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Research ArticleNeuropharmacology

The Unique α4(+)/(−)α4 Agonist Binding Site in (α4)3(β2)2 Subtype Nicotinic Acetylcholine Receptors Permits Differential Agonist Desensitization Pharmacology versus the (α4)2(β2)3 Subtype

J. Brek Eaton, Linda M. Lucero, Harrison Stratton, Yongchang Chang, John F. Cooper, Jon M. Lindstrom, Ronald J. Lukas and Paul Whiteaker
Journal of Pharmacology and Experimental Therapeutics January 2014, 348 (1) 46-58; DOI: https://doi.org/10.1124/jpet.113.208389
J. Brek Eaton
Division of Neurobiology, Barrow Neurologic Institute, Phoenix, Arizona (J.B.E., L.M.L., H.S., Y.C., R.J.L., P.W.); and Department of Neuroscience, Medical School of the University of Pennsylvania, Philadelphia, Pennsylvania (J.F.C., J.M.L.)
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Linda M. Lucero
Division of Neurobiology, Barrow Neurologic Institute, Phoenix, Arizona (J.B.E., L.M.L., H.S., Y.C., R.J.L., P.W.); and Department of Neuroscience, Medical School of the University of Pennsylvania, Philadelphia, Pennsylvania (J.F.C., J.M.L.)
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Harrison Stratton
Division of Neurobiology, Barrow Neurologic Institute, Phoenix, Arizona (J.B.E., L.M.L., H.S., Y.C., R.J.L., P.W.); and Department of Neuroscience, Medical School of the University of Pennsylvania, Philadelphia, Pennsylvania (J.F.C., J.M.L.)
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Yongchang Chang
Division of Neurobiology, Barrow Neurologic Institute, Phoenix, Arizona (J.B.E., L.M.L., H.S., Y.C., R.J.L., P.W.); and Department of Neuroscience, Medical School of the University of Pennsylvania, Philadelphia, Pennsylvania (J.F.C., J.M.L.)
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John F. Cooper
Division of Neurobiology, Barrow Neurologic Institute, Phoenix, Arizona (J.B.E., L.M.L., H.S., Y.C., R.J.L., P.W.); and Department of Neuroscience, Medical School of the University of Pennsylvania, Philadelphia, Pennsylvania (J.F.C., J.M.L.)
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Jon M. Lindstrom
Division of Neurobiology, Barrow Neurologic Institute, Phoenix, Arizona (J.B.E., L.M.L., H.S., Y.C., R.J.L., P.W.); and Department of Neuroscience, Medical School of the University of Pennsylvania, Philadelphia, Pennsylvania (J.F.C., J.M.L.)
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Ronald J. Lukas
Division of Neurobiology, Barrow Neurologic Institute, Phoenix, Arizona (J.B.E., L.M.L., H.S., Y.C., R.J.L., P.W.); and Department of Neuroscience, Medical School of the University of Pennsylvania, Philadelphia, Pennsylvania (J.F.C., J.M.L.)
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Paul Whiteaker
Division of Neurobiology, Barrow Neurologic Institute, Phoenix, Arizona (J.B.E., L.M.L., H.S., Y.C., R.J.L., P.W.); and Department of Neuroscience, Medical School of the University of Pennsylvania, Philadelphia, Pennsylvania (J.F.C., J.M.L.)
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Abstract

