Abstract

The α and β subunits of heteromeric neuronal nicotinic acetylcholine receptors (nAChRs) are thought to contribute “principal” and “complementary” components to the agonist binding site, respectively. At least six loops of amino acid sequence (A, B, and C from α; D, E, and F from β) are involved. We demonstrated previously that receptors containing the β2 subunit had consistently higher affinities for a variety of agonists than β4-containing receptors. For example, the affinity of the α2β2 receptor for epibatidine, ACh, nicotine, and dimethylphenylpiperazinium (DMPP) exceeds that of α2β4 by 9-, 61-, 87-, and 120-fold, respectively. Using saturation and competition analysis of receptors formed by chimeric β subunits coexpressed with α2 inXenopuslaevis oocytes, we have now identified sequence segment 54–63 (corresponding to loop D) as a major determinant of affinity for epibatidine, ACh, nicotine, and DMPP. We then analyzed a series of mutant β2 subunits in which each residue that differs between β2 and β4 in this region was changed from what occurs in β2 to what occurs in β4. The N55S, V56I, and E63T mutations each resulted in a loss of affinity for ACh and nicotine of 3- to 4-fold, whereas the T59K mutation resulted in a 7-fold loss of ACh and nicotine affinity. These mutations had little or no effect on epibatidine and DMPP affinity. The positive charge introduced by the T59K mutation does not appear to underlie loss of agonist affinity, because a similar loss of affinity was observed when a negative charge (T59D) was introduced at this position.