Throughout all investigated isoforms, the onset of drug effect developed rapidly. A steady state of sodium channel block at a given concentration of AJA was obtained during 40 to 60 seconds of drug application. During washout, currents reached 50% to 70 % of the control current value. The blocking effect remained stable and was left unchanged even when the respective concentration of AJA was applied longer than 10 minutes.
We first examined the effect of AJA on Nav1.7. Tonic block was investigated on resting (Fig. 2A) and inactivated channels (Fig. 2B). AJA induced a concentration-dependent tonic block of Nav1.7, yielding IC50 values of 5.0 ± 0.7 (n = 13) for resting channels and 2.7 ± 0.3 μmol/L (n = 9) for inactivated channels (Table 1). Tonic block was significantly different compared with the other channel isoforms except for Nav1.8 (P = 0.7); see section below (statistical results of ANOVA).
We next investigated further effects of 3 μmol/L AJA on Nav1.7; this concentration induced a robust inhibition of resting channels. As demonstrated in Figure 2D, 3 μmol/L AJA induced a small and not significant use-dependent block at 10 Hz (the last 20 of the applied repetitive pulses were compared by ANOVA P = 0.065; n = 6). We did not observe any effect of 3 μmol/L AJA on the current-voltage curve of Nav1.7 (n = 6) (Fig. 2F). In contrast, 3 μmol/L AJA induced robust effects on both fast (Fig. 2G) and slow inactivation (Fig. 2H). AJA 3 μmol/L induced a prominent hyperpolarizing shift of the steady-state inactivation curve from V0.5 −71 ± 1 mV in control solution to V0.5 −90 ± 1 mV in 3 μmol/L AJA (fast inactivation) (n = 8) (Table 2). Regarding slow inactivation, 3 μmol/L AJA induced a shift of the midpoint (V0.5) from −63 ± 1 mV in control solution to −73 ± 1 mV (n = 9) (Fig. 2H).
As is illustrated in Figure 3, A–J, AJA exhibited a tonic block on all investigated subunits. The IC50 values obtained by the Hill fit are shown in Table 1. Except for Nav1.8, all subunits were investigated as described above for Nav1.7. Statistical analysis generally yielded significant differences in IC50 values for tonic block of the investigated channel isoforms Nav1.2, 1.3, 1.4, 1.5, 1.7, and 1.8 (ANOVA, F(5, 49) = 69.051, P < 0.0001; for post hoc analysis, the Tukey HSD test was used). According to the post hoc test, IC50 values for block of resting channels by AJA were significantly lower in Nav1.4, 1.5, 1.7, 1.8 compared with Nav1.2 and 1.3 (P was 0.0001 for Nav1.4, 1.5, 1.7, and 1.8, respectively, in comparison with both Nav1.2 and Nav1.3, while P was 0.07 for Nav1.2 and 1.3 when comparing these 2 isoforms). IC50 values for Nav1.4 and Nav1.5 did not significantly differ (P = 0.99), the same as for Nav1.7 and Nav1.8 (P = 0.7). In comparison to the rest of the investigated isoforms, the respective IC50 value of all these 4 channels were significantly different (P < 0.001).
In the overall view, in all investigated channel isoforms, AJA exhibited half-maximal inhibition of sodium currents at low micromolar concentrations (approximately 2–9 μmol/L, as shown in Table 1). AJA 3 μmol/L induced a strong hyperpolarizing shift of the steady-state inactivation on Nav1.2, Nav1.4, Nav1.5, 1.5NN406K, 1.5F1760A, and 1.7 (Table 2).
Tonic block of resting channels by the local-anesthetic insensitive Nav1.5-mutants N406K and F1760A revealed IC50 values of 3 ± 0.1 μmol/L (n = 9) for N406K (Fig. 4, A and B) and 4 ± 0.3 μmol/L (n = 8) for F1760A (Fig. 4, C and D, Table 1). Thus, both mutants displayed a preserved sensitivity to block by AJA. Statistical analysis by ANOVA (F(2,21) = 5.8267, P < 0.01) followed by the Tukey HSD post hoc test yielded significant differences in IC50 when Nav1.5 (wild-type) was compared with the mutant F1760A (P = 0.009); however, no significant difference in IC50 values was determined comparing the wild-type channel Nav1.5 with the mutant N406K (P = 0.7).
