Vol. 60, Issue 2, 373-381, August 2001

Modeling and Mutational Analysis of a Putative Sodium-Binding Pocket on the Dopamine D₂ Receptor

Kim A. Neve, Medhane G. Cumbay, Kimberly R. Thompson, Rui Yang, David C. Buck, Val J. Watts, Curtiss J. DuRand, and Martha M. Teeter

Portland Veterans Affairs Medical Center and Department of Behavioral Neuroscience, Oregon Health Sciences University, Portland, Oregon (K.A.N., K.R.T., D.C.B.); Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, Indiana (M.G.C., V.J.W.); ENRM Veterans Affairs Medical Center, Bedford, Massachusetts (C.J.D.); and Merkert Chemistry Center, Boston College, Chestnut Hill, Massachusetts (M.M.T.)

A homology model of the dopamine D₂ receptor was constructed based on the crystal structure of rhodopsin. A putative sodium-binding pocket identified in an earlier model (PDB 1I15) was revised. It is now defined by Asn-419 backbone oxygen at the apex of a pyramid and Asp-80, Ser-121, Asn-419, and Ser-420 at each vertex of the planar base. Asn-423 stabilizes this pocket through hydrogen bonds to two of these residues. Highly conserved Asn-52 is positioned near the sodium pocket, where it hydrogen-bonds with Asp-80 and the backbone carbonyl of Ser-420. Mutation of three of these residues, Asn-52 in helix 1, Ser-121 in helix 3, and Ser-420 in helix 7, profoundly altered the properties of the receptor. Mutants in which Asn-52 was replaced with Ala or Leu or Ser-121 was replaced with Leu exhibited no detectable binding of radioligands, although receptor immunoreactivity in the membrane was similar to that in cells expressing the wild-type D_2L receptor. A mutant in which Asn-52 was replaced with Gln, preserving hydrogen-bonding capability, was similar to D_2L in affinity for ligands and ability to inhibit cAMP accumulation. Mutants in which either Ser-121 or Ser-420 was replaced with Ala or Asn had decreased affinity for agonists (Ser-121), but increased affinity for the antagonists haloperidol and clozapine. Interestingly, the affinity of these Ser-121 and Ser-420 mutants for substituted benzamide antagonists showed little or no dependence on sodium, consistent with our hypothesis that Ser-121 and Ser-420 contribute to the formation of a sodium-binding pocket.