title: Functional characterization of mouse alpha4beta2 nicotinic acetylcholine receptors stably expressed in HEK293T cells.
abstract: Mouse alpha4beta2 nicotinic acetylcholine receptors (nAchRs) were stably expressed in HEK293T cells. The function of this stable cell line, termed mmalpha4beta2, was assessed using an aequorin-based luminescence method that measures agonist-evoked changes in intracellular calcium. Agonist-elicited changes in intracellular calcium were due primarily to direct entry of calcium through the alpha4beta2 channel, although release of calcium from intracellular stores contributed approximately 28% of the agonist-evoked response. Agonist pharmacologies were very similar between the mmalpha4beta2 cells and most cell lines that stably express human alpha4beta2 nAchRs. Based on agonist profiles and sensitivity to the antagonist dihydro-beta-erythroidine (DHbetaE), the predominant alpha4beta2 nAchR expressed in the mmalpha4beta2 cells exhibits a pharmacology that most resembles the DHbetaE-sensitive component of 86Rb+ efflux from mouse brain synaptosomes. However, when evaluated with the aequorin assay, the mmalpha4beta2 nAchR was found to be atypically sensitive to blockade by the presumed alpha7-selective antagonist methyllycaconitine (MLA), exhibiting an IC50 value of 31 +/- 0.1 nm. Similar IC50 values have been reported for the MLA inhibition of nicotine-stimulated dopamine release, a response that is mediated by beta2-subunit-containing nAchRs and not alpha7-subunit-containing nAchRs. Consequently, at low nanomolar concentrations, MLA may not be as selective for alpha7-containing nAchRs as previously thought.
authors.affiliation: Department of Pharmacology, University of Michigan Medical School, Ann Arbor, Michigan, USA.
authors.forename: Mark S
authors.initials: MS
authors.lastname: Karadsheh
authors.forename: M Salman
authors.initials: MS
authors.lastname: Shah
authors.forename: Xin
authors.initials: X
authors.lastname: Tang
authors.forename: Robert L
authors.initials: RL
authors.lastname: Macdonald
authors.forename: Jerry A
authors.initials: JA
authors.lastname: Stitzel
mesh_terms: D000109:Acetylcholine / Q000494:pharmacology; D000331:Aequorin / Q000378:metabolism; D000818:Animals; D001921:Brain / Q000166:cytology / Q000187:drug effects; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000493:pharmacokinetics / Q000494:pharmacology; D019256:Cadmium Chloride / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005110:Extracellular Space / Q000187:drug effects / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D020128:Inhibitory Concentration 50; D047028:Macrocyclic Compounds; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D051379:Mice; D018722:Nicotinic Agonists / Q000493:pharmacokinetics / Q000494:pharmacology; D018733:Nicotinic Antagonists / Q000494:pharmacology; D010080:Oxazoles / Q000494:pharmacology; D018408:Patch-Clamp Techniques / Q000379:methods; D011725:Pyridines / Q000493:pharmacokinetics / Q000494:pharmacology; D011869:Radioligand Assay / Q000379:methods; D011978:Receptors, Nicotinic / Q000235:genetics / Q000502:physiology*; D012414:Rubidium Radioisotopes / Q000378:metabolism; D012433:Ryanodine / Q000494:pharmacology; D013574:Synaptosomes / Q000187:drug effects / Q000378:metabolism; D014162:Transfection / Q000379:methods
chemical_list: D019086:Bridged Bicyclo Compounds, Heterocyclic; D004791:Enzyme Inhibitors; D047028:Macrocyclic Compounds; D018722:Nicotinic Agonists; D018733:Nicotinic Antagonists; D010080:Oxazoles; D011725:Pyridines; D011978:Receptors, Nicotinic; D012414:Rubidium Radioisotopes; C403531:nicotinic receptor alpha4beta2; C108644:xestospongin A; D012433:Ryanodine; D000331:Aequorin; D019256:Cadmium Chloride; C082748:epibatidine; D000109:Acetylcholine; D002118:Calcium