Selected nicotinic agonists were used to activate and desensitize high-sensitivity (HS) (α4)2(β2)3) or low-sensitivity (LS) (α4)3(β2)2) isoforms of human α4β2-nicotinic acetylcholine receptors (nAChRs). Function was assessed using 86Rb+ efflux in a stably transfected SH-EP1-hα4β2 human epithelial cell line, and two-electrode voltage-clamp electrophysiology in Xenopus laevis oocytes expressing concatenated pentameric HS or LS α4β2-nAChR constructs (HSP and LSP). Unlike previously studied agonists, desensitization by the highly selective agonists A-85380 [3-(2(S)-azetidinylmethoxy)pyridine] and sazetidine-A (Saz-A) preferentially reduced α4β2-nAChR HS-phase versus LS-phase responses. The concatenated-nAChR experiments confirmed that approximately 20% of LS-isoform acetylcholine-induced function occurs in an HS-like phase, which is abolished by Saz-A preincubation. Six mutant LSPs were generated, each targeting a conserved agonist binding residue within the LS-isoform-only α4(+)/(−)α4 interface agonist binding site. Every mutation reduced the percentage of LS-phase function, demonstrating that this site underpins LS-phase function. Oocyte-surface expression of the HSP and each of the LSP constructs was statistically indistinguishable, as measured using β2-subunit–specific [125I]mAb295 labeling. However, maximum function is approximately five times greater on a “per-receptor” basis for unmodified LSP versus HSP α4β2-nAChRs. Thus, recruitment of the α4(+)/(−)α4 site at higher agonist concentrations appears to augment otherwise-similar function mediated by the pair of α4(+)/(−)β2 sites shared by both isoforms. These studies elucidate the receptor-level differences underlying the differential pharmacology of the two α4β2-nAChR isoforms, and demonstrate that HS versus LS α4β2-nAChR activity can be selectively manipulated using pharmacological approaches. Since α4β2 nAChRs are the predominant neuronal subtype, these discoveries likely have significant functional implications, and may provide important insights for drug discovery and development.

Footnotes

    • Received July 29, 2013.
    • Accepted November 1, 2013.
  • J.B.E. and L.M.L. contributed equally to this work.

  • This research was primarily supported by the National Institutes of Health National Institute on Drug Abuse [Grant R21-DA026627 (to P.W.)]. Additional funding was provided by the National Institutes of Health National Institute on Drug Abuse [Grant R01-DA012242 (to P.W.); R01-DA015389, R01-DA017980, U19-DA019375, and U19-DA019377 (to R.J.L.)]; the National Institutes of Health National Institute of Mental Health [Grant R01-MH085193 (to J.M.L)]; the National Institutes of Health National Institute of Neurological Disorders and Stroke [Grant R01-NS011323 (to J.M.L.)]; and the National Institutes of Health National Institute of General Medical Sciences [Grant R01-GM085237 (to Y.C.)]. The contents of this article do not necessarily reflect the views of the aforementioned awarding agencies.

  • dx.doi.org/10.1124/jpet.113.208389.

  • ↵Embedded ImageThis article has supplemental material available at jpet.aspetjournals.org.

  • Copyright © 2013 by The American Society for Pharmacology and Experimental Therapeutics
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Journal of Pharmacology and Experimental Therapeutics: 348 (1)
Journal of Pharmacology and Experimental Therapeutics
Vol. 348, Issue 1
1 Jan 2014
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The Unique α4(+)/(−)α4 Agonist Binding Site in (α4)3(β2)2 Subtype Nicotinic Acetylcholine Receptors Permits Differential Agonist Desensitization Pharmacology versus the (α4)2(β2)3 Subtype
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Research ArticleNeuropharmacology

HS vs. LS α4β2 Nicotinic Receptor Agonist Desensitization

J. Brek Eaton, Linda M. Lucero, Harrison Stratton, Yongchang Chang, John F. Cooper, Jon M. Lindstrom, Ronald J. Lukas and Paul Whiteaker
Journal of Pharmacology and Experimental Therapeutics January 1, 2014, 348 (1) 46-58; DOI: https://doi.org/10.1124/jpet.113.208389

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Research ArticleNeuropharmacology

HS vs. LS α4β2 Nicotinic Receptor Agonist Desensitization

J. Brek Eaton, Linda M. Lucero, Harrison Stratton, Yongchang Chang, John F. Cooper, Jon M. Lindstrom, Ronald J. Lukas and Paul Whiteaker
Journal of Pharmacology and Experimental Therapeutics January 1, 2014, 348 (1) 46-58; DOI: https://doi.org/10.1124/jpet.113.208389
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