α7β2 Nicotinic Acetylcholine Receptors Assemble, Function, and Are Activated Primarily via Their α7-α7 Interfaces

  1. Teresa A. Murray,
  2. Daniel Bertrand,
  3. Roger L. Papke,
  4. Andrew A. George,
  5. Rigo Pantoja,
  6. Rahul Srinivasan,
  7. Qiang Liu,
  8. Jie Wu,
  9. Paul Whiteaker,
  10. Henry A. Lester and
  11. Ronald J. Lukas
  1. Barrow Neurological Institute, Phoenix, Arizona (T.A.M., A.A.G., Q.L., J.W., P.W., R.J.L.); Biomedical Engineering Department, Louisiana Tech University, Ruston, Louisiana (T.A.M.); Division of Biology, California Institute of Technology, Pasadena, California (R.P., R.S., H.A.L.); Department of Neuroscience, Centre Médical Universitaire, and HiQScreen Sàrl, Geneva, Switzerland (D.B.); and Department of Pharmacology and Therapeutics, College of Medicine, University of Florida, Gainesville, Florida (R.L.P.)
  1. Address correspondence to:
    Dr. Teresa A. Murray, Louisiana Tech University, Biomedical Engineering Center, PO Box 10157, Ruston, LA 71272-0046. E-mail: bioengineer1{at}hotmail.com

Abstract

We investigated assembly and function of nicotinic acetylcholine receptors (nAChRs) composed of α7 and β2 subunits. We measured optical and electrophysiological properties of wild-type and mutant subunits expressed in cell lines and Xenopus laevis oocytes. Laser scanning confocal microscopy indicated that fluorescently tagged α7 and β2 subunits colocalize. Förster resonance energy transfer between fluorescently tagged subunits strongly suggested that α7 and β2 subunits coassemble. Total internal reflection fluorescence microscopy revealed that assemblies localized to filopodia-like processes of SH-EP1 cells. Gain-of-function α7 and β2 subunits confirmed that these subunits coassemble within functional receptors. Moreover, α7β2 nAChRs composed of wild-type subunits or fluorescently tagged subunits had pharmacological properties similar to those of α7 nAChRs, although amplitudes of α7β2 nAChR-mediated, agonist-evoked currents were generally ∼2-fold lower than those for α7 nAChRs. It is noteworthy that α7β2 nAChRs displayed sensitivity to low concentrations of the antagonist dihydro-β-erythroidine that was not observed for α7 nAChRs at comparable concentrations. In addition, cysteine mutants revealed that the α7-β2 subunit interface does not bind ligand in a functionally productive manner, partly explaining lower α7β2 nAChR current amplitudes and challenges in identifying the function of native α7β2 nAChRs. On the basis of our findings, we have constructed a model predicting receptor function that is based on stoichiometry and position of β2 subunits within the α7β2 nAChRs.

Footnotes

  • ↵Graphic The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.

  • This work was supported by the National Institutes of Health National Institute on Drug Abuse [Grants DA015389, DA027070, DA012242]; the National Institutes of Health National Institute of Neurological Diseases and Stroke [Grant NS11756]; the National Institutes of Health National Institute of Mental Health [Grant MH086383]; the National Institutes of Health National Institute of General Medical Sciences [Grant GM057481]; a US National Science Foundation Graduate Research Fellowship; the Barrow Neurological Foundation; a Catholic Healthcare West SEED Grant; the Biodesign Institute at Arizona State University, the Swiss National Science Foundation; the EC Neurocypres Grant; and the California Tobacco-Related Disease Research Program.

  • Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.

    http://dx.doi.org/10.1124/mol.111.074088.

  • ABBREVIATIONS:

    nAChR
    nicotinic acetylcholine receptor
    MS/DB
    medial septum-diagonal band
    YFP
    yellow fluorescent protein
    CFP
    cyan fluorescent protein
    GluCl
    glutamate-gated chloride channel
    α4Y
    YFP-tagged nAChR α4 subunit; α7C YFP-tagged nAChR α7 subunit
    α7Y
    CFP-tagged nAChR α7 subunit
    β2C
    CFP-tagged nAChR β2 subunit
    β2Ch
    mCherry-tagged nAChR β2 subunit
    GCαY
    YFP-tagged glutamate-gated chloride channel α subunit
    GCβC
    CFP-tagged glutamate-gated chloride channel β subunit
    LSCM
    laser scanning confocal microscopy
    FRET
    Förster resonance energy transfer
    TIRF
    total internal reflection fluorescence
    BAPTA
    1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid
    MTSEA
    methanethiosulfonate ethylammonium
    ACh
    acetylcholine
    MLA
    methyllycaconitine
    FP
    fusion protein
    ER
    endoplasmic reticulum
    ROI
    region of interest
    DHβE
    dihydro-β-erythroidine
    PNU-282987
    N-(3R)-1-azabicyclo[2.2.2]oct-3-yl-4-chlorobenzamide
    E
    FRET efficiency
    ID
    intensity of donor FP after photodestruction of acceptor
    IDA
    intensity of donor FP in the presence of the unbleached acceptor
    In
    normalized fluorescence intensity.

  • Received June 12, 2011.
  • Accepted October 28, 2011.
| Table of Contents

This Article

  1. Published online before print October 28, 2011, doi: 10.1124/mol.111.074088 Molecular Pharmacology February 2012 vol. 81 no. 2 175-188
  1. » Abstract
  2. Full Text
  3. Full Text (PDF)
  4. Supplemental Data
  5. All Versions of this Article:
    1. mol.111.074088v1
    2. 81/2/175 most recent

Classifications

Responses

  1. Submit a response
  2. No responses published

Current Issue

  1. February 2012, 81 (2)
  1. Current Issue
  1. Alert me to new issues of Molecular Pharmacology
  • ASPET Pharmacology Education Division
Society Logo HighWire Press Logo CLOCKSS logo