Five subtypes of muscarinic acetylcholine receptors that belong to class A of G-protein coupled receptors have been identified 1. The primary response of stimulation of the M₂ and M₄ subtypes of muscarinic receptors is activation of the G_i/o class of G-proteins resulting in inhibition of adenylyl cyclase, whereas stimulation of M₁, M₃, and M₅ receptors leads to activation of the G_q/11 class of G-proteins and stimulation of phospholipase C2. Muscarinic receptors mediate many diverse physiological functions that are selectively mediated by different receptor subtypes 3. This is why discovery of selective ligands is of prime importance for clinical practice. However, due to the very conserved nature of the orthosteric binding site of muscarinic acetylcholine receptors the selectivity of orthosteric agonists is very poor 4. Orthosteric antagonists that bind to less conserved amino acids located close to the orthosteric binding site display better selectivity than orthosteric agonists. Muscarinic allosteric ligands exhibit remarkable selectivity among receptor subtypes 5. They interact mainly with the second and the third extracellular loops that are much less conserved than transmembrane segments creating the orthosteric binding site 678910.

The extraordinary selectivity of allosteric modulators that is due to differences in both affinity and cooperativity 11 has attracted attention of pharmacologists in the past decade. Somewhat paradoxically, most of originally discovered and probably best studied allosteric compounds of muscarinic receptors are neuromuscular blockers 121314. By definition, these are competitive nicotinic acetylcholine receptor antagonists but many of them have high affinities and strong allosteric interactions, particularly at the M₂ subtype of muscarinic receptors.

In clinical practice, different competitive (nondepolarizing) neuromuscular blockers are employed to induce muscle relaxation to facilitate intubation during surgery. The neuromuscular blocker rapacuronium was withdrawn from clinical use due to high incidence of bronchospasm resulting in death 15. Parasympathetic innervation of airways transmits signal via postsynaptic M₃ receptors that mediate acetylcholine-induced contraction and M₂ receptors that inhibit with high potency smooth muscle relaxation mediated by increase in cytoplasmic cAMP 16. M₂ receptors are also located at parasympathetic cholinergic nerve terminals innervating smooth muscle and their stimulation inhibits acetylcholine (ACh) release 17. In functional experiments on the guinea pig trachea preparation it was demonstrated that rapacuronium preferentially antagonizes M₂ over M₃ muscarinic receptors 18. In addition, involvement of allosteric potentiation of ACh binding to muscarinic M₃ receptors in bronchospasm induced by rapacuronium was suggested, but not proven 19. A very recent paper confirmed a unique behavior of rapacuronium compared to other skeletal muscle relaxants in vivo and demonstrated that rapacuronium potentiates bronchoconstriction evoked by both naturally released and exogenous acetylcholine, indicating an important role of postsynaptic M₃ receptors 20.

Because we have been interested in investigations of positive cooperativity of allosteric ligands with ACh binding 1121 and allosteric agonists 22 these findings led us to analyze in detail the interactions of rapacuronium with acetylcholine binding and receptor activation of all subtypes of muscarinic receptors heterologously expressed in membranes of Chinese hamster ovary (CHO) cells. We demonstrate that rapacuronium binds to and exhibits negative cooperativity with ACh binding at all subtypes of muscarinic receptors. Surprisingly, low concentrations of rapacuronium potentiate ACh-induced signaling at the M₁, M₃, and M₅ receptor subtypes and accelerate ACh binding. This striking behavior is unparallel at other neuromuscular blockers.

Saturation binding experiments (Figure 1; Table 1) with 68 pM to 2 nM [³H]NMS in cell membranes showed similar binding capacity (1 to 2 pmol of binding sites per mg of protein) and affinity (equilibrium dissociation constant (K_D) ranging from 205 pM at M₄ to 320 pM at M₂ receptors) for all receptor subtypes (Figure. 1; Table 1). Significant depletion (up to 34% at M₁ for 68 pM [³H]NMS) occurred despite the use of 0.8 ml incubation volume in the binding assays. Thus, free concentrations of [³H]NMS were calculated and used in Eq. 1. Saturation binding experiments with 3.4 nM to 100 nM [³H]ACh showed similar high affinity binding among all subtypes with K_D around 20 nM. Rapacuronium concentration dependently decreased affinity for [³H]NMS and [³H]ACh at all subtypes without change in maximum binding capacity (B_MAX). Competition experiments of unlabeled ACh vs. [³H]NMS displayed high and low binding sites for ACh at all subtypes with higher proportion of high affinity binding sites at even-numbered subtypes (Figure 2). Equilibrium dissociation constants (K_I) of ACh high-affinity binding derived from competition experiments with [³H]NMS (pK_D = 7.32 ± 0.06, 7.59 ± 0.03, 7.79 ± 0.05, 7.69 ± 0.04, 7.68 ± 0.05, mean ± SE for M₁ to M₅ receptor) correspond to those measured in [³H]ACh saturation experiments (Table 1). In the presence of 10 μM GTPγS to uncouple receptors and G-proteins ACh low affinity binding was similar at all five subtypes with equilibrium dissociation constant (K_I) ranging from 25.5 μM at M₄ to 46.8 μM at M₁.

