Selectivity profile of muscarinic toxin 3 in functional assays of cloned and native receptors.
By using acetylcholine-induced stimulation of [35S]guanosine-5'-O-(3-thio)triphosphate ([35S]GTPgammaS) 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 (pA2 = 8.33) much higher than those displayed at the m1 (pA2 = 6.78), m3 (pA2 = 6.3), and m2 (pA2 < 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 inhibition of adenylyl cyclase with a pA2 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 potency (pA2 = 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 [3H]N-methylscopolamine to NG108-15 cell membranes and by reverse transcription-polymerase chain reaction analysis, revealing 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 receptors coupled to a common response, the toxin constitutes a powerful tool to resolve the relative contribution by each receptor subtype.
Olianas
M C
MC
Section on Biochemical Pharmacology, Departments of Neurosciences, University of Cagliari, Italy.
Ingianni
A
A
Maullu
C
C
Adem
A
A
Karlsson
E
E
Onali
P
P
D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D016466:CHO Cells; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D006224:Cricetinae; D015870:Gene Expression; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D000090023:Metallothionein 3; D051379:Mice; D018727:Muscarinic Antagonists / Q000494:pharmacology*; D019832:N-Methylscopolamine / Q000494:pharmacology; D010455:Peptides / Q000494:pharmacology*; D043643:Receptor, Muscarinic M1; D043585:Receptor, Muscarinic M2; D043588:Receptor, Muscarinic M4; D011976:Receptors, Muscarinic / Q000145:classification / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013462:Sulfur Radioisotopes; D014316:Tritium; D014407:Tumor Cells, Cultured
D036341:Intercellular Signaling Peptides and Proteins; D000090023:Metallothionein 3; C000715430:Mt3 protein, mouse; D018727:Muscarinic Antagonists; D010455:Peptides; D043643:Receptor, Muscarinic M1; D043585:Receptor, Muscarinic M2; D043588:Receptor, Muscarinic M4; D011976:Receptors, Muscarinic; D011994:Recombinant Proteins; D013462:Sulfur Radioisotopes; C089528:muscarinic toxin 3; D014316:Tritium; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000262:Adenylyl Cyclases; D019832:N-Methylscopolamine