Cellular and Molecular Neurobiology

, Volume 29, Issue 1, pp 41–53 | Cite as

Contribution of Position α4S336 on Functional Expression and Up-regulation of α4β2 Neuronal Nicotinic Receptors

  • Gretchen Y. López-Hernández
  • Nilza M. Biaggi-Labiosa
  • Alexis Torres-Cintrón
  • Alejandro Ortiz-Acevedo
  • José A. Lasalde-Dominicci
Original Paper

Abstract

Phosphorylation of the nicotinic acetylcholine receptor (nAChR) is believed to play a critical role in its nicotine-induced desensitization and up-regulation. We examined the contribution of a consensus PKC site in the α4 M3/M4 intracellular loop (α4S336) on the desensitization and up-regulation of α4β2 nAChRs expressed in oocytes. Position α4S336 was replaced with either alanine to abolish potential phosphorylation at this site or with aspartic acid to mimic phosphorylation at this same site. Mutations α4S336A and α4S336D displayed a threefold increase in the ACh-induced response and an increase in ACh EC50. Epibatidine binding revealed a three and sevenfold increase in surface expression for the α4S336A and α4S336D mutations, respectively, relative to wild-type, therefore, both mutations enhanced expression of the α4β2 nAChR. Interestingly, the EC50’s and peak currents for nicotine activation remained unaffected in both mutants. Both mutations abolished the nicotine-induced up-regulation that is normally observed in the wild-type. The present data suggest that adding or removing a negative charge at this phosphorylation site cannot be explained by a simple straightforward on-and-off mechanism; rather a more complex mechanism(s) may govern the functional expression of the α4β2 nAChR. Along the same line, our data support the idea that phosphorylation at multiple consensus sites in the α4 subunit could play a remarkable role on the regulation of the functional expression of the α4β2 nAChR.

Keywords

Whole-mount immunofluorescence assay Epibatidine binding Nicotine Voltage-clamp Confocal imaging 

Notes

Acknowledgments

We thank Mr. José Serrano, Dr. Janice Salas, and Dr. Amelia Rivera for valuable technical assistance. This research was supported by National Institutes of Health Grants NIH 2RO1GM56371-11, SNRP-U54N54301, and GM08102-27. The Research Initiative for Scientific Enhancement-Minority Biomedical Research Support (RISE-MBRS)-NIH program (5R25GM61151) supported Gretchen Y. López-Hernández. Nilza M. Biaggi-Labiosa was supported by the RISE-MBRS-NIH program (2R25GM061151). Alexis Torres-Cintrón was supported by the Minority Access to Research Careers (MARC)-MBRS-NIH program.

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Copyright information

© Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • Gretchen Y. López-Hernández
    • 1
  • Nilza M. Biaggi-Labiosa
    • 2
  • Alexis Torres-Cintrón
    • 3
  • Alejandro Ortiz-Acevedo
    • 4
  • José A. Lasalde-Dominicci
    • 2
  1. 1.Department of Pharmacology and TherapeuticsUniversity of FloridaGainesvilleUSA
  2. 2.Department of BiologyUniversity of Puerto RicoSan JuanUSA
  3. 3.Department of Chemistry and Chemical BiologyCornell UniversityIthacaUSA
  4. 4.Department of Natural SciencesUniversity of Puerto RicoUtuadoUSA

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