Characterization of human recombinant neuronal nicotinic acetylcholine receptor subunit combinations alpha2beta4, alpha3beta4 and alpha4beta4 stably expressed in HEK293 cells.

Article Details

Citation

Stauderman KA, Mahaffy LS, Akong M, Velicelebi G, Chavez-Noriega LE, Crona JH, Johnson EC, Elliott KJ, Gillespie A, Reid RT, Adams P, Harpold MM, Corey-Naeve J

Characterization of human recombinant neuronal nicotinic acetylcholine receptor subunit combinations alpha2beta4, alpha3beta4 and alpha4beta4 stably expressed in HEK293 cells.

J Pharmacol Exp Ther. 1998 Feb;284(2):777-89.

PubMed ID
9454827 [ View in PubMed
]
Abstract

Human embryonic kidney (HEK293) cells were transfected with cDNA encoding the human beta4 neuronal nicotinic acetylcholine (ACh) receptor subunit in pairwise combination with human alpha2, alpha3 or alpha4 subunits. Cell lines A2B4, A3B4.2 and A4B4 were identified that stably express mRNA and protein corresponding to alpha2 and beta4, to alpha3 and beta4 and to alpha4 and beta4 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 with Bmax (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: alpha2beta4, (+)epibatidine (Epi) > Cyt > suberyldicholine (Sub) = Nic = DMPP; alpha3beta4, Epi > DMPP = Cyt = Nic = Sub; alpha4beta4, 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-beta-erythroidine (DHbetaE) was highest at alpha3beta4 compared with alpha2beta4 or alpha4beta4 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 alpha2beta4, alpha3beta4 and alpha4beta4 neuronal nicotinic receptors.

DrugBank Data that Cites this Article

Drug Targets
DrugTargetKindOrganismPharmacological ActionActions
EpibatidineNeuronal acetylcholine receptor subunit alpha-2ProteinHumans
Unknown
Agonist
Details
EpibatidineNeuronal acetylcholine receptor subunit alpha-3ProteinHumans
Unknown
Agonist
Details
EpibatidineNeuronal acetylcholine receptor subunit alpha-4ProteinHumans
Unknown
Agonist
Details
EpibatidineNeuronal acetylcholine receptor subunit beta-4ProteinHumans
Unknown
Agonist
Details
Binding Properties
DrugTargetPropertyMeasurementpHTemperature (°C)
MecamylamineNeuronal acetylcholine receptor subunit alpha-2Ki (nM)3060N/AN/ADetails
NicotineNeuronal acetylcholine receptor subunit alpha-2Ki (nM)9900N/AN/ADetails