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β‐Funaltrexamine, a gauge for the recognition site of wildtype and mutant H297Q μ‐opioid receptors
Author(s) -
Spivak Charles E.,
Beglan Carol L.,
Zöllner Christian,
Surratt Christopher K.
Publication year - 2003
Publication title -
synapse
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.809
H-Index - 106
eISSN - 1098-2396
pISSN - 0887-4476
DOI - 10.1002/syn.10205
Subject(s) - receptor , g protein coupled inwardly rectifying potassium channel , mutant , wild type , chemistry , binding site , opioid receptor , agonist , microbiology and biotechnology , biophysics , biology , biochemistry , g protein , gene
The antagonist β‐funaltrexamine (β‐FNA), known to bind covalently to μ‐opioid receptors by a two‐step, doubly discriminating sequence, was used as a sensitive gauge to compare wildtype to mutant H297Q μ‐opioid receptors. We addressed whether this mutation, which enhances the intrinsic activities of alkaloid μ‐receptor agents, affects both the reversible and covalent phases of β‐FNA binding. Such altered binding serves as a reporter for the dimensions and topography of the receptor's recognition site. Using the voltage‐clamped Xenopus oocyte expression system with coexpressed GIRK potassium channels, we found that β‐FNA blocked the wildtype and mutant H297Q receptors both reversibly and irreversibly, indicating overall conserved tertiary structure in the mutant. The mutant H297Q receptor, however, was more resistant to both phases of blockade, indicating some disturbance of the mutant H297Q receptor recognition site. β‐FNA acted as a partial agonist at the mutant H297Q receptor expressed in both oocytes, as measured by the activation of GIRK channels, and in COS‐7 cells assayed by GTPγ 35 S binding. β‐FNA showed no activity at the wildtype receptor expressed in oocytes, but surprisingly induced binding of GTPγ 35 S in transfected COS‐7 cells. Thus, the topography of the mutant H297Q receptor recognition site is sufficiently conserved to allow the selective binding of β‐FNA, but the decrease in binding affinity and increase in efficacy in oocytes demonstrates clear differences from the wildtype receptor. Synapse 49:55–60, 2003. © 2003 Wiley‐Liss, Inc.