title: Study of the calcium dynamics of the human alpha4beta2, alpha3beta4 and alpha1beta1gammadelta nicotinic acetylcholine receptors.
abstract: In this study three major subtypes of nicotinic acetylcholine receptors were characterized pharmacologically using the calcium influx through the ion channel as a robust functional assay system. Human alpha3beta4 receptors and alpha4beta2 receptors were cloned and stably expressed in HEK293 cells. [(125)I]epibatidine saturation binding yielded a B(max) of 4420+/-840 fmol/mg protein for the alpha4beta2 receptor ( n=4) and 518+/-15 fmol/mg protein for the alpha3beta4 receptor ( n=4). As a source for muscle type of nicotinic receptor, the TE671 cell line was used which expresses endogenously the human fetal alpha1beta1gammadelta subtype of nicotinic receptor. Stimulation of these nicotinic receptor subtypes in the different cell lines led to calcium transients that peaked 5-10 s after agonist application and declined thereafter. Eleven agonists were tested in this study and their efficacy and potency at the three nicotinic receptor subtypes were determined (epibatidine, ABT594, anatoxin, ABT418, nicotine, DMPP, cytisine, ABT089, choline, GTS21, AAR17779). This pharmacological characterization of agonist-induced elevation of intracellular free Ca(2+) revealed a distinct rank order of agonist potency for each receptor subtype. Epibatidine showed at all three subtypes the highest potency and was a full agonist. The agonist-elicited response could be blocked by co-incubation of different antagonists from which mecamylamine did not display a strong subtype specificity. These data illustrate that the assessment of calcium transients upon receptor stimulation is a powerful tool for rapid examination of the functional properties of nicotinic receptors.
authors.affiliation: Nervous System Research, Novartis Pharma AG, 4002 Basel, Switzerland.
authors.forename: Sandra
authors.initials: S
authors.lastname: Michelmore
authors.forename: Kim
authors.initials: K
authors.lastname: Croskery
authors.forename: Joachim
authors.initials: J
authors.lastname: Nozulak
authors.forename: Daniel
authors.initials: D
authors.lastname: Hoyer
authors.forename: Rémy
authors.initials: R
authors.lastname: Longato
authors.forename: Anja
authors.initials: A
authors.lastname: Weber
authors.forename: Rochdi
authors.initials: R
authors.lastname: Bouhelal
authors.forename: Dominik
authors.initials: D
authors.lastname: Feuerbach
mesh_terms: D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005453:Fluorescence; D006801:Humans; D007668:Kidney / Q000378:metabolism; D018722:Nicotinic Agonists / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology; D011869:Radioligand Assay; D011978:Receptors, Nicotinic / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*
chemical_list: D019086:Bridged Bicyclo Compounds, Heterocyclic; D015220:Calcium Channels; D018722:Nicotinic Agonists; D011725:Pyridines; D011978:Receptors, Nicotinic; C403530:nicotinic receptor alpha3beta4; C403531:nicotinic receptor alpha4beta2; C082748:epibatidine; D002118:Calcium