Divergence of allosteric effects of rapacuronium on binding and function of muscarinic receptors

Abstract

Background: Many neuromuscular blockers act as negative allosteric modulators of muscarinic acetylcholine receptors by decreasing affinity and potency of acetylcholine. The neuromuscular blocker rapacuronium has been shown to have facilitatory effects at muscarinic receptors leading to bronchospasm. We examined the influence of rapacuronium on acetylcholine (ACh) binding to and activation of individual subtypes of muscarinic receptors expressed in Chinese hamster ovary cells to determine its receptor selectivity.

Results: At equilibrium rapacuronium bound to all subtypes of muscarinic receptors with micromolar affinity (2.7-17 microM) and displayed negative cooperativity with both high- and low-affinity ACh binding states. Rapacuronium accelerated [3H]ACh association with and dissociation from odd-numbered receptor subtypes. With respect to [35S]GTPgammaS binding rapacuronium alone behaved as an inverse agonist at all subtypes. Rapacuronium concentration-dependently decreased the potency of ACh-induced [35S]GTPgammaS binding at M2 and M4 receptors. In contrast, 0.1 microM rapacuronium significantly increased ACh potency at M1, M3, and M5 receptors. Kinetic measurements at M3 receptors showed acceleration of the rate of ACh-induced [35S]GTPgammaS binding by rapacuronium.

Conclusions: Our data demonstrate a novel dichotomy in rapacuronium effects at odd-numbered muscarinic receptors. Rapacuronium accelerates the rate of ACh binding but decreases its affinity under equilibrium conditions. This results in potentiation of receptor activation at low concentrations of rapacuronium (1 microM) but not at high concentrations (10 microM). These observations highlight the relevance and necessity of performing physiological tests under non-equilibrium conditions in evaluating the functional effects of allosteric modulators at muscarinic receptors. They also provide molecular basis for potentiating M3 receptor-mediated bronchoconstriction.

Figure 1

Saturation binding of [³H]NMS and [³H]Ach. Specific binding of [³H]NMS (circles) and [³H]ACh (squares) to membranes from CHO cells expressing individual subtypes of muscarinic receptors is plotted against the concentration of free radioligand. Binding of radioligand in the absence (closed symbols) and presence of 10 μM (open symbols) or 100 μM (hatched symbols) rapacuronium, respectively. Data are means ± SE from 3 independent experiments performed in quadruplicates. Curves are fits of Eq. 1 to data. Binding parameters are summarized in Table 1.

Figure 2

Effects of 10 μM GTPγS on ACh competition with [³H]NMS binding. Binding of 1 nM [³H]NMS to the membranes from CHO cells expressing individual subtypes of muscarinic receptors in the absence (closed circles) or presence (open circles) of 1 μM GTPγS is expressed as per cent of control binding and are plotted against concentration of ACh. Data are means ± SE of 3 independent experiments performed in quadruplicates. Binding parameters are described in the Results.

Figure 3

Effects of 100 μM rapacuronium on dissociation of [³H]NMS binding. Binding of [³H]NMS to membranes from CHO cells expressing individual subtypes of muscarinic receptors at different times after the addition of 10 μM NMS (closed circles) or a mixture of 10 μM NMS and 100 μM rapacuronium (open circles). Specific binding is expressed as percent of binding at time 0. Data are means ± SE from 3 independent experiments performed in quadruplicates. Binding parameters are summarized in Table 2.

Figure 4

Effects of rapacuronium on high-affinity [³H]ACh binding. Binding of 20 nM [³H]ACh to membranes from CHO cells expressing individual subtypes of muscarinic receptors (circles, M₁; squares, M₂; diamonds, M₃; up-triangles, M₄; down-triangles, M₅) was determined in the presence of rapacuronium at the concentrations indicated on the x-axis and is expressed as percent of specific binding in the absence of rapacuronium. Data are means ± SE from 3 independent experiments performed in quadruplicates. Curves are fits of Eq. 3 to data. Binding parameters are summarized in Table 3.

