The Loop C Region of the Murine 5-HT 3A Receptor Contributes to the Differential Actions of 5-Hydroxytryptamine and m-Chlorophenylbiguanide † Asha Suryanarayanan, ‡,§ Prasad R. Joshi, ‡,§ Zsolt Bika ´di, | Muthalagi Mani, ⊥ Trupti R. Kulkarni, ⊥ Chandra Gaines, ⊥ and Marvin K. Schulte* ,‡ Department of Chemistry and Biochemistry, The UniVersity of Alaska, Fairbanks, Alaska 99775, Department of Molecular Pharmacology, Institute of Chemistry, Chemical Research Center, P.O. Box 17, Budapest H-1525, Hungary, and Department of Basic Pharmaceutical Sciences, College of Pharmacy, The UniVersity of Louisiana, Monroe, Louisiana 71209 ReceiVed April 11, 2005; ReVised Manuscript ReceiVed May 5, 2005 ABSTRACT: Sequence and predicted structural similarities between members of the Cys loop superfamily of ligand-gated ion channel receptors and the acetylcholine binding protein (AChBP) suggest that the ligand-binding site is formed by six loops that intersect at subunit interfaces. We employed site-directed mutagenesis to investigate the role of amino acids from the loop C region of the murine 5-HT 3AS R in interacting with two structurally different agonists, serotonin (5-HT) and m-chlorophenylbiguanide (mCPBG). Mutant receptors were evaluated using radioligand binding, two-electrode voltage clamp, and immunofluorescence studies. Electrophysiological assays were employed to identify changes in response characteristics and relative efficacies of mCPBG and the partial agonist, 2-methyl 5-HT (2-Me5-HT). We have also constructed novel 5-HT and mCPBG docked models of the receptor binding site based on homology models of the AChBP. Both ligand-docked models correlate well with results from mutagenesis and electrophysiological assays. Four key amino acids were identified as being important to ligand binding and/or gating of the receptor. Among these, I228 and D229 are specific for effects mediated by 5-HT compared to mCPBG, indicating a differential interaction of these ligands with loop C. Residues F226 and Y234 are important for both 5-HT and mCPBG interactions. Mutations at F226, I228, and Y234 also altered the relative efficacies of agonists, suggesting a role in the gating mechanism. The serotonin type 3 receptor (5-HT 3 R) is a member of the Cys loop superfamily of ligand-gated ion channel (LGIC) receptors that includes nicotinic acetylcholine, GABA A , GABA C , and glycine receptors (1). These receptors are membrane-bound ion channel-coupled receptors that mediate fast synaptic transmission in both peripheral and central nervous systems. 5-HT 3A subunits form homopentamers that yield characteristic inward currents with rapid onset and desensitization upon exposure to agonist. The ligand-binding site is present in the extracellular amino-terminal domain, at the subunit interface. 5-HT 3A R 1 antagonists are clinically used for the treatment of chemotherapy-induced emesis, and are being evaluated for several other conditions, including alcoholism (2). Sequence and predicted structural similarities between LGIC receptors and the acetylcholine binding protein (AChBP) suggest that the ligand-binding site is formed by six loops, A-F, which intersect at the subunit interfaces (3- 6). Several important residues in the 5-HT 3A R binding domain have been identified (7-12). Earlier work from our laboratory identified three important tyrosine residues (Y141, Y143, and Y153) in the loop E region (13). The development of AChBP-based homology models of the 5-HT 3 R binding domain has also greatly improved our understanding of binding interactions of agonists and antagonists (4, 6, 14). Previously, residues in the loop C region have been shown to contribute to interspecies differences in potencies of various ligands. The loop C region contributes to the higher potency of d-tubocurarine (d-TC) at the mouse 5-HT 3 R compared to human receptors (15). This region has also been shown to contribute to the selective potency of 1-phenyl- biguanide (PBG) at human 5-HT 3 Rs (16). Similarly, the loop C region has been implicated in the higher potency of m-chlorophenyl biguanide (mCPBG) compared to serotonin (5-HT) at rat 5-HT 3 Rs (17). 5-HT and mCPBG have been shown to exhibit distinct profiles on the murine 5-HT 3A R. Specifically, mCPBG exhibits a higher apparent affinity, slower association rates, and a higher affinity for the desensitized state compared to 5-HT (18-20). However, the molecular basis of these differences is not known. The primary focus of this study was to evaluate the interactions of 5-HT and mCPBG with each residue in the loop C region † This work was supported by the National Science Foundation (NSF CAREER Grant 9985077), the American Heart Association (AHA Grant 0151065B), and Alaska INBRE (Grant P20 RR016466). A.S. and P.R.J. are Alaska INBRE graduate research fellows. * To whom correspondence should be addressed: Department of Chemistry and Biochemistry, 146, Natural Sciences Facility, P.O. Box 756160, The University of Alaska, Fairbanks, AK 99775. Phone: (907) 474-5237. Fax: (907) 474-5640. E-mail: ffmks@uaf.edu. ‡ The University of Alaska. § These authors contributed equally to this work and are co-first authors. | Chemical Research Center. ⊥ The University of Louisiana. 1 Abbreviations: 5-HT, serotonin; 5-HT3AR, serotonin type 3A receptor; nAChR, nicotinic acetylcholine receptor; AChBP, acetylcho- line binding protein; mCPBG, m-chlorophenylbiguanide; WT, wild type. 9140 Biochemistry 2005, 44, 9140-9149 10.1021/bi050661e CCC: $30.25 © 2005 American Chemical Society Published on Web 06/01/2005