Activation and Inhibition of Human Muscular and Neuronal Nicotinic Acetylcholine Receptors by Succinylcholine
Jonsson, Malin M.D.*; Dabrowski, Michael Ph.D.†; Gurley, David A. M.S.‡; Larsson, Olof Ph.D.§; Johnson, Edwin C. Ph.D.‡; Fredholm, Bertil B. M.D., Ph.D.∥; Eriksson, Lars I. M.D., Ph.D.#
Background: Succinylcholine is one of the most widely used muscle relaxants in clinical anesthesia and emergency medicine. Although the clinical advantages and cardiovascular side effects are well known, its mechanism of action within the human nicotinic cholinergic receptor system remains to be understood. The aim of this study was to investigate the effect of succinylcholine on human muscle and neuronal nicotinic acetylcholine receptor (nAChR) subtypes.
Methods: Xenopus laevis oocytes were injected with human messenger RNA for muscle and neuronal nAChR subunits. Receptor activation, desensitization, and inhibition induced by the natural ligand acetylcholine or by succinylcholine was studied using a multichannel two-electrode voltage clamp setup. Responses were measured as peak current and net charge.
Results: Succinylcholine concentration-dependently activated the muscle-type nAChR with an EC50 value of 10.8 μm (95% confidence interval, 9.8–11.9 μm), and after the initial activation, succinylcholine desensitized the muscle-type nAChR. Succinylcholine did not activate the neuronal nAChR subtypes α3β2, α3β4, α4β2, or α7 at concentrations up to 1 mm and was a poor inhibitor at these receptor subtypes, with IC50 values above 100 μm.
Conclusion: Succinylcholine activates the muscle-type nAChR followed by desensitization. The observation that succinylcholine does not inhibit the presynaptic α3β2 autoreceptor at clinically relevant concentrations provides a possible mechanistic explanation for the typical lack of tetanic fade in succinylcholine-induced neuromuscular blockade. Finally, cardiovascular side effects (e.g., tachyarrhythmias) of succinylcholine are not mediated via direct activation of the autonomic ganglionic α3β4 subtype because succinylcholine does not activate the neuronal nAChRs.
SUCCINYLCHOLINE has been used for more than 50 yr in clinical anesthesia and emergency medicine, and it is still one of the most widely used muscle relaxants. Succinylcholine is composed of two acetylcholine molecules, linked end to end at the acetyl side, and has the potential to exert acetylcholine-like effects at nicotinic acetylcholine receptors (nAChRs).
The nAChRs are members of a neurotransmitter-gated ion channel superfamily. They are composed of five transmembrane subunits with a central cation pore, and the stoichiometry of subunits gives each receptor its unique properties.1
To date, 17 nicotinic subunits have been cloned in vertebrates: the muscle α1, β1, δ, γ, and ε subunits and the neuronal α2–10 and β2–4 subunits.2
The fetal muscle nAChR consists of two α1, one β1, one δ, and one γ subunit, but in the adult muscle nAChR, the γ subunit is replaced by ε.3,4
The neuronal nAChRs include both of homomeric and heteromeric receptors, with the α7–9 subunits forming homomeric nAChRs. The heteromeric receptors are formed by a combination of α2–6 and β2–4.1
Although there are many potential combinations of heteromeric neuronal nAChRs, to date, only a few have been found to be of biologic importance.5,6
The muscle nAChR is present at the postsynaptic muscle membrane in the neuromuscular junction, whereas the neuronal nAChRs are found both presynaptically and postsynaptically in the central and peripheral nervous system as well as in extraneuronal tissues and cells, such as keratinocytes, muscle, lymphocytes, macrophages, carotid bodies, and neurosecretory cells.1,2,7
There is evidence for nicotinic autoreceptors at the presynaptic terminal in the neuromuscular junction,8,9
and it has recently been shown that a block of α3β2 nicotinic receptors produce tetanic fade by interruption of nicotine-mediated autofacilitation of acetylcholine release.10,11
The major autonomic ganglionic receptor is α3β4, but functional α3β2 and, to a lesser extent, α7 receptors have been detected in ganglionic neurons.6,12,13
In the central nervous system, nAChRs are widespread: α4β2 is the most common nAChR, but α3β2 and α7 are also present and functional.6,14
In clinical anesthesia, administration of succinylcholine activates the muscle-type nAChR, which is seen as fasciculations of skeletal muscle. This is followed by a block or desensitization, seen as flaccidity. Succinylcholine-induced paralysis lasts for only 5–10 min after a single dose, because of the rapid degradation of succinylcholine to succinylmonocholine and choline by plasma butyrylcholinesterase.15
In contrast to nondepolarizing neuromuscular blocking agents, succinylcholine does not produce tetanic fade and therefore seems to lack interaction with presynaptic feedback control of transmitter release mediated via
the α3β2 nAChR subtype.8,10
Succinylcholine has many side effects, of which the cardiovascular effects are more serious.16
It has been suggested that some cardiovascular side effects caused by succinylcholine arise from an interaction with neuronal nAChRs in autonomic ganglia17
; however, this has not been confirmed at the molecular level. Furthermore, the mechanism of action of succinylcholine within the neuromuscular junction is not clear, and the putative interaction with the presynaptic α3β2 nAChR has not been studied previously.
