Selectivity Profile of Muscarinic Toxin 3 in Functional Assays of Cloned and Native Receptors 1 MARIA C. OLIANAS, ANGELA INGIANNI, CARLO MAULLU, ABDU ADEM, EVERT KARLSSON and PIERLUIGI ONALI Section on Biochemical Pharmacology, Departments of Neurosciences (M.C.O., P.O.) and Medical Sciences (A.I., C.M.), University of Cagliari, Italy; and Department of Clinical Neuroscience, Geriatric Section, Karolinska Institute, Stockholm, Sweden (A.A., E.K.) Accepted for publication June 25, 1998 This paper is available online at http://www.jpet.org ABSTRACT By using acetylcholine-induced stimulation of [ 35 S]guanosine- 5'-O-(3-thio)triphosphate ([ 35 S]GTPS) binding to membrane G proteins as a functional assay of the cloned human m1–m4 muscarinic receptor subtypes stably expressed in Chinese hamster ovary cells, muscarinic toxin 3 (MT3) was found to block the m4 receptor with a potency (pA 2 = 8.33) much higher than those displayed at the m1 (pA 2 = 6.78), m3 (pA 2 = 6.3), and m2 (pA 2  6.3) subtypes. In N1E-115 cells, which have been reported to express m4 receptors coupled to inhibition of cAMP, MT3 potently antagonized the carbachol-induced inhi- bition of adenylyl cyclase with a pA 2 of 8.81 and displayed monophasic inhibitory curves. Unexpectedly, in NG108-15 cells, known to express only m4 receptors, MT3 counteracted the carbachol inhibition of adenylyl cyclase with a lower po- tency (pA 2 = 7.60) and showed a biphasic inhibitory curve, suggesting the participation of both m4 and m2 receptors. This possibility was supported by radioligand binding data showing that MT3 failed to completely displace the binding of [ 3 H]N- methylscopolamine to NG108-15 cell membranes and by re- verse transcription-polymerase chain reaction analysis, reveal- ing the presence of mRNAs for both m4 and m2 receptor subtypes. These data demonstrate that MT3 possesses a high functional receptor selectivity for both the cloned and native m4 receptors and that in cell systems containing m4 and m2 re- ceptors coupled to a common response, the toxin constitutes a powerful tool to resolve the relative contribution by each recep- tor subtype. In the central nervous system and in peripheral tissues, many actions of acetylcholine (ACh) occur through the acti- vation of muscarinic receptors. Molecular biology studies have led to the identification of five distinct molecular forms of the muscarinic receptors, named m1–m5, and the artificial expression of the cloned receptor subtypes in host cells has allowed the characterization of their signal transduction pathways (Bonner et al., 1987; Peralta et al., 1987; Hulme et al., 1990). Thus, it has been shown that m1, m3, and m5 receptors are predominantly coupled to phospholipase C through G proteins of G q/11 type, whereas m2 and m4 recep- tors preferentially regulate adenylyl cyclase and ion channel activities by coupling to G i /G o proteins (Peralta et al., 1988). However, a great limitation to the study of the physiological role played by each receptor subtype in the different tissues is the lack of highly selective ligands. Indeed, the muscarinic receptor antagonists currently available display an affinity for one receptor subtype that is 10-fold greater than that for the other subtypes (Caufield, 1993). The limited selectivity of the drugs complicates the receptor characterization in tis- sues expressing a heterogeneous muscarinic receptor popu- lation. In addition, none of the classic antimuscarinic drugs bind with high selectivity to the m4 or m5 receptor subtype (Caufield, 1993). The need of m4-selective ligands is partic- ularly critical because the m4 receptor is almost exclusively expressed in neurons (Wood et al., 1996; Mieda et al., 1997) and is preferentially localized in central neuronal pathways regulating motor and cognitive functions (Levey et al., 1991; Ferrari-Dileo et al., 1994). Recently, Karlsson and collaborators (1994) reported the isolation of a new peptide toxin, named MT3, from green mamba venom. In radioligand binding studies using Chinese hamster ovary (CHO) cells separately expressing the five cloned muscarinic receptors, muscarinic toxin 3 (MT3) showed a high affinity for the m4 (pK i = 8.70), a lower affinity for the m1 (pK i = 7.11), and a very low affinity for the m2, m3, and m5 subtypes (pK i  6.0) (Jolkkonen et al., 1994). The distribution of m4 receptors in the rat brain has been studied by using radiolabeled [ 125 I]MT3 (Adem et al., 1995; Received for publication April 15, 1998. 1 This work was supported by Grant CHRXCT940689 from the European Communities (to P.O.) and by MURST. ABBREVIATIONS: ACh, acetylcholine chloride; CCh, carbachol chloride; CHO, Chinese hamster ovary; BSA, bovine serum albumin; EGTA, ethylene glycol bis(-aminoethyl ether)-N,N,N',N'-tetraacetic acid; GTPS, guanosine-5'-O-(3-thio)triphosphate; HEPES, 4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid; MT3, muscarinic toxin 3; NMS, N-methylscopolamine; PACAP, pituitary adenylate cyclase activating polypeptide; RT, reverse transcription; PCR, polymerase chain reaction. 0022-3565/99/2881-0164$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 288, No. 1 Copyright © 1999 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 288:164 –170, 1999 164 at ASPET Journals on November 29, 2014 jpet.aspetjournals.org Downloaded from