of the conducting channel and how this might influence cation versus anion selectivity, we performed a series of single-channel selectivity measurements at different pH values. At pH 7.0 and below, only anion-selective channels were found with P(K(+))/P(Cl(-)) = 0. 25. From pH 8-10, a mixture of anion-selective, non-selective, and cation-selective channels was found. At pH > 11 only cation-selective channels were found with P(K(+))/P(Cl(-)) = 4. In contrast, native alamethicin-Q18 channels (with Gln in place of Lys at position 18) were cation-selective (P(K(+))/P(Cl(-)) = 4) at all pH values. Continuum electrostatics calculations were then carried out using an octameric model of the alm-K18 channel embedded in a low dielectric slab to simulate a membrane. Although the calculations can account for the apparent pK(a) of the channel, they fail to correctly predict the degree of selectivity. Although a switch from cation- to anion-selectivity as the channel becomes protonated is indicated, the degree of anion-selectivity is severely overestimated, suggesting that the continuum approach does not adequately represent some aspect of the electrostatics of permeation in these channels. Side-chain conformational changes upon protonation, conformational changes, and deprotonation caused by permeating cations and counterion binding by lysine residues upon protonation are considered as possible sources of the overestimation.

Full Text

The Full Text of this article is available as a PDF (392.9 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Adcock C., Smith G. R., Sansom M. S. Electrostatics and the ion selectivity of ligand-gated channels. Biophys J. 1998 Sep;75(3):1211–1222. doi: 10.1016/S0006-3495(98)74040-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Antosiewicz J., McCammon J. A., Gilson M. K. Prediction of pH-dependent properties of proteins. J Mol Biol. 1994 May 6;238(3):415–436. doi: 10.1006/jmbi.1994.1301. [DOI] [PubMed] [Google Scholar]
  3. Bashford D., Karplus M. pKa's of ionizable groups in proteins: atomic detail from a continuum electrostatic model. Biochemistry. 1990 Nov 6;29(44):10219–10225. doi: 10.1021/bi00496a010. [DOI] [PubMed] [Google Scholar]
  4. Bell T. W. Carriers and channels: current progress and future prospects. Curr Opin Chem Biol. 1998 Dec;2(6):711–716. doi: 10.1016/s1367-5931(98)80108-7. [DOI] [PubMed] [Google Scholar]
  5. Borisova M. P., Brutyan R. A., Ermishkin L. N. Mechanism of anion-cation selectivity of amphotericin B channels. J Membr Biol. 1986;90(1):13–20. doi: 10.1007/BF01869681. [DOI] [PubMed] [Google Scholar]
  6. Bowen K. A., Tam K., Colombini M. Evidence for titratable gating charges controlling the voltage dependence of the outer mitochondrial membrane channel, VDAC. J Membr Biol. 1985;86(1):51–59. doi: 10.1007/BF01871610. [DOI] [PubMed] [Google Scholar]
  7. Breed J., Biggin P. C., Kerr I. D., Smart O. S., Sansom M. S. Alamethicin channels - modelling via restrained molecular dynamics simulations. Biochim Biophys Acta. 1997 Apr 26;1325(2):235–249. doi: 10.1016/s0005-2736(96)00262-3. [DOI] [PubMed] [Google Scholar]
  8. Cafiso D. S. Alamethicin: a peptide model for voltage gating and protein-membrane interactions. Annu Rev Biophys Biomol Struct. 1994;23:141–165. doi: 10.1146/annurev.bb.23.060194.001041. [DOI] [PubMed] [Google Scholar]
  9. Cescatti L., Pederzolli C., Menestrina G. Modification of lysine residues of Staphylococcus aureus alpha-toxin: effects on its channel-forming properties. J Membr Biol. 1991 Jan;119(1):53–64. doi: 10.1007/BF01868540. [DOI] [PubMed] [Google Scholar]
  10. Corringer P. J., Bertrand S., Galzi J. L., Devillers-Thiéry A., Changeux J. P., Bertrand D. Mutational analysis of the charge selectivity filter of the alpha7 nicotinic acetylcholine receptor. Neuron. 1999 Apr;22(4):831–843. doi: 10.1016/s0896-6273(00)80741-2. [DOI] [PubMed] [Google Scholar]