Our aim was to study the effect of succinylcholine on human muscle and neuronal nAChRs expressed in Xenopus oocytes, to describe activation, desensitization, and inhibition of the nAChRs in interaction with the natural ligand, acetylcholine.
Materials and Methods
The human nAChR subunits α1, α3–4, α7, β1, β2, β4, δ, and ε were cloned from a human complementary DNA (cDNA) library. GenBank (Bethesda, MD) access numbers for the cDNA nucleotide sequences are as follows: NM 000079 (α1), NM 000747 (β1), NM 000751 (δ), NM 000080 (ε), HSU62432 (α3), L35901 (α4), Y08420 (α7), Y08415 (β2), and NM 000750 (β4). The cDNAs were subcloned into different expression vectors, pBluescript II SK (−) (Stratagene, La Jolla, CA; α7), pKGem (AstraZeneca, Wilmington, DE; α1, α3, β1, β2, δ, and ε), and pBSTA (University of California, Irvine, CA; α4 and β4). Messenger RNA (mRNA) was transcribed in vitro using the mMessage mMachine® T7 kit (Ambion, Austin, TX) and analyzed using a bioanalyzer (Agilent Technologies, Palo Alto, CA).
Xenopus Oocyte Injection
The study was approved by the local animal ethics committee at Karolinska Institutet, Stockholm, Sweden. Xenopus laevis oocytes were isolated by partial ovariectomy from frogs anesthetized with 0.2% tricaine (3-aminobenzoic acid ethyl ester, 2 g/l added to the water). The incision was sutured, and the animals were monitored during the recovery period before being returned to their tank. The ovaries were mechanically dissected to smaller lumps and digested in OR-2 buffer (82.5 mm NaCl, 2 mm KCl, 2 1 mm MgCl, 5 mm HEPES, pH adjusted to 7.5 with NaOH) containing 1.5 mg/ml collagenase (type 1A; Sigma, St. Louis, MO) for 90 min to remove the follicular epithelia from the oocytes. Injection electrodes were made from 3.5-in Drummond tubes (#3-000-203-G/X; Drummond Scientific Company, Broomall, PA) and pulled using a DMZ-Universal Puller (Zeitz-Instrumente GmbH, Munich, Germany). After 1–24 h, the oocytes were injected with 0.2–18 ng mRNA in a total volume of 30–40 nl/oocyte. Multiple subunit combinations were injected at a 1:1 ratio (α1β1δε or αx βy), except for α4β2, where the injection ratio was 1:9. The oocytes were maintained in Leibovitz L-15 medium (Sigma) diluted 1:1 with Millipore (Billerica, MA) filtered double distilled water and 80 μg/ml gentamicin, 100 units/ml penicillin and 100 μg/ml streptomycin added. Oocytes were incubated at 18°–19°C for 3–7 days after injection before being studied.
