Leucine codon 282 (CTC) in exon 5 was changed to serine (AGC) (p.L282S) and an FRT site flanked neomycin resistance gene cassette was inserted into intron 4. The neo cassette was removed through subsequent Flp-mediated recombination. The mutation, at Leu 9' in the transmembrane region 2 (TM2) of the encoded peptide, modifies the
physiological response of the receptor to nicotine and acetylcholine.