The Journal of Membrane Biology

, Volume 214, Issue 3, pp 131–138 | Cite as

The Polarity of Lipid-Exposed Residues Contributes to the Functional Differences between Torpedo and Muscle-Type Nicotinic Receptors

  • Gisila R. Guzmán
  • Alejandro Ortiz-Acevedo
  • Ariamsi Ricardo
  • Legier V. Rojas
  • José A. Lasalde-Dominicci
Article

Abstract

A comparison between the Torpedo and muscle-type acetylcholine receptors (AChRs) reveals differences in several lipid-exposed amino acids, particularly in the polarity of those residues. The goal of this study was to characterize the role of eight lipid-exposed residues in the functional differences between the Torpedo and muscle-type AChRs. To this end, residues αS287, αC412, βY441, γM299, γS460, δM293, δS297 and δN305 in the Torpedo AChR were replaced with those found in the muscle-type receptor. Mutant receptor expression was measured in Xenopus oocytes using [125I]-α-bungarotoxin, and AChR ion channel function was evaluated using the two-electrode voltage clamp. Eight mutant combinations resulted in an increase (1.5- to 5.2-fold) in AChR expression. Four mutant combinations produced a significant 46% decrease in the ACh 50% inhibitory concentration (EC50), while three mutant combinations resulted in 1.7- to 2-fold increases in ACh EC50. Finally, seven mutant combinations resulted in a decrease in normalized, ACh-induced currents. Our results suggest that these residues, although remote from the ion channel pore, (1) contribute to ion channel gating, (2) may affect trafficking of AChR into specialized membrane domains and (3) account for the functional differences between Torpedo and muscle-type AChR. These findings emphasize the importance of the lipid-protein interface in the functional differences between the Torpedo and muscle-type AChRs.

Keywords

Acetylcholine receptor Site-directed mutagenesis Xenopus oocyte Lipid-exposed residue Torpedo californica Muscle-type receptor 

Notes

Acknowledgement

This work was supported in part by grants from the National Institutes of Health: 2RO1GM5637-10 and GM08102-27, as well as UPR Institutional Funds for Research awarded to J.A. Lasalde-Dominicci. G. Guzman was supported by NSF-AGEP grant HRD-9817642 and A. Ricardo by NIH-MARC grant 5T34GM07821.