All recordings were performed at room temperature (20°–22°C). During recording, the oocytes were continuously perfused with ND-96 (96.0 mm NaCl, 2.0 mm KCl, 1.8 mm CaCl2, 1.0 mm MgCl2, 5.0 mm HEPES, pH 7.4 adjusted with NaOH). Oocyte recordings were performed using an integrated system that provides automated impalement of up to eight oocytes studied in parallel with two-electrode voltage clamp, and current measurements were automatically coordinated with fluid delivery throughout the experiment (OpusXpress™ 6000A; Molecular Devices, Union City, CA). Electrodes made from 1.5-mm borosilicate tubes (World Precision Instruments Inc, Sarasota, FL) were pulled using a micropipette puller (PP-83; Narishige Scientific Instrument Lab, Tokyo, Japan) and filled with 3 m KCl (0.5–2.5 MΩ resistance). The oocytes were voltage clamped at a holding potential of −60 mV.
Oocytes were continuously perfused with ND-96 at a rate of 2 ml/min in a 150-μl chamber. Drugs were delivered from a 96-well plate using disposable tips and administrated at a rate of 2 ml/min for the first 2 s, and thereafter at 1 ml/min. In activation experiments, acetylcholine and succinylcholine were applied for 20 s. To determine whether succinylcholine inhibited acetylcholine-induced currents, succinylcholine was coapplied and preapplied with acetylcholine for 55 s before a 20-s application of both acetylcholine and succinylcholine. Between each drug application, there was a 6-min washout period to allow clearance of the drugs and to avoid desensitization of the channels. Before and after each concentration-response experiment, three control responses were recorded using an EC50 acetylcholine concentration for each receptor subtype to exclude desensitization (precontrol and postcontrol). Experiments were rejected if the postcontrol response was less than 80% of the precontrol response. To adjust for the level of channel expression, the responses in agonist concentration-response experiments were normalized to peak response in each oocyte. For inhibition experiments, responses in each oocyte were normalized to the mean of the second and third acetylcholine precontrols.
Succinylcholine, acetylcholine, and butyrylcholinesterase were purchased from Sigma. Chemicals used in buffers were purchased from Sigma or Merck (Nottingham, United Kingdom) unless otherwise stated. Stock solutions of 1 mm acetylcholine and succinylcholine were prepared in ND-96 buffer and frozen. All drugs were then diluted in ND-96 immediately before use.
Data Analysis and Statistics
Off-line analyses were made using Clampfit 9.2 (Molecular Devices). Changes in currents were studied both as peak and net charge responses (area under the curve); however, for α7, only net charge analysis was used, as previously described.18,19
The baseline current immediately before drug application was subtracted from the response, and the analysis region for peak and net charge analysis was 20 s, i.e.
, during the time of agonist application. Concentration-response relations for agonists (acetylcholine and succinylcholine) were fitted by nonlinear regression (Prism 4.0; GraphPad, San Diego, CA) to the four parameter logistic equation: Y = Bottom + (Top − Bottom)/(1 + 10∧((Log EC50
− X) * Hill Slope)), wherein Y is the normalized response, X is the logarithm of concentration, and EC50
is the concentration of agonist eliciting half-maximal response. When succinylcholine-induced inhibition was studied, the same equation was used, and EC50
was replaced by IC50
, which is the concentration of antagonist eliciting half maximal inhibition. Unless otherwise stated, data are given as mean ± SEM or 95% confidence interval (CI). Differences in IC50
values were compared by using paired or unpaired two-tailed Student t
test as appropriate. A P
value of less than 0.05 was considered significant.
Acetylcholine Concentration-Response Relations for Muscular and Neuronal nAChRs
Acetylcholine produced a concentration-dependent inward current in oocytes injected with muscle- and neuronal-type nAChRs and voltage clamped at −60 mV (fig. 1
), whereas uninjected oocytes did not respond to acetylcholine (data not shown). The responses to acetylcholine of the nAChR subtypes are consistent with previous reports20–22
in terms of kinetics and EC50
values (fig. 1
and table 1
), thus confirming the expression of the individual nAChRs in our receptor model. However, there is a lack of published data for comparison of net charge in human nAChRs, the only exception being for the α7 nAChR subtype.19
As shown in figure 1B
, the α1β1δε, α3β4, and α4β2 nAChR concentration-response relations based on net charge analysis correlate well with peak currents, with almost identical EC50
and Hill coefficients (table 1
). However, the α7-subtype nAChR displays unique properties, with very fast desensitization kinetics (fig. 1A
), which gives a different concentration-response relation depending on whether peak response or net charge was measured (fig. 1B
). Therefore, EC50
is significantly lower if calculated from net charge analysis (table 1
< 0.0001), in agreement with Papke et al.18,19
Succinylcholine and Activation of Muscular nAChRs
Succinylcholine produced a concentration-dependent inward current in voltage clamped oocytes expressing adult muscle-type (α1β1δε) nAChR (fig. 1
). Succinylcholine caused an increasing activation of the α1β1δε nAChR in concentrations up to 100 μm; however, higher concentrations resulted in a reduced response to succinylcholine, likely because of a more rapid channel desensitization by higher succinylcholine concentrations (fig. 1B
). In most of these succinylcholine concentration-response experiments, we never saw full recovery despite trying both extended washout periods and application of the succinylcholine degrading enzyme, butyrylcholinesterase. Butyrylcholinesterase did not seem to have any effect by itself in the range of 100–500 μm (data not shown). The maximal response to succinylcholine tended to be 30–40% of that produced by acetylcholine, but detailed comparisons were not performed to calculate efficacy, because the controls did not return at the end of the experiments.