Effects of 100 μM rapacuronium on the rate of ([³H]NMS) dissociation were measured in membranes from CHO cell expressing individual subtypes of muscarinic receptors after 60 min preincubation with 1 nM [³H]NMS. Dissociation was evoked by addition of 10 μM unlabeled NMS. Rapacuronium slowed dissociation of [³H]NMS from all subtypes of muscarinic ACh receptors (Figure 3, Table 2). This is an established hallmark of allosteric receptor modulation. It had the strongest effect at M₂ receptors (7-fold decrease in rate of dissociation) and weakest effect at M₃ and M₅ receptors (40% decrease). While dissociation evoked by NMS was monophasic (Figure 3 closed symbols) it became biphasic in the presence of 100 μM rapacuronium with the exception of the M₅ subtype.

Displacement radioligand binding experiments with either 20 nM [³H]ACh (Figure 4) or 1 nM [³H]NMS (Figure 5, circles) and increasing concentrations of rapacuronium showed that rapacuronium binds equally well to all five muscarinic receptor subtypes. Equilibrium dissociation constants (pK_A; Table 3) for rapacuronium derived from experiments with [³H]NMS and [³H]ACh were virtually the same with a rank order of affinity of M₂>M₄>M₁>M₅>M₃ (range from 2.6-17.8 μM). Rapacuronium displayed negative cooperativity with [³H]NMS in binding to all subtypes, as evidenced by a maximal limit to its effects on the affinity of the radioligand that differed as a function of radioligand concentration. These effects were strongest at the M₅ subtype (35-fold decrease in affinity) and weakest at the M₂ subtype (6.8-fold decrease in affinity; Figure 5, closed circles). While cooperativity of rapacuronium with high- (pα) and low-affinity (pβ) ACh binding was essentially the same at individual subtypes (Table 3, row-wise comparison) it was slightly different among subtypes (e.g. 16-fold decrease in ACh low-affinity binding at M₃ receptors vs. 36-times decrease at M₄ receptors; Table 3, column-wise comparison).

Rapacuronium alone concentration dependently lowered [³⁵S]GTPγS binding to membranes (Figure 6, closed squares; Table 4) with a maximal effect of approximately 25% at odd-numbered subtypes and 15% at even-numbered subtypes, with similar half-effective concentrations (EC₅₀) ranging from 28 μM at M₂ receptors to 76 μM at M₃ receptors. While the EC₅₀ values of rapacuronium in inhibiting [³⁵S]GTPγS binding at individual subtypes correlated with affinities measured in binding experiments with [³H]ACh (R² = 0.76) they were lower (4- to 12-fold) at all subtypes. We could not test the involvement of muscarinic receptors in the effects of rapacuronium on [³⁵S]GTPγS binding using orthosteric antagonists, since 10 μM NMS or 10 μM atropine by themselves decreased [³⁵S]GTPγS binding by more than 30% at all receptor subtypes. Inhibitory effects of rapacuronium were not additive to those of NMS or atropine (not shown). However, rapacuronium did not decrease [³⁵S]GTPγS binding in membranes from nontransfected CHO cells (Figure 6, bottom row right).

As expected, ACh concentration-dependently stimulated [³⁵S]GTPγS binding to membranes from cells expressing all individual subtypes of muscarinic receptors (Table 4 and Figure 6, closed circles). The maximal effect of ACh (E_MAX) was about two-fold increase in basal binding at odd-numbered receptors and three-fold increase at even-numbered receptors with a rank order of efficacy of M₂>M₄>M₁>M₅>M₃ (range from 3.12 to 1.99-fold increase). In control conditions ACh EC₅₀ values were lower at even-numbered subtypes than at odd-numbered subtypes with a rank order of potency of M₂>M₄>M₃>M₅>M₁ (range from 0.25 to 6.31 μM) (Table 5). While the EC₅₀ of ACh-stimulated [³⁵S]GTPγS binding was less than that of its low-affinity binding conformation by 178-times at M₂ and 23-times at M₄ receptors it was only 7.4-, 5.4-, and 4.7-times lower at M₁, M₃, and M₅ receptors, respectively. In comparison with its high-affinity binding, the EC₅₀ of ACh-stimulated [³⁵S]GTPγS binding was only 10-times higher at M₂ and 55-times at M₄ receptors but 130-260- and 300-times higher at M₁, M₅ and M₃ receptors, respectively. E_MAX was about two-fold increase in basal binding at odd-numbered receptors and three-fold increase at even-numbered receptors with a rank order of efficacy of M₂>M₄>M₁>M₅>M₃ (range from 3.12 to 1.99-fold increase) (Table 5).

