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OtherCELLULAR AND MOLECULAR PHARMACOLOGY

Characterization of Human Recombinant Neuronal Nicotinic Acetylcholine Receptor Subunit Combinations α2β4, α3β4 and α4β4 Stably Expressed in HEK293 Cells

Kenneth A. Stauderman, L. Scott Mahaffy, Michael Akong, Gönül Veliçelebi, Laura E. Chavez-Noriega, James H. Crona, Edwin C. Johnson, Kathryn J. Elliott, Alison Gillespie, Richard T. Reid, Pamala Adams, Michael M. Harpold and Janis Corey-Naeve
Journal of Pharmacology and Experimental Therapeutics February 1998, 284 (2) 777-789;
Kenneth A. Stauderman
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L. Scott Mahaffy
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Michael Akong
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Gönül Veliçelebi
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Laura E. Chavez-Noriega
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James H. Crona
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Edwin C. Johnson
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Kathryn J. Elliott
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Alison Gillespie
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Richard T. Reid
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Pamala Adams
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Michael M. Harpold
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Janis Corey-Naeve
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Abstract

Human embryonic kidney (HEK293) cells were transfected with cDNA encoding the human β4 neuronal nicotinic acetylcholine (ACh) receptor subunit in pairwise combination with human α2, α3 or α4 subunits. Cell lines A2B4, A3B4.2 and A4B4 were identified that stably express mRNA and protein corresponding to α2 and β4, to α3 and β4 and to α4 and β4 subunits, respectively. Specific binding of [3H]epibatidine was detected in A2B4, A3B4.2 and A4B4 cells with Kd (mean ± S.D. in pM) values of 42 ± 10, 230 ± 12 and 187 ± 29 and withBmax (fmol/mg protein) values of 1104 ± 338, 2010 ± 184 and 3683 ± 1450, respectively. Whole-cell patch-clamp recordings in each cell line demonstrated that (−)nicotine (Nic), ACh, cytisine (Cyt) and 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP) elicit transient inward currents. The current-voltage (I-V) relation of these currents showed strong inward rectification. Pharmacological characterization of agonist-induced elevations of intracellular free Ca++concentration revealed a distinct rank order of agonist potency for each subunit combination as follows: α2β4, (+)epibatidine (Epi) > Cyt > suberyldicholine (Sub) = Nic = DMPP; α3β4, Epi > DMPP = Cyt = Nic = Sub; α4β4, Epi > Cyt = Sub > Nic > DMPP. The noncompetitive antagonists mecamylamine and d-tubocurarine did not display subtype selectivity. In contrast, the Kb value for the competitive antagonist dihydro-β-erythroidine (DHβE) was highest at α3β4 compared with α2β4 or α4β4 receptors. These data illustrate that the A2B4, A3B4.2 and A4B4 stable cell lines are powerful tools for examining the functional and pharmacological properties of human α2β4, α3β4 and α4β4 neuronal nicotinic receptors.

Footnotes

  • Send reprint requests to: Kenneth A. Stauderman, SIBIA Neurosciences, Inc., 505 Coast Blvd. So., Suite 300, La Jolla, CA 92037-4641.

  • Abbreviations:
    [Ca++]i
    intracellular free Ca++ concentration
    DHβE
    dihydro-β-erythroidine
    HBK
    HEPES-buffered Krebs-saline
    PBS
    phosphate-buffered saline
    SDS
    sodium dodecyl sulfate
    ACh
    acetylcholine
    Epi
    (+)epibatidine
    Nic
    (−)nicotine
    DMPP
    1,1-dimethyl-4-phenylpiperazinium iodide
    Cyt
    cytisine
    Sub
    suberyldicholine
    EC50
    concentration of agonist producing half-maximal effect
    IC50
    concentration of antagonist producing half-maximal inhibition
    • Received June 24, 1997.
    • Accepted October 15, 1997.
  • The American Society for Pharmacology and Experimental Therapeutics
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Journal of Pharmacology and Experimental Therapeutics
Vol. 284, Issue 2
1 Feb 1998
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OtherCELLULAR AND MOLECULAR PHARMACOLOGY

Characterization of Human Recombinant Neuronal Nicotinic Acetylcholine Receptor Subunit Combinations α2β4, α3β4 and α4β4 Stably Expressed in HEK293 Cells

Kenneth A. Stauderman, L. Scott Mahaffy, Michael Akong, Gönül Veliçelebi, Laura E. Chavez-Noriega, James H. Crona, Edwin C. Johnson, Kathryn J. Elliott, Alison Gillespie, Richard T. Reid, Pamala Adams, Michael M. Harpold and Janis Corey-Naeve
Journal of Pharmacology and Experimental Therapeutics February 1, 1998, 284 (2) 777-789;

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OtherCELLULAR AND MOLECULAR PHARMACOLOGY

Characterization of Human Recombinant Neuronal Nicotinic Acetylcholine Receptor Subunit Combinations α2β4, α3β4 and α4β4 Stably Expressed in HEK293 Cells

Kenneth A. Stauderman, L. Scott Mahaffy, Michael Akong, Gönül Veliçelebi, Laura E. Chavez-Noriega, James H. Crona, Edwin C. Johnson, Kathryn J. Elliott, Alison Gillespie, Richard T. Reid, Pamala Adams, Michael M. Harpold and Janis Corey-Naeve
Journal of Pharmacology and Experimental Therapeutics February 1, 1998, 284 (2) 777-789;
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