Methocarbamol blocks muscular Nav1.4 channels and decreases isometric force of mouse muscles

The muscle relaxant methocarbamol is widely used for the treatment of muscle spasms and pain syndromes. To elucidate molecular mechanisms of its action, we studied its influence on neuromuscular transmission, on isometric muscle force, and on voltage‐gated Na+ channels.

LBP, methocarbamol can reduce pain intensity and pain-related disability and improve lumbar mobility. 14 Although methocarbamol has been used clinically for more than 50 y, 16 its mechanism of action is still unclear. Some studies suggested a central mode of action of methocarbamol on spinal interneurons. 17,18 The reduction of reflex contractions and the mono-and polysynaptic components of ventral root potentials by methocarbamol are consistent with an effect of the drug at higher centers concerned with motor function. 19 However, some early reports raise doubts about this view. Crankshaw and Raper reported that methocarbamol caused a prolongation of the refractory period of cat tibialis muscles 20 and showed a suppression of polysynaptic reflex contractions in this preparation. They discussed their results in terms of depressant actions of the drug on α-motor neurons and muscle fibers. Since the refractory period of muscle fibers is mainly determined by voltagegated Na + channels, 21 Na v 1.4 channels are potential molecular targets of methocarbamol. In contrast to the Na v 1.4 that is almost exclusively expressed by extrafusal muscle fibers, peripheral nociceptive neurons express Na v 1.7 channels, which are essential for pain perception.
Thus, the pain-relieving effect of methocarbamol could also be explained by interference with Na v 1.7 channels of nociceptive nerve fibers.
The current study was designed to investigate effects of methocarbamol on neuromuscular transmission, muscle force, and voltagegated sodium channels.

| Analysis of neuromuscular transmission by extracellular recordings
Electrophysiological recordings were essentially done as previously described. 23,24 Isolated diaphragm-phrenic nerve preparations were maintained in Liley's solution, gassed with carbogen, and kept at room temperature. The recording chamber had a volume of approximately 1 mL and was perfused at a rate of 1 mL/min. The nerve was drawn up into a suction electrode for stimulation with pulses of 0.1 ms duration.
The preparation was placed on the stage of a Zeiss Axio Examiner Z1 microscope fitted with incident light fluorescence illumination with filters for red (Zeiss filter set 20) fluorescing fluorophore (Carl Zeiss Micro-Imaging, Göttingen, Germany). At the beginning of the experiment, the compound muscle action potential (CMAP) was recorded using a micropipette with a tip diameter of~10 μm, filled with bathing solution. The electrode was positioned so that the latency of the major negative peak was minimized. The electrode was then positioned 100 μm above the surface of the muscle and CMAP was recorded. Unless stated otherwise, a concentration of 2 mM methocarbamol was used in all experiments, since in pilot experiments this concentration appeared to result in maximal effects. Muscles were incubated for 20 min with methocarbamol before CMAPs were measured.

| Intracellular recordings of muscle fibers and data analysis
To block muscle action potentials, so that endplate potentials (EPPs) and endplate currents (EPCs) could be recorded 25 To study the effects of methocarbamol on EPPs and EPCs, the muscle was incubated after the μ-conotoxin GIIIB solution for 20 min with 2 mM methocarbamol. Afterward the recording was started with freshly gassed Liley's solution containing 2 mM methocarbamol, which was renewed every 20 min. Records were analyzed using pCLAMP 10. Spontaneous events were extracted using the "template search" facility and edited by eye to remove obvious artifacts. Events recorded from each NMJ were averaged and the amplitude, rise time and single exponential decay time determined.

| Force measurements on soleus muscles
After dissection, muscles were transferred into a recording chamber and fixed as described previously. 27 Experiments were performed at 25 C.
Supramaximal electrical stimuli of 40 V and 0.2 ms duration were applied through two platinum electrodes. Stimulation and data acquisition were performed with a special software (Myodat, MyoTronic UG, Heidelberg, Germany) and data evaluated and plotted with SigmaPlot. Before recordings were started, optimal muscle length was determined as described. 27 Isometric forces in response to single stimuli (twitches) and to 500-ms tetanic stimuli of 10, 50, and 120 Hz were recorded ("1st run"). Then the control group of muscles remained in Krebs-Henseleit solution and the methocarbamol group was exposed to a solution containing 2 mM methocarbamol. A second series of experiments was performed after 10 min ("2nd run"). Time to peak (time from stimulation start to the time point of maximum force) and half relaxation time (time from maximum force to 50% decline of twitch force, or, for tetani, time from end of stimulation to 50% decline of force) were calculated. Finally, a so-called fatigue protocol was applied consisting of 500-ms tetani at 50 Hz repeated every 2 s for a total time of 400 s. The half fatigue time was taken, that is, the time between stimulation start and decline of force to 50% of the initial force.
After the experiments, muscles were weighed and optimal length and weight were used to calculate muscle cross-sectional area (CSA). 28,29 Specific muscle fore was defined as force/CSA. Chinese hamster ovary (CHO) cells stably expressing the α-subunit of the human Na v 1.7 were obtained from Genionics AG (Zürich, Switzerland). They were grown in a medium composed of 90% DMEM/F12 (1:1, v:v, Gibco/Thermo Fisher Scientific) an 10% FCS. To positively select for cells expressing the Na + channels, the media contained 800 μg/mL of the antibiotic geneticin (G418, Gibco/Thermo Fisher Scientific) or 500 μg/mL hygromycin (InvivoGen, Toulouse, France).

