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.

    * Copyright © 2012 The American Society for Pharmacology and Experimental Therapeutics
