The loop C region of the murine 5-HT3A receptor contributes to the differential actions of 5-hydroxytryptamine and m-chlorophenylbiguanide.
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.
Suryanarayanan
Asha
A
Department of Chemistry and Biochemistry, The University of Alaska, Fairbanks, Alaska 99775, USA.
Joshi
Prasad R
PR
Bikádi
Zsolt
Z
Mani
Muthalagi
M
Kulkarni
Trupti R
TR
Gaines
Chandra
C
Schulte
Marvin K
MK
D000595:Amino Acid Sequence; D000818:Animals; D001645:Biguanides / Q000737:chemistry / Q000378:metabolism*; D002460:Cell Line; D004594:Electrophysiology; D006801:Humans; D008745:Methylation; D051379:Mice; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D017434:Protein Structure, Tertiary; D011869:Radioligand Assay; D044406:Receptors, Serotonin, 5-HT3 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D012701:Serotonin / Q000737:chemistry / Q000378:metabolism*; D058827:Serotonin 5-HT3 Receptor Agonists; D017366:Serotonin Receptor Agonists / Q000737:chemistry / Q000378:metabolism*; D014982:Xenopus laevis
D001645:Biguanides; D044406:Receptors, Serotonin, 5-HT3; D058827:Serotonin 5-HT3 Receptor Agonists; D017366:Serotonin Receptor Agonists; D012701:Serotonin; C065734:1-(3-chlorophenyl)biguanide