A NOVEL M(3) MUSCARINIC ACETYLCHOLINE-RECEPTOR IS EXPRESSED IN CHICK ATRIUM AND VENTRICLE

Prior studies have suggested that heart expresses only the M(2) isofor m of the muscarinic receptor (Peralta, E. G., Ashkenazi, A., Winslow, J. W., Smith, D. H., Ramachandran, J., and Capon, D, J. (1987) EMBO J. 6, 3923-3929). Tietje and Nathanson (Tietje, K. M., and Nathanson, N. M. (1991) J. Biol. Chem. 266, 17382-17387) have recently demonstrated that the chick heart may be unique since it expresses both the M(2) a nd M(4) isoforms of the muscarinic receptor. In this study, in order t o determine whether other isoforms of the muscarinic receptor were pre sent in the chick heart, a chick M(3) muscarinic recep tor was cloned, characterized, and its expression in chick tissues determined. Using a human M(3) muscarinic receptor cDNA as a probe, a 2.4-kilobase pair cDNA was isolated from a chick brain cDNA library which contained an o pen reading frame coding for a 639 amino acid protein. This protein de monstrated an 87 and 86% homology to the human and rat M(3) muscarinic receptor, respectively. Chinese hamster ovary (CHO-GRA) cells were st ably transfected with the chick M(3) muscarinic receptor and one clone (CHO-CM3) expressed the M(3) receptor, as measured by the binding of quinuclidinly benzilate at 116 +/- 14 (+/-S.E., n = 3) fmol/mg protein with a K-d of 76 +/- 17 pM. This receptor demonstrated a rank order o f potency for muscarinic antagonist binding characteristic for the M(3 ) receptor: with high affinity binding for hexahydrosiladifenidol, K-d : 16 +/- 2 nM (+/-S.E., n = 3); intermediate affinity for pirenzepine, K-d: 383 +/- 47 nM, and low affinity for methoctramine, K-d: 533 +/- 185 nM (+/-S.E., n = 3). Carbamylcholine stimulation of CHO-CM3 cells resulted in a 1.6-fold increase in cyclic AMP accumulation and a 3.5-f old increase in a pertussis toxin-insensitive inositol phosphate relea se. These data demonstrate that the chick M(3) muscarinic receptor has the properties characteristic of M(3) receptors from other species. R Nase protection studies demonstrated the presence of M(3) muscarinic r eceptor mRNA in the brain, atria, and ventricle of chicks 17 days in o vo. Hence, the chick heart appears to have the unique capacity to expr ess mRNAs coding not only for the M(2) and M(4) muscarinic receptors b ut also for the M(3) muscarinic receptor.