Muscarinic allosteric modulation: M-2/M-3 subtype selectivity of gallamineis independent of G-protein coupling specificity

Among the five subtypes of muscarinic acetylcholine receptors, the sensitiv ity towards allosteric modulation is generally higher in M-2 and M-4 recept ors that preferentially couple to inhibitory G-proteins of the G(i/o) type than in M-1, M-3, and M-5 that preferentially couple to stimulatory G-prote ins such as G(q/11). We aimed to check whether the high allosteric sensitiv ity of the M-2 receptor compared to M-3 is related to the differential G-pr otein coupling preference. As the third intracellular loop (i3) is known to be the major determinant in receptor G-protein coupling specificity, we us ed wild-type M-2 and M-3 receptors and the related chimeric constructs with exchanged i3-loops, i.e., M-2 containing M-3-i3 (M-2/M-3-i3) and M-3 conta ining M-2-i3 (M-3/M-2-i3). The allosteric effect of the archetypal modulato r gallamine on the dissociation and the equilibrium binding of [H-3]N-methy lscopolamine ([H-3]NMS) was measured in membranes of mouse A9L cells stably expressing the wild-type and the chimeric receptors (4 mM Na2HPO4, 1 mM KH 2PO4, pH 7.4, 23 degreesC). The dissociation of [3H]NMS was monophasic unde r all conditions studied. Control values of t(1/2), were (means SEM, n=4-7) : M-2: 3.8 +/-0.2 min, M-2/M-3-i3: 4.8 +/-0.3 min. M-3: 43.3 +/-4.2 min. M- 3/M-2-i3: 41.1 +/-3.6 min. At M-2 receptors, 0.2 muM gallamine allosterical ly reduced the apparent rate constant of dissociation k(-1) to 51 +/-5% of the control value (n=5). At M2/M-3-i3 the allosteric potency of gallamine w as not significantly changed (0.2 muM gallamine --> k-1=61 +/-4%, n=7). At M-3, a 20-fold higher concentration was required for an equieffective allos teric action (10 muM gallamine ---> k-1=51 5%, n=5). The potency of gallami ne at M-3/M-2-i3 was not increased compared with M3 receptors (10 muM galla mine -> k(-1)=73 +/-2%, n=4) but even significantly diminished. [H-3]NMS eq uilibrium binding experiments revealed that neither the binding constants o f gallamine at free receptor subtypes (pK(A,M2): 7.57 +/-0.04, n=4; pK(A,M3 ): 5.56 +/-0.13, n=3) nor the factors of negative cooperativity with [H-3]N MS (alpha (M2)=31 +/-1, alpha (M3)=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, alpha (M2/M3-i3)= 30 +/-2, alpha (M3/M2-i2)=3 +/-0.7). In conclusion, the different sensitivities Of M2 and M3 receptors towards a llosteric modulation by gallamine are not related to the G-protein coupling specificity of the receptors.