| Whole cell recording of Na + currents
Whole-cell Na + currents were recorded at room temperature using an  3.59 ± 0.14 (n = 12) Note: The values represent the mean ± SEM of three mice; n represents the number of measured fibers. Significance was determined by unpaired t-test. responses to 0.5-Hz repetitive 10-ms pulses before, during and after application of the test solution.
To determine the voltage-dependence of activation of the currents, a cyclic pulse program was applied, each cycle consisting of a constant 100-ms prepulse to −135 mV and a 10-ms test pulse, varied from −80 mV to +40 mV in 5-mV steps. The peak currents were plotted against the test potential (current/voltage curves). The voltage dependence of steady-state activation of the Na + channels was derived from these plots and Boltzmann equations were fitted to the data points. 31 To study the voltage dependence of steady-state inactivation of the Na + channels, another cyclic pulse program was applied with each cycle consisting of a 100-ms prepulse that was varied between −120 mV and − 10 mV in 5-mV steps followed by test pulses to −10 mV (10 ms). The obtained current peaks were normalized and plotted against the prepulse potential. Finally, Boltzmann curves were fitted to the data points. To investigate the recovery of the Na + channels from inactivation, a cyclic pulse program was used with each cycle consisting of a 100-ms voltage pulse to −140 mV, followed by an inactivating pulse to −10 mV for 10 ms.
Then a prepulse of −105 mV was applied, the duration ranging from 1 ms to 3.2 s. Finally, a 10-ms test pulse to −10 mV was given to test for the fraction of channels that had recovered from inactivation. Current amplitudes were plotted against the prepulse dura- coefficients, and τ 1 and τ 2 the time constants of fast and slow recovery, respectively. 32 3 | RESULTS

| Methocarbamol modulates neuromuscular transmission
We studied neuromuscular transmission in phrenic nerve-diaphragm explants. While most parameters of miniature EPPs were unaffected, the decay times of the EPCs and those of the EPPs induced by the phrenic nerve stimulation were significantly increased at 2 mM methocarbamol ( Table 1, Supporting Information Figure S1, which is available online). At 5-Hz stimulation, the EPP decrement was not affected by methocarbamol (Table 1).
In contrast, the CMAP amplitude of the diaphragm-phrenic nerve preparation was markedly reduced by methocarbamol ( Figure 1A). Furthermore, the decrement of the CMAP was larger in the presence of the drug, both at 5 and 50-Hz stimulation ( Figure 1, Table 2).

| Methocarbamol affects muscle force
Application of methocarbamol had no effect on amplitudes of twitches (Figure 2A,B). Kinetics of twitches was slightly faster in the presence of the muscle relaxant, but the same effect occurred in the control group (Table 3, 2nd run). However, during high frequency tetanic stimulation, methocarbamol decreased tetanic force moderately, but significantly ( Figure 2D,E,F). At 50-Hz stimulation, the reduction was about 4% and at 120 Hz about 9%, while in the control group both parameters appeared identical ( Table 3, results from first and second run). Since twitch amplitudes were unaffected, the tetanus/twitch ratio was reduced by methocarbamol by about 9% ( Figure 2C, Table 3). Decline of force during sustained repetitive tetanic stimulation (fatigue protocol) was slightly, but not significantly aggravated in the presence of methocarbamol (Supporting Information Figure S2, open circles,

