Vol. 299, Issue 1, 385-391, October 2001

Determinants of Agonist Binding Affinity on Neuronal Nicotinic Receptor  Subunits

Michael J. Parker¹, Scott C. Harvey² and Charles W. Luetje

Department of Molecular and Cellular Pharmacology, University of Miami School of Medicine, Miami, Florida

The  and  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 ; D, E, and F from ) are involved. We demonstrated previously that receptors containing the 2 subunit had consistently higher affinities for a variety of agonists than 4-containing receptors. For example, the affinity of the 22 receptor for epibatidine, ACh, nicotine, and dimethylphenylpiperazinium (DMPP) exceeds that of 24 by 9-, 61-, 87-, and 120-fold, respectively. Using saturation and competition analysis of receptors formed by chimeric  subunits coexpressed with 2 in Xenopus laevis 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 2 subunits in which each residue that differs between 2 and 4 in this region was changed from what occurs in 2 to what occurs in 4. 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.