Molecular cloning of m3 muscarinic acetylcholine receptor in rat iris

Muscarinic receptor subtypes that involved in cholinergic responses in rat iris were identified by reverse transcription-polymerase chain reaction (RT-PCR) analysis. mRNAs encoding m2, m3, and m4 subtypes were abundantly expressed in iris, whereas m1 and m5 subtypes were not detected. Selective amplification of the coding regions of m2, m3, and m4 subtypes in iris was carried out using specific primers based on the sequence of each subtype previously cloned from rat brain and heart by RT-PCR. The amino acid sequence for iris m2 was different from published heart and genomic m2 by nine and one residue(s), respectively. It was also found that the sequence for m2 that in brain, heart, and several smooth muscles determined in the present study is completely identical to that in iris but not to that reported previously in heart. The sequence for iris m4 was completely identical to that for m4 in brain. The sequence identity between m3 subtype in iris and that in brain is 99.3%, with four amino acid substitutions at the sites of the position 165 and 184 in the edge of second intracellular loop and the sites of the position 337 and 406 in the central of i3 loop. It was found that iris m3 is slightly but substantially different in amino acid sequence from that in brain of the rat.