Figure 5

Effects of rapacuronium on low-affinity ACh binding and [³H]NMS binding. Binding of 1 nM [³H]NMS to membranes from CHO cells expressing individual subtypes of muscarinic receptors was measured in the presence of rapacuronium at the concentrations indicated on the x-axis. Data are expressed as percent of specific binding in the absence of rapacuronium. Symbols represent binding of [³H]NMS (circles), and [³H]NMS in the presence of 10 μM GTPγS and 100 μM (squares) or 200 μM (triangles) ACh. Binding of [³H]NMS was decreased by 100 μM ACh to 71, 64, 59, 60 and 56% and by 200 μM ACh to 55, 47, 42, 43 and 39% at M₁ to M₅ receptors, respectively. Bigger divergence of curves denotes stronger negative cooperativity between ACh and rapacuronium binding. Data are means ± SE from 3 independent experiments performed in quadruplicates. Curves are fits of Eq. 3 (circles) and Eq. 4 (squares and triangles) to data prior to normalization. Binding parameters are summarized in Table 3.

Figure 6

Effects of rapacuronium on ACh-induced [³⁵S]GTPγS binding to membranes. Membranes from CHO cells expressing individual subtypes of muscarinic receptors or nontransfected CHO-K1 cells were preincubated for 60 min with buffer (closed symbols) or with rapacuronium (open symbols). [³⁵S]GTPγS binding was induced by rapacuronium alone (closed squares), ACh alone (closed circles), or by ACh in the presence of rapacuronium (0.1 μM (open circles), 1 μM (open squares), or 10 μM (open triangles)). Data are expressed as fold increase of basal [³⁵S]GTPγS binding and are presented as means ± SE from 3 independent experiments performed in quadruplicates. Curves are fits of Eq. 5 to data. Parameters are summarized in Tables 4 and 5.

Figure 7

Effects of rapacuronium on kinetics of [³⁵S]GTPγS binding. Membranes were preincubated for 60 min in the presence (open symbols) or absence (closed symbols) of 1 μM rapacuronium. Then [³⁵S]GTPγS was added simultaneously with buffer (circles) or 10 μM ACh (squares). Incubations were terminated at the times indicated on the x-axis. The increase of specific [³⁵S]GTPγS binding is expressed as fmol per μg of protein (left) and as fold increase of specific binding under basal conditions (right). Data are means ± SE of values from 3 independent experiments performed in quadruplicates.

Figure 8

Effects of rapacuronium on [³H]ACh association. Membranes from CHO cells expressing individual subtypes of muscarinic receptors were preincubated 60 min with buffer (closed circles) or 1 μM (open circles) or 10 μM (hatched circles) rapacuronium and then [³H]ACh was added to a final concentration of 40 nM at time 0. Incubations were terminated at the times indicated on the x-axis. Specific [³H]ACh binding is expressed as fmol per mg of proteins. Data are means ± SE of values from 3 independent experiments performed in quadruplicates. Binding parameters are shown in Table 6.

Figure 9

Effect of rapacuronium on the time course of [³H]ACh dissociation. Membranes from CHO cells expressing individual subtypes of muscarinic receptors were prelabeled with 40 nM [³H]ACh for 60 min. At time zero 40 μM unlabeled ACh was added alone (closed circles) or as a mixture with 1 μM rapacuronium (open circles) or 10 μM rapacuronium (hatched circles). Incubations were terminated at the times indicated on the x-axis. Specific [³H]ACh binding is expressed as percent of specific binding at time 0 on x-axis. Data are means ± SE of values from 3 independent experiments performed in quadruplicates. Binding parameters are shown in Table 6.

Figure 10

Effects of alcuronium and gallamine on [³H]ACh binding and ACh-stimulated [³⁵S]GTPγS binding to M₃ membranes. Effects of the reference allosteric modulators alcuronium (left) and gallamine (right) on kinetics of [³H]ACh binding (top) and ACh-stimulated [³⁵S]GTPγS binding (middle) and concentration response of [³⁵S]GTPγS binding to ACh stimulation (bottom) at M₃ receptors were measured after preincubation of membranes for 60 min with buffer (closed circles) or with 1 μM (open squares) or 10 μM (open triangles) rapacuronium. Then either [³H]ACh (top) or [³⁵S]GTPγS simultaneously with 10 μM ACh (middle and bottom) was added. Incubation was terminated at the times indicated on the x-axis (top and middle) or after 20 min (bottom). Binding is expressed as fmol per μg of protein of specific [³H]ACh (top) or [³⁵S]GTPγS (middle) binding or as fold increase of basal [³⁵S]GTPγS binding (bottom). Data are means ± SE from 3 to 6 independent experiments performed in quadruplicates. Parameters are summarized in Table 7.