Measurements of ACh-stimulated [³⁵S]GTPγS binding in the presence of 0.1, 1 and 10 μM rapacuronium showed differential effects of rapacuronium on receptor activation by an orthosteric agonist at individual receptor subtypes (Figure 6 open symbols). At even-numbered subtypes 1 μM and 10 μM rapacuronium significantly increased ACh EC₅₀, with lowering of E_MAX at 10 μM rapacuronium. These results are in line with the effects of rapacuronium on ACh binding. In contrast, the effects of rapacuronium on activation of odd-numbered subtypes were more complex. At these subtypes rapacuronium had the strongest effect on activation of the M₃ subtype. At this subtype 0.1 and 1 μM rapacuronium caused a significant 2-fold decrease in ACh EC₅₀ and approximately 60% and 35% increase in E_MAX, respectively. Rapacuronium at 10 μM increased ACh EC₅₀ by about 3-fold without a significant change in E_MAX. Rapacuronium (0.1 - 10 μM) had no effect on ACh efficacy at the M₁ and M₅ subtypes but decreased the EC₅₀ of ACh in stimulating [³⁵S]GTPγS binding by 1.5- and 4-fold, respectively, at concentrations of 0.1 and 1 μM. However, this effect was not evident at 10 μM rapacuronium (Figure 6).

Kinetics of [³⁵S]GTPγS binding to membranes from CHO-M₃ cells (where rapacuronium has the most pronounced effects) were measured after 5 min preincubation with 5 μM GDP and 60 min with 1 μM rapacuronium followed by simultaneous addition of [³⁵S]GTPγS and ACh, with or without rapacuronium (Figure 7). Under basal conditions ([³⁵S]GTPγS + buffer, Figure 7, closed circles) [³⁵S]GTPγS bound to membranes with an observed rate constant (k_obs) of 0.0269 ± 0.0022 min^-1. Ten μM ACh accelerated the rate of [³⁵S]GTPγS binding to 0.0583 ± 0.047 min^-1 (Figure 7, closed squares) and 1 μM rapacuronium further increased the rate to 0.166 ± 0.013 min^-1 (Figure 7, open squares). While 10 μM ACh alone produced only 2-fold increase in [³⁵S]GTPγS binding at 5 min incubation it caused nearly 5-fold increase in the presence of 1 μM rapacuronium. Estimated [³⁵S]GTPγS equilibrium binding (B_eq) was the same for all 3 treatments (10,000 ± 300, 10,100 ± 100, and 9,980 ± 120 cpm per well for basal, ACh and ACh with rapacuronium, respectively; mean ± SE, n = 3). Rapacuronium alone slightly decreased the rate of [³⁵S]GTPγS binding (Figure 7, open circles).

Effects of rapacuronium on the association rate of high-affinity (40 nM) [³H]ACh binding were measured after 60 min preincubation of membranes with rapacuronium. Association of [³H]ACh is complex and consists of an initial very fast step in the range of seconds followed by a slower phase (Figure 8, first time point is 5 s). Under control conditions (Figure 8, closed circles) the slower phase of [³H]ACh association was characterized by an observed rate (k_obs) in the range of 1.43 (M₃) to 3.5 (M₅) min^-1. While the presence of 1 μM rapacuronium had marginal effects on [³H]ACh association (Figure 8, open circles) the association binding curve became more complex and showed a peak in the presence of 10 μM rapacuronium (Figure 8, hatched circles). This peak occurred at 15 to 20 seconds at odd-numbered receptors and at 30 to 50 seconds at the M₄ and around 90 seconds at the M₂ receptor. Peak binding was higher than control binding at odd-numbered receptors, the same as control binding at M₄ and lower than control binding at M₂ receptor. An increase in [³H]ACh binding after extended incubation (hours) occurred at M₃, M₄, and M₅ receptors.

