The Seventh Transmembrane Domains of the δ and κ Opioid Receptors Have Different Accessibility Patterns and Interhelical Interactions

Abstract

We applied the substituted cysteine accessibility method (SCAM) to map the residues of the transmembrane helices (TMs) 7 of δ and κ opioid receptors (δOR and κOR) that are on wateraccessible surface of the binding-site crevices. Twenty five consecutive residues (except C7.38) in the TMs7 were mutated to Cys, one at a time, and each mutant was expressed in HEK 293 cells. Most mutants displayed similar binding affinity for [ 3 H]diprenorphine, an antagonist, as the wildtypes. Pretreatment with (2-aminoethyl) methanethiosulfonate (MTSEA) inhibited [ 3 H] diprenorphine binding to eight δOR and eight κOR mutants. All mutants except δOR L7.52(317)C were protected by naloxone from MTSEA effect, indicating that the side chains of V7.31(296), A7.34 (299), I7.39(304), L7.41(306), G7.42(307), P7.50(315) and Y7.53(318) of δOR and S7.34(311), F7.37(314), I7.39(316), A7.40(317), L7.41(318), G7.42(319), Y7.43(320) and N7.49(326) of κOR are on water-accessible surface of the binding pockets. Combining the SCAM data with rhodopsinbased molecular models of the receptors led to the following conclusions. i) The residues of the extracellular portion of TM7 predicted to face TM1 are sensitive to MTSEA in κOR, but are not in δOR. Thus, TM1 may be closer to TM7 in δOR than in κOR. ii) MTSEA-sensitive mutants start at position 7.31(296) in δOR and at 7.34(311) in κOR, suggesting that TM7 in δOR may have an additional helical turn (from 7.30 to 7.33). iii) There is a conserved H-bond network linking D2.50 of the NLxxxD motif in TM2 with W6.48 of the CWxP motif in TM6. iv) The NPxxY motif in TM7 interacts with TM2, TM6, and helix 8 to maintain receptors in inactive states. To the best of our knowledge, this represents the first such comparison of the structures of two highly homologous GPCRs.More than one thousand G protein-coupled receptors (GPCRs) are present in the human genome. GPCRs play important roles in physiological functions, including smell, taste, light perception, neurotransmission, functions of endocrine and exocrine glands and immune functions. In addition, GPCRs are targets of numerous clinically used drugs. It is, therefore, of great interests to understand their structures at the molecular level. Based on structural characteristics, GPCRs are classified into multiple families and the rhodopsin family is by far the largest in number. To date, rhodopsin is the only GPCR of which high-resolution crystal *Address correspondence to: Dr. Lee-Yuan Liu-Chen, Department of Pharmacology, Temple University School of Medicine, 3420 N. Broad St., Philadelphia, PA 19140, phone: (215) 707−4188; fax: (215) 707−7068; e-mail: E-mail: lliuche@temple.edu. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2009 June 1. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript structures have been elucidated (1-3). Rhodopsin has an extracellular N-terminal domain, seven transmembrane helices (TMs) connected by alternating intracellular and extracellular hydrophilic loops and an in...

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