The role of tyrosine residues at the mouse 5-HT3A receptor lihand binding site investigated by unnatural amino acid mutagenesis K.L. Price1, D.L. Beene2, D.A. Dougherty2, H.A. Lester2 & S.C.R.Lummis1. 1Dept. of Biochemistry, Tennis Court Road, Cambridge CB2 1GA. 2Divisions of Chemistry and Chemical Engineering and Biology, California Institute of Technology, Pasadena, California 91125.

Print abstract Search PubMed for: Price KL Beene DL Dougherty DA Lester HA Lummis SCR

The 5-HT₃ receptor (5-HT₃R) is a member of the Cys-loop family of ligand-gated ion channels and shares a high degree of homology with nicotinic acetylcholine, GABA_A/C, glycine and GluCl receptors. Previous data has shown that the amino acids involved in ligand binding comprise six non-contiguous loops (A-F). Tyrosine residues from binding loops C (Y234) and E (Y141, Y143 and Y153) are important for ligand binding and/or receptor gating transitions in the 5-HT₃R (Price et al. 2001, Venkataraman et al. 2002). To characterise the role of these residues further, we have used in vivo nonsense suppression to incorporate unnatural amino acids site-specifically in 5-HT₃Rs expressed in Xenopus oocytes.

Mutant mouse 5-HT_3AsR subunits with TAG codons in place of tyrosine codons were made using the Kunkel (1985) method, subcloned into pGEMHE (Reeves et al., 2001), and then used for in vitro transcription reactions (Ambion). Unnatural amino acids coupled to the dinucleotide dCA were chemically synthesised, enzymatically ligated to 74-mer THG73 tRNA_CUA (Nowak et al., 1998) and co-injected with mRNA into Xenopus oocytes. Voltage clamp recordings were performed 18-36h post-injection, in some cases on Opus Express (Axon) and pEC₅₀ values calculated using the Hill equation (Table 1).

Table 1. pEC₅₀s ± s.e. mean (n=3) for 5-HTR₃s mutated at positions 141, 143, 153 and 234. NR = no response, - = not attempted. Bold = significantly different (P<0.01; ANOVA, Dunnett post test) to Tyr (WT).

The results indicate that the -OH groups of Y143 and Y153 are critical for binding and/or function, whilst that of Y141 is not, as aromatic substitutions are well tolerated. Aromatic substitutions of Y234 increase 5-HT EC₅₀s only modestly, but substituents at the 4 position seem to be advantageous. These data provide support for a homology model of the 5-HT₃R extracellular domain (Reeves et al., 2003).

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Nowak et al. (1998) Methods Enzymol. 293: 504-529.
Price et al. (2001) Br. J. Pharmacol. 134: 143.
Reeves et al. (2001) J. Biol. Chem. 276 (45): 42035-42042.
Reeves et al. (2003) Biophys. J. 84: 2338-2344.
Venkataraman et al. (2002) BMC Biochem. 3 (1):15.

Supported by the Wellcome Trust (SCRL), MRC (KLP), and NIH grants NS-11756 (HAL) and NS-34407 (DAD).