Previously, a missense polymorphism was identified in the mouse nicotinic receptor alpha4 subunit gene, Chrna4. This polymorphism leads to an Ala/Thr variation at amino acid position 529 of the alpha4 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 alpha4 subunit confer functional differences when expressed with the beta2 subunit in a heterologous system. alpha4beta2 receptors containing the T529 variant of the alpha4 subunit exhibited a higher EC(50) value for the high affinity receptor population and an apparent reduced sensitivity to blockade by DHbetaE relative to alpha4beta2 receptors containing the A529 variant of the alpha4 subunit. Moreover, the proportion of the total agonist-elicited current contributed by the high affinity alpha4beta2 receptor population was greater for alpha4beta2 receptors containing the alpha4(T529) variant (64%) than the alpha4beta2 receptors containing the alpha4(A529) variant (41%). These data suggest that the polymorphism in the mouse alpha4 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 alpha4beta2 receptors.