  11. Darveau R. P., Hancock R. E., Benz R. Chemical modification of the anion selectivity of the PhoE porin from the Escherichia coli outer membrane. Biochim Biophys Acta. 1984 Jul 11;774(1):67–74. doi: 10.1016/0005-2736(84)90275-x. [DOI] [PubMed] [Google Scholar]
  12. Dieckmann G. R., Lear J. D., Zhong Q., Klein M. L., DeGrado W. F., Sharp K. A. Exploration of the structural features defining the conduction properties of a synthetic ion channel. Biophys J. 1999 Feb;76(2):618–630. doi: 10.1016/S0006-3495(99)77230-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Dorman V., Partenskii M. B., Jordan P. C. A semi-microscopic Monte Carlo study of permeation energetics in a gramicidin-like channel: the origin of cation selectivity. Biophys J. 1996 Jan;70(1):121–134. doi: 10.1016/S0006-3495(96)79554-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Doyle D. A., Morais Cabral J., Pfuetzner R. A., Kuo A., Gulbis J. M., Cohen S. L., Chait B. T., MacKinnon R. The structure of the potassium channel: molecular basis of K+ conduction and selectivity. Science. 1998 Apr 3;280(5360):69–77. doi: 10.1126/science.280.5360.69. [DOI] [PubMed] [Google Scholar]
  15. Dutzler R., Rummel G., Albertí S., Hernández-Allés S., Phale P., Rosenbusch J., Benedí V., Schirmer T. Crystal structure and functional characterization of OmpK36, the osmoporin of Klebsiella pneumoniae. Structure. 1999 Apr 15;7(4):425–434. doi: 10.1016/s0969-2126(99)80055-0. [DOI] [PubMed] [Google Scholar]
  16. Fahlke C., Yu H. T., Beck C. L., Rhodes T. H., George A. L., Jr Pore-forming segments in voltage-gated chloride channels. Nature. 1997 Dec 4;390(6659):529–532. doi: 10.1038/37391. [DOI] [PubMed] [Google Scholar]
  17. Franciolini F., Nonner W. Anion and cation permeability of a chloride channel in rat hippocampal neurons. J Gen Physiol. 1987 Oct;90(4):453–478. doi: 10.1085/jgp.90.4.453. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Guinamard R., Akabas M. H. Arg352 is a major determinant of charge selectivity in the cystic fibrosis transmembrane conductance regulator chloride channel. Biochemistry. 1999 Apr 27;38(17):5528–5537. doi: 10.1021/bi990155n. [DOI] [PubMed] [Google Scholar]
  19. Hall J. E., Vodyanoy I., Balasubramanian T. M., Marshall G. R. Alamethicin. A rich model for channel behavior. Biophys J. 1984 Jan;45(1):233–247. doi: 10.1016/S0006-3495(84)84151-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hancock R. E., Benz R. Demonstration and chemical modification of a specific phosphate binding site in the phosphate-starvation-inducible outer membrane porin protein P of Pseudomonas aeruginosa. Biochim Biophys Acta. 1986 Sep 11;860(3):699–707. doi: 10.1016/0005-2736(86)90569-9. [DOI] [PubMed] [Google Scholar]
  21. Honig B., Nicholls A. Classical electrostatics in biology and chemistry. Science. 1995 May 26;268(5214):1144–1149. doi: 10.1126/science.7761829. [DOI] [PubMed] [Google Scholar]
  22. Huyghues-Despointes B. M., Scholtz J. M., Baldwin R. L. Effect of a single aspartate on helix stability at different positions in a neutral alanine-based peptide. Protein Sci. 1993 Oct;2(10):1604–1611. doi: 10.1002/pro.5560021006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Jaikaran D. C., Biggin P. C., Wenschuh H., Sansom M. S., Woolley G. A. Structure-function relationships in helix-bundle channels probed via total chemical synthesis of alamethicin dimers: effects of a Gln7 to Asn7 mutation. Biochemistry. 1997 Nov 11;36(45):13873–13881. doi: 10.1021/bi9716130. [DOI] [PubMed] [Google Scholar]
  24. Karshikoff A., Spassov V., Cowan S. W., Ladenstein R., Schirmer T. Electrostatic properties of two porin channels from Escherichia coli. J Mol Biol. 1994 Jul 22;240(4):372–384. doi: 10.1006/jmbi.1994.1451. [DOI] [PubMed] [Google Scholar]
  25. Kasianowicz J. J., Bezrukov S. M. Protonation dynamics of the alpha-toxin ion channel from spectral analysis of pH-dependent current fluctuations. Biophys J. 1995 Jul;69(1):94–105. doi: 10.1016/S0006-3495(95)79879-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lanyi J. K., Duschl A., Váro G., Zimányi L. Anion binding to the chloride pump, halorhodopsin, and its implications for the transport mechanism. FEBS Lett. 1990 Jun 4;265(1-2):1–6. doi: 10.1016/0014-5793(90)80869-k. [DOI] [PubMed] [Google Scholar]