Succinylcholine and Inhibition of Muscular nAChRs
To investigate succinylcholine as a blocker at the α1β1δε-subtype nAChR, succinylcholine was applied before (preapplication) and together with acetylcholine (coapplication). Two concentrations of acetylcholine were studied, 1 μm and 5 μm. As shown in figure 2A
, the concentration-response curve for succinylcholine in the presence of 1 μm acetylcholine was similar to the concentration-response curve for succinylcholine alone (fig. 1B
), but slightly shifted to the right with an EC50
of 19.3 μm (95% CI, 5.1–73.2 μm) and a Hill coefficient of 1.12 ± 0.59 (n = 8). In contrast, the succinylcholine concentration-response curve in presence of 5 μm acetylcholine (fig. 2B
) was bell shaped. The responses to low added concentrations of succinylcholine (0.1–1 μm) were not different from the 5 μm acetylcholine control response, whereas 1–100 μm succinylcholine caused a concentration-dependent potentiation of the acetylcholine current. However, at succinylcholine concentrations above 100 μm, the potentiation decreased similar to the activation curve elicited by succinylcholine alone. The kinetics were unchanged at succinylcholine up to 100 μm for both 1 and 5 μm acetylcholine. Preapplication of succinylcholine for 55 s before 5 μm acetylcholine concentration-dependently inhibited the receptor acetylcholine response (fig. 2C
). To calculate an IC50
for the succinylcholine-dependent decrease in acetylcholine activation, the peak acetylcholine response in presence of succinylcholine minus the base current after 54 s of succinylcholine incubation was normalized to the acetylcholine preapplication current. This yielded an IC50
of 126 μm (95% CI, 47.8–334 μm) and a Hill coefficient of −1.23 ± 0.93 (n = 11).
As for the succinylcholine activation concentration-response relations, the 5 μm acetylcholine controls did not recover fully at the end of the experiments in either the coapplication or the preapplication protocol. In contrast, in succinylcholine concentration-response experiments with 1 μm acetylcholine, the acetylcholine controls displayed full recovery, indicating receptor desensitization at only repeated higher acetylcholine applications. In previous studies investigating the effect of succinylcholine on the α1β1δε nAChR, precontrol and postcontrol responses to the agonist have not been measured,23
or a mean of the controls before and after succinylcholine has been used as reference, and it has not been stated whether the controls where stable during the experiment.24
Succinylcholine and Activation of Neuronal nAChRs
In contrast to the muscle-type nAChR, the neuronal types did not respond to succinylcholine in concentrations up to 1 mm (fig. 1
). In the range of 1–10 mm succinylcholine, the neuronal α3β2 and α3β4 displayed no succinylcholine activated current, and the α4β2 and α7 displayed very small inward currents, with a very low efficacy compared with acetylcholine (fig. 1A
). The lack of activation of the neuronal nAChRs is not explained by receptor desensitization, inhibition, or oocyte deterioration, because we obtained identical EC50
acetylcholine control values before and after the experiments.