Effects of 10 μM rapacuronium on the dissociation rate of high-affinity [³H]ACh binding were measured after 60 min preincubation of membranes with 40 nM [³H]ACh. Dissociation was evoked by the addition of unlabeled ACh at a final concentration of 40 μM, either alone or mixed with 10 μM rapacuronium (Figure 9, Table 6). [³H]ACh dissociation curves consisted of a very rapid phase followed by a slow one, both in the absence (Figure 9, closed circles) and in the presence (Figure 9, open and hatched circles) of rapacuronium. The slower phase of [³H]ACh dissociation displayed a rate (k_off) in the range of 0.112 (M₅) to 0.507 (M₂) min^-1 (Figure 9, closed circles). While effects of 1 μM rapacuronium on [³H]ACh dissociation were marginal, 10 μM rapacuronium either accelerated (odd-numbered), did not change (M₄) or slowed (M₂) the rate of [³H]ACh dissociation (Figure 9, hatched circles).

The effects of rapacuronium and the two prototypic allosteric modulators alcuronium and gallamine on ACh-stimulated [³⁵S]GTPγS binding and kinetics of [³H]ACh binding were compared at M₃ receptors where the effects of rapacuronium were most pronounced. Measurements of ACh-stimulated [³⁵S]GTPγS binding in the presence of 1 and 10 μM alcuronium or 1 and 10 μM gallamine, showed a small concentration-dependent increase in the EC₅₀ of ACh without change in E_MAX values (Figure 10, Table 7). Both alcuronium and gallamine concentration dependently slowed [³⁵S]GTPγS association stimulated by 10 μM ACh. At 10 μM concentrations they also decreased [³⁵S]GTPγS equilibrium binding (Figure 10, Table 7). Alcuronium and gallamine slowed down association of 40 nM [³H]ACh and decreased its equilibrium binding at M₃ receptors (Figure 10, Table 7).

Our results clearly demonstrate that the neuromuscular blocker rapacuronium binds to all muscarinic receptor subtypes at physiologically relevant concentrations 18 and displays micromolar affinity and slight selectivity towards M₂ receptor. This selectivity is smaller than that of other neuromuscular blockers such as alcuronium, gallamine and pancuronium [23, 24, Jakubík, unpublished data]. Like the majority of this class of compounds, rapacuronium acts as a negative allosteric modulator (alters dissociation kinetics and incompletely inhibits binding of orthosteric ligands) with respect to binding of both the natural agonist ACh (Figures 1, 4, 5, 8 and 9) and the classical antagonist NMS (Figures 1, 3, and 5). Rapacuronium exhibits complex effects on the kinetics of ACh binding (Figures 8 and 9) and subsequent receptor activation estimated from stimulation of [³⁵S]GTPγS binding (Figures 6 and 7). Functional effects differ from those of the prototypic negative allosteric modulators alcuronium and gallamine (Figure 10, Table 7).

Our observation of an allosteric mode of interaction between rapacuronium and muscarinic receptors is in agreement with reported slowing-down of NMS dissociation from M₂ and M₃ receptors by this drug 19. The observed biphasic dissociation of NMS under non-equilibrium conditions in the presence of an allosteric modulator such as rapacuronium was described earlier 24.

Although rapacuronium exerts negative cooperativity with binding of ACh to all muscarinic receptor subtypes at equilibrium it accelerates the rate of ACh binding at odd numbered subtypes. At concentrations below 10 μM, it increases the potency and efficacy of ACh in increasing the rate of [³⁵S]GTPγS binding at odd-numbered subtypes. The time between acetylcholine release and termination of its action by acetylcholinesterase is in the range of a fraction of a second. Therefore, the effects of allosteric modulators in the early non-equilibrium stage of receptor signaling are therapeutically more important than effects on acetylcholine equilibrium binding, as the latter conditions do not occur in vivo. Our study demonstrates a case of dichotomous effects of the allosteric modulator rapacuronium on ACh equilibrium binding on the one hand and on the kinetics of ACh binding on the other. Our observations emphasize the necessity to employ fast functional assays in screening for potential allosteric modulators of neurotransmission that much better simulate physiological conditions than long-lasting equilibrium binding experiments.

Ach: acetylcholine; ANOVA: analysis of variance; CHO: Chinese hamster ovary; GTPγS: guanosine 5'-O-(3-thio)triphosphate; NMS: N-methylscopolamine; TCM: ternary complex model.

JJ carried out [³H]ACh and [³H]NMS binding studies and performed statistical analysis. AR carried out [³⁵S]GTPγS binding studies. All authors participated in experimental design and draft manuscript. All authors read and approved final manuscript.

JJ is scientist, AR PhD student and VD head of Department of Neurochenistry. EEE is Professor at the University of Minnesota Medical School. JJ, VD and EEE have decades-long experience in the research of muscarinic receptors and neurochemistry.