Skip to main content
SpringerLink
Log in

Diffusion Pathways to Critical Cysteines in the Vesicular Acetylcholine Transporter of Torpedo

  • Published:
Neurochemical Research Aims and scope Submit manuscript

Abstract

Previous work had demonstrated that organomercurial-mediated modification of two cysteine residues in the vesicular acetylcholine transporter (VAChT) from Torpedo californica inhibits binding of vesamicol. The cysteines are protected by acetylcholine and vesamicol (Keller et al. 2000. J. Neurochem. 74:1739–1748). Modified “cysteine 1” is accessible to glutathione from the cytoplasmic surface, whereas modified “cysteine 2” is not. Different organomercurials and aqueous environments were used here to characterize diffusion pathway(s) leading to the cysteines. para-Chloromercuriphenylsulfonate modifies VAChT much more slowly than do more hydrophobic p-chloromercuribenzoate and phenylmercury chloride. Permeabilization of vesicles with cholate detergent increases the rate of modification by p-chloromercuriphenylsulfonate. Permeabilization does not affect the ability of glutathione to reverse modification by p-chloromercuriphenylsulfonate. Higher ionic strength causes about four-fold increase in the rate of modification. The results suggest that hydrophobic and electrostatic barriers inhibit modification of Torpedo VAChT by negatively charged organomercurials and glutathione cannot reach cysteine 2 from either side of the membrane.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

REFERENCES

  1. Keller, J. E., Bravo, D. T., and Parsons, S. M. 2000. Modification of cysteines reveals linkage to acetylcholine and vesamicol binding sites in the vesicular acetylcholine transporter of Torpedo californica. J. Neurochem. 74:1739–1748.

    PubMed  Google Scholar 

  2. Gracz, L. M. and Parsons, S. M. 1996. Purification of active synaptic vesicles from the electric organ of Torpedo californica and comparison to reserve vesicles. Biochim. Biophys. Acta 1292:293–302.

    PubMed  Google Scholar 

  3. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248–254.

    PubMed  Google Scholar 

  4. Habeeb, A. F. S. A. 1972. Reaction of protein sulfhydryl groups with Ellman's reagent. Methods Enzymol. 25:457–464.

    Google Scholar 

  5. Rowland, R. L. 1952. Mercurial diuretics: VI. Ionization of organic mercurials. J. Am. Chem. Soc. 74:5482–5484.

    Google Scholar 

  6. Selwyn, M. J. 1972. Permeability effects of organomercurials. Biochem. J. 130:65p–67p.

    PubMed  Google Scholar 

  7. Simpson, R. B. 1961. Association constants of methylmercury with sulfhydryl and other bases. J. Am. Chem. Soc. 83:4711–4717.

    Google Scholar 

  8. Vansteveninck, J., Weed, R., and Rothstein, A. 1965. Localization of erythrocyte membrane sulfhydryl groups essential for glucose transport. J. Gen. Physiol. 48:617–632.

    PubMed  Google Scholar 

  9. Knauf, P. A. and Rothstein, A. 1971a. Chemical modification of membranes: I. Effects of sulfhydryl and amino reactive reagents on anion and cation permeability of the human red blood cell. J. Gen. Physiol. 58:190–210.

    PubMed  Google Scholar 

  10. Knauf, P. A. and Rothstein, A. 1971b. Chemical modification of membranes: II. Permeation paths for sulfhydryl agents. J. Gen. Physiol. 58:211–223.

    PubMed  Google Scholar 

  11. Hellman, B., Lernmark, A., Sehlin, J., Soderberg, M., and Taljedal, I. 1973. The pancreatic beta-cell recognition of insulin secretagogues: VII. binding and permeation of chloromercuribenzene-p-sulphonic acid in the plasma membrane of pancreatic beta-cells. Arch. Biochem. Biophys. 158:434–441.

    Google Scholar 

  12. Shapiro, B., Kollmann, G., and Martin, D. 1970. The diversity of sulfhydryl groups in the human erythrocyte membrane. J. Cell Physiol. 75:281–292.

    PubMed  Google Scholar 

  13. Rothstein, A. 1970. Sulfhydryl groups in membrane structure and function. Curr. Top. Memb. Transp. 1:135–176.

    Google Scholar 

  14. Zhu, H., Duerr, J. S., Varoqui, H., McManus, J. R., Rand, J. B., and Erickson, J. D. 2001. Analysis of point mutants in the Caenorhabditis elegans vesicular acetylcholine transporter reveals domains involved in substrate translocation. J. Biol. Chem. 276:41580–41587.

    PubMed  Google Scholar 

  15. Wilkinson, G., Stone, F. G. A., and Abel, E. W. eds. 1982. Comprehensive Organometallic Chemistry, Vol. 2, Pages 931–933, Pergamon Press, Oxford, New York.

    Google Scholar 

  16. Waugh, T. D., Walton, F. H., and Laswick, J. A. 1955. Ionization constants of some organomercuric hydroxides and halides. J. Phys. Chem. 59:395–399.

    Google Scholar 

  17. Merickel, A., Kaback, H. R., and Edwards, R. H. 1997. Charged residues in transmembrane domains II and XI of a vesicular monoamine transporter form a charge pair that promotes high affinity substrate recognition. J. Biol. Chem. 272:5403–5408.

    PubMed  Google Scholar 

  18. Kim, M.-H., Lu, M., Lim, E.-J., Chai, Y.-G., and Hersh, L. B. 1999. Mutational analysis of aspartate residues in the transmembrane regions and cytoplasmic loops of rat vesicular acetylcholine transporter. J. Biol. Chem. 274:673–680.

    PubMed  Google Scholar 

  19. Ripoll, D. R., Faerman, C. H., Axelsen, P. H., Silman, I., and Sussman, J. L. 1993. An electrostatic mechanism for substrate guidance down the aromatic gorge of acetylcholinesterase. Proc. Natl. Acad. Sci. USA 90:5128–5132.

    PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Chemistry and Biochemistry and The Neuroscience Research Institute, University of California, Santa Barbara, California, 93106

    James E. Keller & Stanley M. Parsons

Authors
  1. James E. Keller
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Stanley M. Parsons
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Keller, J.E., Parsons, S.M. Diffusion Pathways to Critical Cysteines in the Vesicular Acetylcholine Transporter of Torpedo . Neurochem Res 28, 477–482 (2003). https://doi.org/10.1023/A:1022856919926

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1023/A:1022856919926

Search

Navigation