Succinylcholine and Inhibition of Neuronal nAChRs
To investigate whether succinylcholine inhibits acetylcholine-induced nAChR responses by an interaction with the nAChR site or by succinylcholine-induced desensitization, succinylcholine was given both as a preapplication for 55 s before acetylcholine and as a coapplication with acetylcholine. Concentration-response relations were established with increasing succinylcholine concentrations for each nAChR subtype. For each receptor subtype a fixed acetylcholine concentration was applied (approximately EC50
), being 300 μm for α3β2 and α3β4, 5 μm for α4β2, and 100 μm for α7. Succinylcholine reversibly and concentration-dependently inhibited acetylcholine-induced currents in the neuronal nAChRs tested (fig. 3
). There was no significant difference in IC50
values whether succinylcholine was preapplied or coapplied with acetylcholine (table 2
). The kinetics of the activated current were unchanged with increased succinylcholine concentrations.
To determine the possible mechanism of succinylcholine-induced inhibition of the α3β4 nAChR, we investigated the effect of 150 μm succinylcholine on the acetylcholine concentration-response curve. As shown in figure 4
, 150 μm succinylcholine reduced acetylcholine-induced currents and peak responses to approximately 70% of the maximal acetylcholine response, and the EC50
was 304 μm (95% CI, 200–463 μm) for acetylcholine and 529 μm (95% CI, 83–3,359 μm), for acetylcholine plus 150 μm succinylcholine (P
< 0.05). Because the succinylcholine-induced inhibition on the α3β4 subtype seems not to be a pure competitive displacement of acetylcholine at the acetylcholine-binding site, we cannot rule out inhibition by channel block or allosteric inhibition.
This study demonstrates that succinylcholine desensitizes the muscular α1β1δε nAChR after initial activation. In contrast, succinylcholine does not activate or desensitize neuronal α3β2, α3β4, α4β2, or α7 nAChRs and is moreover a low potency inhibitor of these receptor subtypes.
Succinylcholine concentration-dependently activates the human α1β1δε nAChR heterologously expressed in Xenopus
oocytes with an EC50
of 10.8 μm. This indicates that the human receptor may be slightly more sensitive compared with the mouse receptor, for which an EC50
value of 33 μm 24
has been reported. Succinylcholine has been described as a partial agonist at the muscle nAChR,23
but because of receptor desensitization after repeated applications of acetylcholine at EC50
, we were unable to properly compare the potency of succinylcholine with acetylcholine. Coapplication of succinylcholine with 1 and 5 μm acetylcholine slightly right shifted the concentration-response activation curve of succinylcholine, and in addition, 5 μm acetylcholine produced a decidedly bell-shaped curve. Preapplication of succinylcholine before 5 μm acetylcholine concentration-dependently inhibited the response of the α1β1δε receptor subtype to acetylcholine with an IC50
of 126 μm. Our results support the hypothesis that succinylcholine prevents receptor activation at least partly by desensitization of the α1β1δε nAChR and are in line with a previous report.25
We also demonstrate that succinylcholine does not activate the human neuronal nAChRs, α3β2, α3β4, α4β2, or α7, in concentrations up to 1 mm and furthermore is a low-potency antagonist at these receptor subtypes. Succinylcholine has previously been shown to be a weak agonist at the rat α7 subtype, and furthermore, it has been reported that the rat α3β4 nAChR is not activated by succinylcholine in concentrations up to 1 mm.23
Interestingly, insertion of a point mutation in the second transmembrane domain of the α7 receptor, T244F (Thr to Phe), dramatically changes both biophysical and pharmacologic properties toward those of the muscle receptor.23
The mutation results in succinylcholine having increased potency and efficacy: Although being a partial agonist at the wild-type α7 receptor, succinylcholine is a full agonist at the T244F mutant α7 receptor.23
There is more than 80% homology between human and rodent DNA for a given nAChR subunit.1
However, a small difference in amino acid sequence at some regions can cause significant changes in biophysical and pharmacologic properties of the receptors.18,20,26
Here, we show that succinylcholine is a partial agonist at the human muscle nAChR; however, we could not calculate the exact efficacy because of apparent receptor desensitization. In contrast, either succinylcholine does not act as an agonist or it is a very weak partial agonist at the neuronal nAChR subtypes. The efficacies were extremely low at these receptor subtypes. Furthermore, no desensitization was seen, because the acetylcholine control responses after the succinylcholine concentration-response curve were preserved, and there were no differences in inhibition whether succinylcholine was preapplied or coapplied with acetylcholine. Altogether, succinylcholine does not desensitize the neuronal nAChRs in contrast to the muscle subtype, and the succinylcholine-induced inhibition on the α3β4 subtype seems not to be a pure competitive displacement of acetylcholine at the acetylcholine-binding site. We speculate that inhibition by channel block or allosteric inhibition may also be involved in the inhibition. This has obviously to do with binding site, but this is beyond the scope of this article.
