Cell autonomy, receptor autonomy, and thermodynamics in nicotine receptor up-regulation

Abstract

Chronic nicotine exposure, in smokers or in experimental rodents administered nicotine, produces elevated levels of nicotinic acetylcholine receptors in several brain regions. However, there are few data on up-regulation of receptors in specific neuronal subtypes. We tested whether functional up-regulation of nicotinic responses occurs in cultured GABAergic neurons of the ventral midbrain. Fura-2 measurements of nicotinic responses were made on ventral midbrain neurons from knock-in mice heterozygous for the α4-M2 domain Leu9′Ala mutation, which confers nicotine hypersensitivity. Chronic nicotine exposure at a concentration (10 nM for 3 days) that activates only the hypersensitive α4* (Leu9′Ala) receptors, but not wild-type receptors, resulted in significant potentiation of ACh (100 μM)-elicited responses. Experiments were also performed on midbrain neuronal cultures heterozygous for the α4* (Leu9′Ala) mutation as well as for a GFP protein fused to a GABA transporter that reliably reveals GABAergic neurons. In cultures chronically treated with 10 nM nicotine, there was significantly increased α4* nicotinic-induced Ca 2+ influx elicited by low concentration of ACh (3 μM). Furthermore, chronic exposure to the competitive antagonist dihydro-β-erythroidine, but not to the noncompetitive antagonist mecamylamine, induced up-regulation of ACh elicited nicotinic responses. These results suggest that occupation of α4* nicotinic receptor binding site(s), at the interface between two subunits, is sufficient to promote assembly and/or up-regulation of functional receptors in GABAergic neurons. Up-regulation in neurons is both “cell-autonomous”, occurring at the cell itself, and “receptor autonomous”, occurring at the receptor itself, and may be a thermodynamic necessity of ligand–protein interactions.