  27. Latorre R., Alvarez O. Voltage-dependent channels in planar lipid bilayer membranes. Physiol Rev. 1981 Jan;61(1):77–150. doi: 10.1152/physrev.1981.61.1.77. [DOI] [PubMed] [Google Scholar]
  28. Levitt D. G. General continuum theory for multiion channel. I. Theory. Biophys J. 1991 Feb;59(2):271–277. doi: 10.1016/S0006-3495(91)82220-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Levitt D. G. General continuum theory for multiion channel. II. Application to acetylcholine channel. Biophys J. 1991 Feb;59(2):278–288. doi: 10.1016/S0006-3495(91)82221-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Malashkevich V. N., Kammerer R. A., Efimov V. P., Schulthess T., Engel J. The crystal structure of a five-stranded coiled coil in COMP: a prototype ion channel? Science. 1996 Nov 1;274(5288):761–765. doi: 10.1126/science.274.5288.761. [DOI] [PubMed] [Google Scholar]
  31. Moczydlowski E. Chemical basis for alkali cation selectivity in potassium-channel proteins. Chem Biol. 1998 Nov;5(11):R291–R301. doi: 10.1016/s1074-5521(98)90288-5. [DOI] [PubMed] [Google Scholar]
  32. Nicholls A., Sharp K. A., Honig B. Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins. 1991;11(4):281–296. doi: 10.1002/prot.340110407. [DOI] [PubMed] [Google Scholar]
  33. Nilges M., Brünger A. T. Successful prediction of the coiled coil geometry of the GCN4 leucine zipper domain by simulated annealing: comparison to the X-ray structure. Proteins. 1993 Feb;15(2):133–146. doi: 10.1002/prot.340150205. [DOI] [PubMed] [Google Scholar]
  34. O'Keeffe F., Shamsi S. A., Darcy R., Schwinté P., Warner I. M. A persubstituted cationic beta-cyclodextrin for chiral separations. Anal Chem. 1997 Dec 1;69(23):4773–4782. doi: 10.1021/ac970370e. [DOI] [PubMed] [Google Scholar]
  35. Perutz M. F., Shih D. T., Williamson D. The chloride effect in human haemoglobin. A new kind of allosteric mechanism. J Mol Biol. 1994 Jun 17;239(4):555–560. doi: 10.1006/jmbi.1994.1394. [DOI] [PubMed] [Google Scholar]
  36. Pfeiffer S., Fushman D., Cowburn D. Impact of Cl- and Na+ ions on simulated structure and dynamics of betaARK1 PH domain. Proteins. 1999 May 1;35(2):206–217. [PubMed] [Google Scholar]
  37. Prod'hom B., Pietrobon D., Hess P. Direct measurement of proton transfer rates to a group controlling the dihydropyridine-sensitive Ca2+ channel. Nature. 1987 Sep 17;329(6136):243–246. doi: 10.1038/329243a0. [DOI] [PubMed] [Google Scholar]
  38. Rink T., Bartel H., Jung G., Bannwarth W., Boheim G. Effects of polycations on ion channels formed by neutral and negatively charged alamethicins. Eur Biophys J. 1994;23(3):155–165. doi: 10.1007/BF01007607. [DOI] [PubMed] [Google Scholar]
  39. Root M. J., MacKinnon R. Two identical noninteracting sites in an ion channel revealed by proton transfer. Science. 1994 Sep 23;265(5180):1852–1856. doi: 10.1126/science.7522344. [DOI] [PubMed] [Google Scholar]
  40. Sansom M. S. Alamethicin and related peptaibols--model ion channels. Eur Biophys J. 1993;22(2):105–124. doi: 10.1007/BF00196915. [DOI] [PubMed] [Google Scholar]
  41. Sansom M. S. Models and simulations of ion channels and related membrane proteins. Curr Opin Struct Biol. 1998 Apr;8(2):237–244. doi: 10.1016/s0959-440x(98)80045-6. [DOI] [PubMed] [Google Scholar]
  42. Sansom M. S. Structure and function of channel-forming peptaibols. Q Rev Biophys. 1993 Nov;26(4):365–421. doi: 10.1017/s0033583500002833. [DOI] [PubMed] [Google Scholar]
  43. Sansom M. S. The biophysics of peptide models of ion channels. Prog Biophys Mol Biol. 1991;55(3):139–235. doi: 10.1016/0079-6107(91)90004-c. [DOI] [PubMed] [Google Scholar]