For the neuronal nAChRs, a stoichiometry of 2 α and 3 β has been found when DNA/RNA have been injected into Xenopus
oocytes at a ratio of 1:1 (α:β).27–29
However, it has recently been shown that the α4β2 subtype, when injected in a subunit ratio of 1:9, displays a subtype that is more sensitive to activation and desensitizes more slowly compared with the 1:1 subtype.30
In addition, human embryonic kidney cells transfected with the human cDNA for the α4 and β2 subunits displayed the more sensitive subtype if transfected with additional β2 subunits or if they were exposed to nicotine or low temperature.31
In the current study, we investigated this more sensitive α4β2 subtype, and the EC50
(1.63 μm) was similar to those reported in previous studies (1.8 μm).30,32
We analyzed both peak current and net charge after acetylcholine-induced nAChR activation and restricted the analysis to 20 s (during agonist application) because we believe that this better reflects the effects of agonist and eliminates confounding factors such as rundown and washout conditions. Net charge analysis has convincingly been shown to be a more accurate method to analyze activation of the α7 nAChR subtype, which desensitizes very rapidly.18,19
For the α3β2, α3β4, α4β2, and α1β1δε subtypes, the pharmacologic properties are almost identical independent of the method used. The very small difference between peak current and net charge present in the α3β2 nAChR subtype might reflect the rapid initial desensitization of this receptor subtype.
There is a long-standing controversy regarding the mechanism of action of succinylcholine, i.e.
, whether succinylcholine blocks presynaptic or postsynaptic nAChRs or other structures in the neuromuscular junction.33
Here, we confirm that succinylcholine initially activates the adult muscle nAChR with an EC50
of 10.8 μm, in reasonable accordance with the clinical EC50
of 2.6 μm.34
Thereafter, succinylcholine causes a desensitization of the receptor with an apparent IC50
of 126 μm on the acetylcholine response. However, we found that succinylcholine is not an effective competitive antagonist at the human muscle-type nAChR, because succinylcholine, when coapplied with acetylcholine, does not block the receptor. There is a discrepancy between the clinical EC50
of 2.6 μm and the IC50
in our oocyte preparation, and in addition, nerve stimulation using an in vitro
nerve-muscle preparation indicates an EC50
of 21.3 μm for succinylcholine.35
The latter preparation, however, involves activation through direct innervation rather than through application of exogenous acetylcholine. Based on this, we believe that succinylcholine after initial receptor activation shifts the receptor population toward a desensitized state, rendering fewer receptors available for acetylcholine activation. Because of the high receptor density in the oocytes, there will still be receptors in both open and closed states available for acetylcholine activation, and what we actually measure after succinylcholine preapplication is the “nondesensitized” receptor population in the oocytes. Alternatively, in the neuromuscular junction, activation of approximately 25% of the muscle nAChRs may be sufficient to depolarize and subsequently desensitize the muscle tissue.36
Given this view, the EC50
of 10.8 μm at the recombinant adult muscle nAChR agrees very closely with the clinical determined EC50
of 2.6 μm. In addition, desensitization of a part of the receptor population on the muscle tissue could be sufficient for inducing a neuromuscular block due to subthreshold muscle depolarization. Finally, we cannot rule out the possibility that accessory proteins associated with the muscle nAChR might be involved, yielding a more potent succinylcholine neuromuscular inhibition in vivo
Tetanic fade is caused by an interaction with cholinergic presynaptic autoreceptors mediating acetylcholine release from the motor nerve end.8
For many years, measurement of twitch tension and tetanic fade has served as the base for clinical monitoring of neuromuscular blockade.37
In contrast to nondepolarizing neuromuscular agents, succinylcholine-induced neuromuscular block is characterized by a lack of tetanic fade. The mechanism behind tetanic fade is not known, and furthermore, it is not known how neuromuscular blocking agents interact with this receptor at a molecular level. It has recently been shown that a block of the presynaptic α3β2 nAChR produces tetanic fade due to inhibition of an acetylcholine-mediated α3β2 autofacilitation.10