References

  1. Akabas M.H., Karlin A. 1995. Identification of acetylcholine receptor channel-lining residues in the M1 segment of the alpha-subunit. Biochemistry 34:12496–12500PubMedCrossRefGoogle Scholar
  2. Blanton M.P., Cohen J.B. 1992. Mapping the lipid-exposed regions in the Torpedo californica nicotinic acetylcholine receptor. Biochemistry 31:3738–3750. Erratum in Biochemistry 31:5951PubMedCrossRefGoogle Scholar
  3. Blanton M.P., Cohen J.B. 1994. Identifying the lipid-protein interface of the Torpedo nicotinic acetylcholine receptor: Secondary structure implications. Biochemistry 33:2859–2872PubMedCrossRefGoogle Scholar
  4. Blanton M.P., Dangott L.J., Raja S.K., Lala A.K., Cohen J.B. 1998. Probing the structure of the nicotinic acetylcholine receptor ion channel with the uncharged photoactivable compound 3H-diazofluorene. J. Biol. Chem. 273:8659–8668PubMedCrossRefGoogle Scholar
  5. Bouzat C., Bren N., Sine S.M. 1994. Structural basis of the different gating kinetics of fetal and adult acetylcholine receptors. Neuron 13:1395–1402PubMedCrossRefGoogle Scholar
  6. Bouzat C., Roccamo A.M., Garbus. I., Barrantes F.J. 1998. Mutations at lipid-exposed residues of the acetylcholine receptor affect its gating kinetics. Mol. Pharmacol. 54:146–153PubMedGoogle Scholar
  7. Butler, D.H., Lasalde, J.A., Butler, J.K., Tamamizu, S., Zimmerman, G., McNamee, M.G. 1997. Mouse-Torpedo chimeric alpha-subunit used to probe channel-gating determinants on the nicotinic acetylcholine receptor primary sequence. Cell. Mol. Neurobiol. 17:13–33CrossRefGoogle Scholar
  8. Chothia C. 1975. Structural invariants in protein folding. Nature 254:304–308PubMedCrossRefGoogle Scholar
  9. Couet J., Li S., Okamoto T., Ikezu T., Lisanti M.P. 1997. Identification of peptide and protein ligands for the caveolin-scaffolding domain. Implications for the interaction of caveolin with caveolae-associated proteins. J. Biol. Chem. 272:6525–6533PubMedCrossRefGoogle Scholar
  10. Cruz-Martin A., Mercado J.L., Rojas L.V., McNamee M.G., Lasalde-Dominicci J.A. 2001. Tryptophan substitutions at lipid-exposed positions of the gamma M3 transmembrane domain increase the macroscopic ionic current response of the Torpedo californica nicotinic acetylcholine receptor. J. Membr. Biol. 183:61–70PubMedCrossRefGoogle Scholar
  11. Guzman G.R., Santiago J., Ricardo A., Marti-Arbona R., Rojas L.V., Lasalde-Dominicci J.A. 2003. Tryptophan scanning mutagenesis in the alpha M3 transmembrane domain of the Torpedo californica acetylcholine receptor: Functional and structural implications. Biochemistry 42:12243–12250PubMedCrossRefGoogle Scholar
  12. Lasalde, J.A., Tamamizu, S., Butler, D.H., Vibat, C.R., Hung, B., McNamee, M.G. 1996. Tryptophan substitutions at the lipid-exposed transmembrane segment M4 of Torpedo californica acetylcholine receptor govern channel gating. Biochemistry :14139–14148Google Scholar
  13. Lee Y.H., Li L., Lasalde J., Rojas L., McNamee M., Ortiz-Miranda S.I., Pappone P. 1994. Mutations in the M4 domain of Torpedo californica acetylcholine receptor dramatically alter ion channel function. Biophys. J. 66:646–653PubMedCrossRefGoogle Scholar
  14. Miyazawa A., Fujiyoshi Y., Unwin N. 2003. Structure and gating mechanism of the acetylcholine receptor pore. Nature 423:949–955PubMedCrossRefGoogle Scholar
  15. Naranjo D., Brehm P. 1993. Modal shifts in acetylcholine receptor channel gating confer subunit-dependent desensitization. Science 260:1811–1814PubMedCrossRefGoogle Scholar
  16. Navedo M., Nieves M., Rojas L., Lasalde-Dominicci J.A. 2004. Tryptophan substitutions reveal the role of nicotinic acetylcholine receptor alpha-TM3 domain in channel gating: differences between Torpedo and muscle-type AChR. Biochemistry 43:78–84PubMedCrossRefGoogle Scholar
  17. Noda M., Takahashi H., Tanabe T., Toyosato M., Kikyotani S., Furutani Y., Hirose T., Takashima H., Inayama S., Miyata T., Numa S. 1983. Structural homology of Torpedo californica acetylcholine receptor subunits. Nature 302:528–532PubMedCrossRefGoogle Scholar
  18. Mitra A., Bailey T.D., Auerbach A.L. 2004. Structural dynamics of the M4 transmembrane segment during acetylcholine receptor gating. Structure 12:1909–1918PubMedCrossRefGoogle Scholar
  19. Ortiz-Miranda S.I., Lasalde J.A., Pappone P.A., McNamee M.G. 1997. Mutations in the M4 domain of the Torpedo californica nicotinic acetylcholine receptor alter channel opening and closing. J. Membr. Biol. 158:17–30PubMedCrossRefGoogle Scholar
  20. Otero-Cruz J.D., Báez-Pagán C.A., Caraballo-González I.M., Lasalde-Dominicci J.A. 2006. A lipid-exposed transmembrane domain of ligand-gated ion channel receptor displays a tilted spring motion during channel activation, comparing Torpedo versus muscle-type acetylcholine Receptors. A SPRING MODEL REVEALED. J. Biol. Chem. 282:9162–9171CrossRefGoogle Scholar
  21. Santiago J., Guzman G.R., Rojas L.V., Marti R., Asmar-Rovira G.A., Santana L.F., McNamee M., Lasalde-Dominicci J.A. 2001. Probing the effects of membrane cholesterol in the Torpedo californica acetylcholine receptor and the novel lipid-exposed mutation alpha C418W in Xenopus oocytes. J. Biol. Chem. 276:46523–46532PubMedCrossRefGoogle Scholar
  22. Tamamizu S., Lee Y., Hung B., McNamee M.G., Lasalde-Dominicci J.A. 1999. Alteration in ion channel function of mouse nicotinic acetylcholine receptor by mutations in the M4 transmembrane domain. J. Membr. Biol. 170:157–164. Erratum in J. Membr. Biol. 1999;172:89PubMedCrossRefGoogle Scholar
  23. Tamamizu S., Guzman G.R., Santiago J., Rojas L.V., McNamee M.G., Lasalde-Dominicci J.A. 2000. Functional effects of periodic tryptophan substitutions in the alpha M4 transmembrane domain of the Torpedo californica nicotinic acetylcholine receptor. Biochemistry 39:4666–4673PubMedCrossRefGoogle Scholar
  24. Unwin N. 2005. Refined structure of the nicotinic acetylcholine receptor at 4Å resolution. J. Mol. Biol. 346:967–989PubMedCrossRefGoogle Scholar
  25. Wang H.L., Milone M., Ohno K., Shen X.M., Tsujino A., Batocchi A.P., Tonali P., Brengman J., Engel A.G., Sine S.M. 1999. Acetylcholine receptor M3 domain: Stereochemical and volume contributions to channel gating. Nat. Neurosci. 2:226–233. Erratum in Nat. Neurosci. 1999;2:485PubMedCrossRefGoogle Scholar
  26. Yu, L., Leonard, R.J., Davidson, N., Lester, H.A. 1991. Single-channel properties of mouse-Torpedo acetylcholine receptor hybrids expressed in Xenopus oocytes. Brain Res. Mol. Brain Res. :203–211Google Scholar

Copyright information

© Springer Science+Business Media, LLC 2007

Authors and Affiliations

  • Gisila R. Guzmán
    • 1
  • Alejandro Ortiz-Acevedo
    • 1
  • Ariamsi Ricardo
    • 1
  • Legier V. Rojas
    • 2
  • José A. Lasalde-Dominicci
    • 1
  1. 1.Department of BiologyUniversity of Puerto RicoSan Juan
  2. 2.Department of PhysiologySchool of Medicine, Universidad Central del CaribeBayamón

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