| Species | Original | Mutated to | Mutation |
| Human | Thr 81 |
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| Rat Equivalent | Thr 80 | ||
| Mouse Equivalent | Thr 86 | Ala | T86A |
Nine residues (Thr 86, Thr 87, Tyr 88, Ile 89, Tyr 91, Arg 92, Gln 93, Tyr 94, Trp 95) were mutated individually to alanine (alanine scanning mutagenesis). [3H] granisetron, curare, and serotonin were used to examine binding.
W90F was analyzed instead of W90A, and it proved to be the only mutation that significantly affected the interaction of curare with the receptor. R92A was the only substitution that altered the affinity of the agonist serotonin. W90F, R92A, and Y94A all reduced the affinity of [3H] granisetron. The periodicity of the effect suggests the involvement of a beta-strand.