Pharmacological Research 54 (2006) 345–355 Kinetic and molecular evidences that human cardiac muscle express non-M2 muscarinic receptor subtypes that are able to interact themselves Carmen Carlota Nello P´ erez a , Iv´ an Dar´ ıo Bravo Tobar a , Eli´ ezer Jim´ enez a , Darwin Casta ˜ neda a , Maria Bel´ en Rivero a , Juan Lu´ ıs Concepci ´ on b , Miguel ´ Angel Chiurillo a , Rafael Bonfante-Cabarcas a,∗ a Unidad de Bioqu´ ımica “Dr. Jos´ e Antonio Moreno Y´ anes”, Escuela de Medicina “Pablo Acosta Ortiz”, Universidad Centro Occidental “Lisandro Alvarado”, Avenida Libertador con Avenida Andr´ es Bello, Barquisimeto, Estado Lara, C´ odigo 3001, Venezuela b Laboratorio de Enzimologia de Par´ asitos, CIGEN, Facultad de Ciencias, Universidad de los Andes, M´ erida, Venezuela Accepted 3 July 2006 Abstract In heart tissue five isoforms of the muscarinic acetylcholine receptor (mAChR) have been identified, designated m1–m5, of which only M1, M2 and M3 have functional evidences for their role in cardiac physiology. The present study was designed to explore the diversity of mAChR subtypes in human hearts and determine whether these subtypes are able to interact themselves. Expression of mRNAs encoding all five subtypes was readily detected by RT-PCR reaction in both atrial (A) and ventricle (V) samples. Immunoblotting, MABA and ELISA with subtype-specific antibodies confirmed the presence of M1, M2, M3, M4 and M5 proteins in membrane preparations from both A and V. Kinetic characterization using [ 3 H]-QNB shown: (1) atrium had greater B max than did the ventricle, (2) [ 3 H]-QNB behave as an allosteric modulator, inducing cooperativity at high and disclosing heterogeneity at low concentrations, (3) heterogenity was observed in pirenzepine, biperiden and tropicamide competition curves, being the high affinity sites compatible with M1 and M4 muscarinic receptor subtypes and (4) methoctramine competition curves in presence of selective muscarinic receptor subtypes antagonist displayed heterogeneity profile still maintaining cooperativity (n H > 1), indicating muscarinic receptors subtypes are able to form homo- and hetero-oligomers. In conclusion, our results provide molecular and kinetic evidence for the presence of multiple subtypes of mAChR in human hearts, which are able to undergo discrete transitions from a non-cooperative kinetics of non-interacting monomers to a cooperative kinetics of interacting oligomers. © 2006 Elsevier Ltd. All rights reserved. Keywords: Muscarinic receptor subtypes; Human heart; [ 3 H]-QNB binding kinetics; Cooperative interactions; RT-PCR 1. Introduction Muscarinic acetylcholine receptors (mAChR) belong to the super-family of seven transmembrane helix G-proteins cou- pled receptors. Five mAChR’s subtypes: m 1 ,m 2 ,m 3 ,m 4 and m 5 have been described based on molecular cloning studies. Each receptor is encoded by an uninterrupted gene, located in 11q12–13, 7q35–36, 1q43–44, 11p12-11.2 and 15q26 human chromosomes, respectively. Four of the cloned subtypes corre- spond to functional receptors described as M1, M2, M3, and M4, which already have been characterized pharmacologically and functionally. In general, M1, M3, and M5 receptors couple via ∗ Corresponding author. Tel.: +58 251 2591854; fax: +58 251 2591886. E-mail address: rcabarca@ucla.edu.ve (R. Bonfante-Cabarcas). Gq/11 to phospholipase C (PLC) with the subsequent formation of inositol phosphate (IP) and diacylglycerol (DAG). M2 and M4 receptors are coupled via a pertussis toxin (PTX)-sensitive G protein (Gi/Go) to adenylyl cyclase, Ca 2+ channels and K + channels (references in [1,2]). M2 receptor is commonly described as the only functional mAChR subtype in the heart. It mediates a negative chronotropic and inotropic effects. Acetylcholine (ACh) acting on M2 subtype induces either an inward rectifier potassium current (IK ACh ) or inhibited voltage-gated Ca 2+ channels; both effects are mediated via Gi protein coupled pathway (references in [2]). New evidences have emerged indicating that cardiac cells can express another mAChR subtypes. Cloning and functional studies have identified the M1 subtype in mammalian hearts [3–5] and mRNAs encoding M2, M3, and M4 isoforms have been described in chicken heart [6,7]. Furthermore, it has been 1043-6618/$ – see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.phrs.2006.07.001