| Methocarbamol affects muscular sodium channels
When HEK 293 cells, stably expressing the Na v 1.4, were stimulated with square voltage pulses increasing from −85 to −10 mV, they responded with transient inward currents that were stable over time.
Upon application of methocarbamol, the Na + currents were inhibited on average by 37 ± 8% (Figure 3). The inhibition occurred within about 10 s and was reversible upon washout (Supporting Information Figure S3A). Complete solution change was achieved within less than 1 s. Na + current kinetics remained apparently unchanged in the presence of methocarbamol ( Figure 3A and Supporting Information Figure S4).
To investigate whether the methocarbamol effect on Na v 1.4 channels is voltage-dependent, we determined current/voltage relations. These curves were only marginally affected by methocarbamol ( Figure 4A). Voltage dependence of activation of the Na v 1.4 channels was completely unaffected ( Figure 4B). The small decrease of the I/V curve over the whole voltage range ( Figure 4A) can only partly explain the observed Na + current inhibition shown in indicating that methocarbamol stabilized Na v 1.4 channels in a state of inactivation. Therefore, we next studied the effect of methocarbamol on the recovery from inactivation of the Na v 1.4 channels. The Na v 1.4 currents almost completely inactivated within 5 ms at a test potential F I G U R E 4 Voltage dependence of the methocarbamol block and recovery of Na + channels from inactivation. A, Normalized and averaged current/voltages curves recorded in standard external solution (black circles) and in the presence of 2 mM methocarbamol (red triangles); current maxima plotted against the test potential. B, Voltage dependence of activation of the Na + currents; data derived from I/V curves and Boltzmann equations fitted to the data points. C, Voltage dependence of inactivation of Na v 1.4 channels before and after methocarbamol application. Average normalized current maxima are plotted against the prepulse potential. Boltzmann curves were fitted to the data points. D, Recovery of Na v 1.4 channels from inactivation before and during methocarbamol application. Currents were induced by test pulses to −10 mV after an initial inactivating pulse and a re-activating prepulse to −105 mV (recovery). Current maxima were plotted against the variable prepulse duration. Data points were either fitted by an exponential curve with a single time constant (τ 1, black line) or by an exponential equation with two time constants (τ 1, fast and τ 2 slow, red line). Mean values ± SD are given for n = 16 tested cells (standard external solution) and 12 cells (2 mM methocarbamol), respectively [Color figure can be viewed at wileyonlinelibrary.com] of −10 mV ( Figure 3A). To allow recovery from inactivation we applied −105 mV for variable time periods. Recovery of Na + channels from inactivation occurred within a few milliseconds and reached nearly 100% at 10 ms ( Figure 4D). The data points could be fitted by an exponential curve ( Figure 4D, black line), resulting in a recovery time constant (τ 1 ) of 3.2 ± 0.7 ms. In the presence of methocarbamol recovery from inactivation was slowed and interestingly split into a fast (τ 1 ) and a much slower phase (τ 2 , Figure 4D, red line). In agreement with this observation, the data points could not be appropriately fitted with a single exponential curve, but by an equation with two time constants (τ 1 = 4.0 ± 0.9 ms and τ 2 = 55.3 ± 20.1 ms). In summary, these results demonstrate that methocarbamol reversibly affects voltage dependence of inactivation of Na v 1.4 channels.
3.4 | Methocarbamol has no effect on Na v 1.7 currents Methocarbamol (at 2 mM) did not change amplitude and kinetics of Na v 1.7 currents (Supportinng Information Figure S3B). Neither I/Vcurves nor inactivation curves and recovery from inactivation of the Na v 1.7 currents were influenced by the relaxant.

| DISCUSSION
Our study supports the view that methocarbamol has peripheral relaxing effects directly interacting with the neuromuscular system, In the diaphragm-phrenic nerve preparation, methocarbamol prolonged both EPP and EPC decay times. Prolongation of these two parameters could be caused by altered transmitter release, transmitter degradation, or by altered AChR function. 33 However, the underlying effect would not explain the aggravation of the CMAP decrement discussed above.
In addition to its effects on neuromuscular transmission, methocarbamol attenuated isometric force of isolated mouse soleus muscles. The results are in line with the observed inhibition of the CMAP amplitudes by methocarbamol. However, the marked effects on CMAP amplitude and the moderate inhibition of tetanic force (4%-9%) do not indicate a linear relationship between the two parameters.
Probably, the 40% block of Na + channels is not sufficient to suppress action potentials in muscle fibers, but merely reduces action potential amplitudes 34 and CMAP amplitudes without much influence on excitation/contraction coupling. Furthermore, direct supramaximal stimulation of muscles in the presence of methocarbamol may compensate for the weakened indirect stimulation via motoneurons.
At very high frequency stimulation (400 Hz) Crankshaw and Raper 20 showed a collapse of tetanic force of cat muscle in the presence of methocarbamol. The authors concluded that the mechanism by which the drug caused its effect was a prolongation of the refractory period of muscle. 20 Our results support this view, since the inhibition of isometric force by methocarbamol was most prominent at 120 Hz stimulation frequency. As the time course of twitches and tetani were unaffected we have no evidence that methocarbamol influenced the intracellular Ca 2+ cycle during muscle performance.
A key finding of this study is the inhibition of Na + currents by meth- suggesting that plasma levels in the millimolar range are normally not likely to be achieved. In a pharmacokinetic study with rats, the plasma level reached 0.8 mM after IV injection of 150 mg/mL methocarbamol (highest dose). 36 On the other hand, intramuscular injection of methocarbamol at a concentration of 100 mg/mL 37 would locally cause much higher concentrations than the 2 mM used in the present work.
We conclude that the muscle relaxant methocarbamol, in addition to its effects on the CNS, has the potential to modulate neuromuscular transmission, force production of muscles and voltage-gated Na + channels. This study shows that the Na v 1.4 channel is a molecular target of methocarbamol, which explains some of its peripheral effects.

CONFLICT OF INTEREST
Stephan Kröger received research support from Recordati Pharma GmbH, Ulm, Germany and compensation from the company for his lecture activities. None of the other authors has any conflict of interest to disclose.

ETHICAL PUBLICATION STATEMENT
We confirm that we have read the Journal's position on issues involved in ethical publication and affirm that this report is consistent with those guidelines.