Neuropharmacology 45 (2003) 345–354 www.elsevier.com/locate/neuropharm The mouse Chrna4 A529T polymorphism alters the ratio of high to low affinity α4β2 nAChRs Heejeong Kim a , Brody A. Flanagin b , Chuan Qin b , Robert L. Macdonald c , Jerry A. Stitzel b,* a Department of Neurology, University of Michigan Medical School, Ann Arbor, MI 48109-0316, USA b Department of Pharmacology, University of Michigan Medical School, 1500 E. Medical Center Drive CCGC 2150A, Ann Arbor, MI 48109-0930, USA c Department of Neurology, Vanderbilt University Medical School, Nashville, TN 37212-3375, USA Received 21 January 2003; received in revised form 26 March 2003; accepted 7 April 2003 Abstract Previously, a missense polymorphism was identified in the mouse nicotinic receptor α4 subunit gene, Chrna4. This polymorphism leads to an Ala/Thr variation at amino acid position 529 of the α4 subunit. Chrna4 A529T is associated with several measures of acute sensitivity to nicotine as well as with mouse strain differences in nicotine-stimulated 86 Rb + efflux from synaptosomes. Here, we report that the variant forms of the mouse α4 subunit confer functional differences when expressed with the β2 subunit in a heterologous system. α4β2 receptors containing the T529 variant of the α4 subunit exhibited a higher EC 50 value for the high affinity receptor population and an apparent reduced sensitivity to blockade by DHβE relative to α4β2 receptors containing the A529 variant of the α4 subunit. Moreover, the proportion of the total agonist-elicited current contributed by the high affinity α4β2 receptor population was greater for α4β2 receptors containing the α4(T529) variant (64%) than the α4β2 receptors containing the α4(A529) variant (41%). These data suggest that the polymorphism in the mouse α4 subunit is located in a previously unidentified functional domain of the receptor subunit that influences receptor function, including regulation of the affinity population ratio of α4β2 receptors.  2003 Elsevier Science Ltd. All rights reserved. Keywords: Nicotinic acetylcholine receptor; Polymorphism; Nicotine; Mice 1. Introduction Nicotinic cholinergic receptors (nAChRs) are mem- bers of the superfamily of ligand-gated ion channels that include GABA A , GABA C , glycine, and 5-HT3 receptors. nAChRs are located at the neuromuscular junction, in the peripheral nervous system and in the brain. In mam- mals, the neuronal subfamily of nAChRs is comprised of an indeterminate number of pentameric combinations of the subunits α2–α7, α9, α10, β2–β4(Lindstrom, 1997). The most abundant nAChR subtype expressed in the brain is composed of the α4 and β2 subunits (Flores et al., 1992). Like most nAChRs expressed in the brain, * Corresponding author. Tel.: +1-734-763-0642; fax: +1-734-647- 9817. E-mail address: stitzel@umich.edu (J.A. Stitzel). 0028-3908/03/$ - see front matter  2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0028-3908(03)00167-9 the α4β2-subtype appears to be localized primarily pre- synaptically where it modulates the release of various neurotransmitters (Wonnacott, 1997). Mutations in both the α4(CHRNA4)(Steinlein et al., 1995, 1997) and β2(CHRNB2)(Phillips et al., 2001) subunit genes have been implicated in autosomal domi- nant nocturnal frontal lobe epilepsy. In addition, the α4β2 nAChR subtype appears to be significantly reduced in post-mortem brain tissue from patients with neurodegenerative diseases (Zanardi et al., 2002) such as Parkinson’s disease, Alzheimer’s disease and Lewy body dementia when compared to age-matched controls. In smokers, the number of α4β2 receptors are upregul- ated (Benwell et al., 1988; Breese et al., 1997; Perry et al., 1999) although this upregulation in receptor numbers is curiously not observed in smokers with schizophrenia (Breese et al., 2000). Similarly in rodents, chronic treat- ment with nicotine leads to an upregulation of α4β2