title: Muscarinic allosteric modulation: M2/M3 subtype selectivity of gallamine is independent of G-protein coupling specificity.
abstract: Among the five subtypes of muscarinic acetylcholine receptors, the sensitivity towards allosteric modulation is generally higher in M2 and M4 receptors that preferentially couple to inhibitory G-proteins of the Gi/o type than in M1, M3, and M5 that preferentially couple to stimulatory G-proteins such as Gq/11. We aimed to check whether the high allosteric sensitivity of the M2 receptor compared to M3 is related to the differential G-protein coupling preference. As the third intracellular loop (i3) is known to be the major determinant in receptor G-protein coupling specificity, we used wild-type M2 and M3 receptors and the related chimeric constructs with exchanged i3-loops, i.e., M2 containing M3-i3 (M2/M3-i3) and M3 containing M2-i3 (M3/M2-i3). The allosteric effect of the archetypal modulator gallamine on the dissociation and the equilibrium binding of [3H]N-methylscopolamine ([3H]NMS) was measured in membranes of mouse A9L cells stably expressing the wild-type and the chimeric receptors (4 mM Na2HPO4, 1 mM KH2PO4, pH 7.4, 23 degrees C). The dissociation of [3H]NMS was monophasic under all conditions studied. Control values of t 1/2 were (means +/- SEM, n = 4-7): M2: 3.8 +/- 0.2 min, M2/M3-i3: 4.8 +/- 0.3 min, M3:43.3 +/- 4.2 min, M3/M2-i3: 41.1 +/- 3.6 min. At M2 receptors, 0.2 microM gallamine allosterically reduced the apparent rate constant of dissociation k-1 to 51 +/- 5% of the control value (n = 5). At M2/M3-i3 the allosteric potency of gallamine was not significantly changed (0.2 microM gallamine --> k-1 = 61 +/- 4%, n = 7). At M3, a 20-fold higher concentration was required for an equieffective allosteric action (10 microM gallamine --> k-1 = 51 +/- 5%, n = 5). The potency of gallamine at M3/M2-i3 was not increased compared with M3 receptors (10 microM gallamine --> k-1 = 73 +/- 2%, n = 4) but even significantly diminished. [3H]NMS equilibrium binding experiments revealed that neither the binding constants of gallamine at free receptor subtypes (pKA,M2: 7.57 +/- 0.04, n = 4; pKA,M3: 5.56 +/- 0.13, n = 3) nor the factors of negative cooperativity with [3H]NMS (alphaM2 = 31 +/- 1, alphaM3 = 3 +/- 0.4) were affected by the exchanged i3-loops (pKA,M2/M3-i3: 7.65 +/- 0.03, pKA,M3/M2-i2: 5.35 +/- 0.24, alphaM2/M3-i3= 30 +/- 2, alphaM3/M2-i2 = 3 +/- 0.7). In conclusion, the different sensitivities of M2 and M3 receptors towards allosteric modulation by gallamine are not related to the G-protein coupling specificity of the receptors.
authors.affiliation: Department of Pharmacology and Toxicology, Institute of Pharmacy, University of Bonn, Germany. traenkle@uni-bonn.de
authors.forename: C
authors.initials: C
authors.lastname: Tränkle
authors.forename: E
authors.initials: E
authors.lastname: Kostenis
authors.forename: K
authors.initials: K
authors.lastname: Mohr
mesh_terms: D000494:Allosteric Regulation / Q000187:drug effects / Q000502:physiology; D000818:Animals; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D019204:GTP-Binding Proteins / Q000378:metabolism*; D005703:Gallamine Triethiodide / Q000494:pharmacology*; D006801:Humans; D019832:N-Methylscopolamine / Q000378:metabolism; D003473:Neuromuscular Nondepolarizing Agents / Q000494:pharmacology*; D010276:Parasympatholytics / Q000378:metabolism; D051381:Rats; D043585:Receptor, Muscarinic M2; D043587:Receptor, Muscarinic M3; D011976:Receptors, Muscarinic / Q000378:metabolism*
chemical_list: D003473:Neuromuscular Nondepolarizing Agents; D010276:Parasympatholytics; D043585:Receptor, Muscarinic M2; D043587:Receptor, Muscarinic M3; D011976:Receptors, Muscarinic; D019204:GTP-Binding Proteins; D005703:Gallamine Triethiodide; D019832:N-Methylscopolamine