Functional characterization of mouse a4b2 nicotinic acetylcholine receptors stably expressed in HEK293T cells

Mouse a4b2 nicotinic acetylcholine receptors (nAchRs) were stably expressed in HEK293T cells. The function of this stable cell line, termed mma4b2, was assessed using an aequorinbased 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 a4b2 channel, although release of calcium from intracellular stores contributed ~ 28% of the agonistevoked response. Agonist pharmacologies were very similar between the mma4b2 cells and most cell lines that stably express human a4b2 nAchRs. Based on agonist profiles and sensitivity to the antagonist dihydro-b-erythroidine (DHbE), the predominant a4b2 nAchR expressed in the mma4b2 cells exhibits a pharmacology that most resembles the DHbE-sensitive component of Rb efflux from mouse brain synaptosomes. However, when evaluated with the aequorin assay, the mma4b2 nAchR was found to be atypically sensitive to blockade by the presumed a7-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 b2-subunit-containing nAchRs and not a7-subunit-containing nAchRs. Consequently, at low nanomolar concentrations, MLA may not be as selective for a7containing nAchRs as previously thought.