Agonists and antagonists bind to an A-A interface in the heteromeric 5-HT3AB receptor.
The 5-HT3 receptor is a member of the Cys-loop family of transmitter receptors. It can function as a homopentamer (5-HT3A-only subunits) or as a heteropentamer. The 5-HT3AB receptor is the best characterized heteropentamer. This receptor differs from a homopentamer in its kinetics, voltage dependence, and single-channel conductance, but its pharmacology is similar. To understand the contribution of the 5-HT3B subunit to the binding site, we created homology models of 5-HT3AB receptors and docked 5-HT and granisetron into AB, BA, and BB interfaces. To test whether ligands bind in any or all of these interfaces, we mutated amino acids that are important for agonist and antagonist binding in the 5-HT3A subunit to their corresponding residues in the 5-HT3B subunit and vice versa. Changes in [3H]granisetron binding affinity (Kd) and 5-HT EC50 were determined using receptors expressed in HEK-293 cells and Xenopus oocytes, respectively. For all A-to-B mutant receptors, except T181N, antagonist binding was altered or eliminated. Functional studies revealed that either the receptors were nonfunctional or the EC50 values were increased. In B-to-A mutant receptors there were no changes in Kd, although EC50 values and Hill slopes, except for N170T mutant receptors, were similar to those for 5-HT3A receptors. Thus, the experimental data do not support a contribution of the 5-HT3B subunit to the binding pocket, and we conclude that both 5-HT and granisetron bind to an AA binding site in the heteromeric 5-HT3AB receptor.
Lochner
M
M
Department of Chemistry, University of Warwick, Coventry, United Kingdom.
Lummis
S C R
SC
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D017829:Granisetron / Q000378:metabolism; D006801:Humans; D008024:Ligands; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000819:agonists / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D055503:Protein Multimerization; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D044406:Receptors, Serotonin, 5-HT3 / Q000737:chemistry / Q000378:metabolism; D012701:Serotonin / Q000378:metabolism; D058827:Serotonin 5-HT3 Receptor Agonists; D058831:Serotonin 5-HT3 Receptor Antagonists; D040681:Structural Homology, Protein; D014981:Xenopus
D017829:Granisetron; D008024:Ligands; D050505:Mutant Proteins; D021122:Protein Subunits; D044406:Receptors, Serotonin, 5-HT3; D012701:Serotonin; D058827:Serotonin 5-HT3 Receptor Agonists; D058831:Serotonin 5-HT3 Receptor Antagonists
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