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Volume 16, Number 24,
Issue of December 15, 1996
pp. 7880-7891
Copyright ©1996 Society for Neuroscience
Human  4 2 Neuronal Nicotinic Acetylcholine Receptor in HEK
293 Cells: A Patch-Clamp Study
Received Aug. 2, 1996; revised Sept. 16, 1996; accepted Sept. 30, 1996.
Bruno Buisson1,
Murali Gopalakrishnan2,
Stephen P. Arneric2,
James P. Sullivan2, and
Daniel Bertrand1
1 Department of Physiology, Faculty of Medicine,
University of Geneva, CH-1211 Geneva 4, Switzerland, and
2 Neuroscience Research, Abbott Laboratories, Abbott Park,
Illinois 60064-3500
The cloning and expression of genes encoding for the human neuronal
nicotinic acetylcholine receptors (nAChRs) has opened new possibilities
for investigating their physiological and pharmacological properties.
Cells (HEK 293) stably transfected with two of the major brain
subunits,  4 and  2, were characterized electrophysiologically using the patch-clamp technique. Fast application of the natural ligand
ACh can evoke currents up to 3500 pA, with an apparent affinity
(EC50) of 3 µM and a Hill coefficient of 1.2. The rank order of potency of four nAChR ligands to activate human
42 receptors is ( )-nicotine > ACh > ()-cytisine > ABT-418. At saturating concentrations, the
efficacy of these ligands is ABT-418  ()-nicotine > ACh
()-cytisine > GTS-21 (previously named DMXB).
Coapplication of 1 µM ACh with known nAChR inhibitors
such as dihydro--erythroidine and methyllycaconitine reversibly
reduces the current evoked by the agonist with respective
IC50 values of 80 nM and 1.5 µM.
The current-voltage relationship of human 42 displays a strong
rectification at positive potentials. Experiments of ionic
substitutions suggest that human 42 nAChRs are permeable to
sodium and potassium ions. In the ``outside-out'' configuration, ACh
evokes unitary currents (main conductance 46 pS) characterized by a
very fast rundown. Potentiation of the ACh-evoked currents is observed
when the extracellular calcium concentration is increased from 0.2 to 2 mM. In contrast, however, a reduction of the evoked
currents is observed when calcium concentration is elevated above 2 mM.
Key words:
human;
42;
neuronal;
nicotinic;
acetylcholine;
receptor
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