Functional Deactivation of the Major Neuronal Nicotinic Receptor Caused by Nicotine and a Protein Kinase C-Dependent Mechanism

  1. Helge Eilers1,
  2. Eric Schaeffer2,
  3. Philip E. Bickler1 and
  4. John R. Forsayeth3
  1. 1Department of Anesthesia, University of California San Francisco, San Francisco, California 94143-0542 (H.E., P.E.B.), 2Pfizer, Inc., Central Research Division, Groton, Connecticut 06340 (E.S.), and 3Neurex Corporation, Menlo Park, California 94025 (J.R.F.)

    Abstract

    The effect of nicotine on the major human neuronal nicotinic receptor (α4β2 subtype) was studied in permanently transfected HEK 293 cells. Prolonged exposure to low concentrations of nicotine (1 μm) increased epibatidine binding but functionally deactivated the nicotinic receptor, abolishing Ca2+ influx in response to an acute nicotine challenge. Deactivation could also be caused by down-regulating protein kinase C (PKC) activity with 0.5 μm phorbol-12,13-dibutyrate or briefly incubating cells with the PKC inhibitor NPC-15437. Recovery from receptor deactivation caused by either nicotine treatment or PKC inhibition occurred slowly (4–6 hr). Reversal of nicotine-induced deactivation was accelerated by the addition of inhibitors of protein phosphatases 2A and 2B. These data suggest a hypothetical mechanism of nicotine-induced deactivation that involves dephosphorylation of nicotinic receptors at PKC phosphorylation sites.

    Footnotes

    • Send reprint requests to: Dr. John R. Forsayeth, Neurex Corporation, 3760 Haven Avenue, Menlo Park, CA 94025-1012. E-mail:johnf{at}neurex.com

    • This work was supported by a postdoctoral fellowship from the Deutsche Forschungsgemeinschaft (H.E.) and by a grant from Pfizer, Inc. (J.F.).

    • Abbreviations:
      nAChR
      neuronal nicotinic acetylcholine receptor
      HEK
      human embryonic kidney
      PKC
      protein kinase C
      EGTA
      ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid
      PKA
      protein kinase A
      PDBu
      phorbol-12,13-dibutyrate
      RT
      reverse transcription
      PCR
      polymerase chain reaction
      PP
      protein phosphatase
      PBS
      phosphate-buffered saline
      ANOVA
      analysis of variance
      • Received April 18, 1997.
      • Accepted August 16, 1997.
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    1. Molecular Pharmacology December 1, 1997 vol. 52 no. 6 1105-1112
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