Here, we clearly demonstrate that succinylcholine does not activate the human α3β2 nAChR subtype and, furthermore, blocks the receptor only at very high concentrations of succinylcholine (IC50
> 700 μm). The finding that succinylcholine has a very low affinity to the α3β2 nAChR is in accordance with the clinical observation that succinylcholine does not cause tetanic fade, and we can, for the first time, provide a molecular explanation and experimental evidence for the lack of tetanic fade in succinylcholine-induced neuromuscular blockade. In addition, the high IC50
value for succinylcholine at the α3β2 nAChR might also explain the clinical “phase II block,” which is seen when succinylcholine has been overdosed and is characterized by tetanic fade and a partial or complete reversal of the block by anticholinesterase.38,39
We speculate that this phase II block might in part be due to a block of presynaptic α3β2 nAChRs at high succinylcholine concentrations, because peak succinylcholine plasma concentrations at normal dosage have been shown to be approximately 75.5–143.2 μm,34
and a “phase II block” may develop when succinylcholine is given three to five times the normal dose.39
However, other receptors as well as other components of the presynaptic nerve terminal can modulate the tetanic fade response,8,40,41
and therefore, involvement of other mechanisms in the “phase II block” in addition to the inhibition of the presynaptic α3β2 nAChR is possible and must be further evaluated. Altogether, our results support the hypothesis that succinylcholine blocks the transmission in the neuromuscular junction by activation and desensitization of the α1β1δε nAChR, not by an antagonistic effect or block at the presynaptic α3β2 nAChR subtype at normal dosage.
Tachyarrhythmias, mainly of ventricular origin, as a side effect of succinylcholine have been suggested to arise as a result of catecholamine release from the adrenal medulla after stimulation of ganglionic nAChRs.17
The nAChRs present in ganglion are mainly the α3β4 type, but α3β2 and α7 nAChR subtypes are also present.5,14
Here, we show that none of the α3β2 or α3β4 nAChR subtypes were activated by succinylcholine, and the α7 nAChR subtype was activated only at succinylcholine concentrations above 1 mm.
Because succinylcholine is rapidly hydrolyzed to succinylmonocholine and choline, we cannot exclude a possible effect of these breakdown products on neuronal nAChRs. Conformational analysis, however, reveals that succinylmonocholine does not fit very well into the nAChR binding sites.42
The other breakdown product, choline, is known as a selective α7 nAChR agonist,43,44
but as such has low potency, with an EC50
of 400–500 μm, and inhibits the receptor with an IC50
of greater than 1–10 mm.18,45
Interaction of succinylcholine with the M2 muscarinic AChR has also been suggested to cause cardiovascular interactions, but a recent binding study shows that succinylcholine has low affinity for both the M2 and M3 muscarinic AChRs.46
Interestingly, succinylcholine increases the afferent activity in the cerebral cortex as a result of a stimulation of muscle spindles, leading to an arousal effect on the electroencephalogram and increased cerebral blood flow.47,48
The arousal effects on the electroencephalogram have been shown to be similar to those during surgical incision.49
Therefore, it is possible that some succinylcholine-induced cardiovascular side effects are due to an indirect central activation of the sympathetic nervous system, rather than by a direct interaction with cholinergic receptors in peripheral ganglia.
In conclusion, succinylcholine activates the muscle-type nAChR followed by desensitization. Our observation that succinylcholine does not inhibit the presynaptic α3β2 autoreceptor at clinically relevant concentrations provides a possible mechanistic and molecular explanation for the typical lack of tetanic fade in succinylcholine-induced neuromuscular blockade. Finally, cardiovascular side effects (e.g., tachyarrhythmias) of succinylcholine are not mediated via direct activation of the ganglionic α3β4 subtype because succinylcholine does not activate the neuronal nAChRs.
The authors thank Alan Robbins, M.S. (Scientist), Jodi D. Bradley (Associate Scientist), Robert Manning (Scientist), Rich Hastings (Associate Scientist), Geeta Iyer (Contractor), and Tirell Norris, B.S. (Scientist), from AstraZeneca Pharmaceuticals, Wilmington, Delaware, and Wendy Gion (Scientist) from AstraZeneca Pharmaceuticals, Boston, Massachusetts, for DNA cloning and generation of messenger RNA.
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