  44. Schmid B., Maveyraud L., Krömer M., Schulz G. E. Porin mutants with new channel properties. Protein Sci. 1998 Jul;7(7):1603–1611. doi: 10.1002/pro.5560070714. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Schmidtchen Franz P., Berger Michael. Artificial Organic Host Molecules for Anions. Chem Rev. 1997 Aug 5;97(5):1609–1646. doi: 10.1021/cr9603845. [DOI] [PubMed] [Google Scholar]
  46. Sitkoff D., Lockhart D. J., Sharp K. A., Honig B. Calculation of electrostatic effects at the amino terminus of an alpha helix. Biophys J. 1994 Dec;67(6):2251–2260. doi: 10.1016/S0006-3495(94)80709-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Smart O. S., Breed J., Smith G. R., Sansom M. S. A novel method for structure-based prediction of ion channel conductance properties. Biophys J. 1997 Mar;72(3):1109–1126. doi: 10.1016/S0006-3495(97)78760-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Smart O. S., Goodfellow J. M., Wallace B. A. The pore dimensions of gramicidin A. Biophys J. 1993 Dec;65(6):2455–2460. doi: 10.1016/S0006-3495(93)81293-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Starostin A. V., Butan R., Borisenko V., James D. A., Wenschuh H., Sansom M. S., Woolley G. A. An anion-selective analogue of the channel-forming peptide alamethicin. Biochemistry. 1999 May 11;38(19):6144–6150. doi: 10.1021/bi9826355. [DOI] [PubMed] [Google Scholar]
  50. Tabcharani J. A., Linsdell P., Hanrahan J. W. Halide permeation in wild-type and mutant cystic fibrosis transmembrane conductance regulator chloride channels. J Gen Physiol. 1997 Oct;110(4):341–354. doi: 10.1085/jgp.110.4.341. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Tieleman D. P., Berendsen H. J., Sansom M. S. An alamethicin channel in a lipid bilayer: molecular dynamics simulations. Biophys J. 1999 Apr;76(4):1757–1769. doi: 10.1016/s0006-3495(99)77337-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Tieleman D. P., Sansom M. S., Berendsen H. J. Alamethicin helices in a bilayer and in solution: molecular dynamics simulations. Biophys J. 1999 Jan;76(1 Pt 1):40–49. doi: 10.1016/S0006-3495(99)77176-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Van Gelder P., Saint N., van Boxtel R., Rosenbusch J. P., Tommassen J. Pore functioning of outer membrane protein PhoE of Escherichia coli: mutagenesis of the constriction loop L3. Protein Eng. 1997 Jun;10(6):699–706. doi: 10.1093/protein/10.6.699. [DOI] [PubMed] [Google Scholar]
  54. Wang C. T., Zhang H. G., Rocheleau T. A., ffrench-Constant R. H., Jackson M. B. Cation permeability and cation-anion interactions in a mutant GABA-gated chloride channel from Drosophila. Biophys J. 1999 Aug;77(2):691–700. doi: 10.1016/S0006-3495(99)76924-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Warshel A., Papazyan A. Electrostatic effects in macromolecules: fundamental concepts and practical modeling. Curr Opin Struct Biol. 1998 Apr;8(2):211–217. doi: 10.1016/s0959-440x(98)80041-9. [DOI] [PubMed] [Google Scholar]
  56. Woolley G. A., Biggin P. C., Schultz A., Lien L., Jaikaran D. C., Breed J., Crowhurst K., Sansom M. S. Intrinsic rectification of ion flux in alamethicin channels: studies with an alamethicin dimer. Biophys J. 1997 Aug;73(2):770–778. doi: 10.1016/S0006-3495(97)78109-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Woolley G. A., Wallace B. A. Model ion channels: gramicidin and alamethicin. J Membr Biol. 1992 Aug;129(2):109–136. doi: 10.1007/BF00219508. [DOI] [PubMed] [Google Scholar]
  58. You S., Peng S., Lien L., Breed J., Sansom M. S., Woolley G. A. Engineering stabilized ion channels: covalent dimers of alamethicin. Biochemistry. 1996 May 21;35(20):6225–6232. doi: 10.1021/bi9529216. [DOI] [PubMed] [Google Scholar]
  59. van Vlijmen H. W., Schaefer M., Karplus M. Improving the accuracy of protein pKa calculations: conformational averaging versus the average structure. Proteins. 1998 Nov 1;33(2):145–158. doi: 10.1002/(sici)1097-0134(19981101)33:2<145::aid-prot1>3.0.co;2-i. [DOI] [PubMed] [Google Scholar]

Articles from Biophysical Journal are provided here courtesy of The Biophysical Society

RESOURCES