Moreover, we found that 3 μmol/L AJA induced a prominent shift of steady-state fast inactivation of N406K (−87 ± 1 mV in control to −120 ± 1 mV in 3 μmol/L AJA, n = 10) (Fig. 4F) and of F1760A (−76 ± 1 mV in control to −118 ± 1 mV in 3 μmol/L AJA, n = 9) (Fig. 4G, Table 2).
We observed robust resurgent currents by including 100 μmol/L of the Navβ4-peptide into the pipette solution (Fig. 5, A and B). As is demonstrated in Figure 4C, 1 μmol/L AJA induced a robust inhibition of the resurgent current at all potentials. The inhibition of the peak current amplitude was 24 ± 4 % (n = 7). Tonic block of resting Nav1.5 channels by 1 μmol/L AJA was significantly less pronounced (6.6% ± 1%, n = 9) (P = 0.002, unpaired Student t test), indicating that AJA is indeed a potent inhibitor of resurgent currents.
This in vitro study identified the synthetic cannabinoid AJA as a potent blocker of voltage-gated sodium channels. Despite the fact that AJA does not induce cardiac or central nervous side effects in human volunteers, we found that AJA nonspecifically blocked all investigated α-subunits in a low micromolar concentration range.9 The molecular mechanism mediating this property is distinct from that used by classical local anesthetics. Thus, AJA might target a yet unknown binding site of α-subunits of sodium channels. Although speculative, it is possible that substances targeting this binding site can suppress pathological excitability in sensory neurons while leaving normal membrane excitability largely unaffected.
It is generally acknowledged that the redistribution and altered functional expression of voltage-gated sodium channels are implicated in chronic painful neuropathies that can arise from peripheral nerve injury.27,28 Among the α-subunits investigated in this study, primarily Nav1.3, Nav1.7, and Nav1.8 are expressed in sensory neurons. There is strong evidence that Nav1.3 and Nav1.8 have an impact on neuropathic pain-like behavior after nerve injury in rodents, and Nav1.7 has emerged as a key molecule for peripheral pain processing and for certain painful neuropathies in humans.27,29 Although the Nav1.9 channel is thought to play a prominent role in nociception and has the most divergent amino acid sequence among all Nav channel isoforms, it was not studied because it cannot be reliably expressed in heterologous expression systems.30
We found that AJA is an unspecific blocker of investigated α-subunits, excluding the possibility that analgesia induced by this compound involves a selective interaction with 1 sodium channel isoform. Although we did not have any direct reasons to hypothesize this property for AJA, the reported lack of cardiac and central nervous side effects in humans receiving AJA did not indicate an unselective inhibition of cardiac and neuronal α-subunits.8 Batista et al.31 reported that the plasma-concentration of AJA in patients receiving AJA at analgesic effective doses can be up to 2 μmol/. A fact that hinders the direct comparison between the plasma concentration in patients and the effective concentrations in our in vitro experiments is the plasma protein binding of AJA. From animal studies and unpublished phase I studies it is known that AJA is highly bound to plasma proteins in dog (99.5%–99.9%), rat (98.5%–99.8%), and human (97.0%–99.9%) samples (unpublished results of pharmacokinetic studies). Considering our experiments on Nav1.5 (heart) and Nav1.2 (central nervous system), one would definitely expect side effects to occur at these concentrations if the drug were all present in the free state and not bound to plasma proteins.
The state-dependent inhibition of sodium channels found for AJA in this study closely resembles the properties of local anesthetics, that is, our data strongly suggested that AJA also interacts with the local anesthetic binding site. However, both analyzed local anesthetic-insensitive Nav1.5-mutants (N406K and F1760A) displayed a preserved AJA sensitivity. These data more or less exclude a relevant interaction of AJA with the local anesthetics binding site and indicate that AJA targets a yet unknown site to block sodium channels. A more critical analysis of our data indeed reveals further properties of AJA that support this notion: Use-dependent block by 3 μmol/L AJA was minimal and probably not relevant. Use-dependent block is likely to arise from a higher affinity of a blocker to the open and inactivated states, and is a prototypical effect for most sodium channel blockers interacting with the local anesthetic binding site.32 Blockers with a high cardiotoxic potential, such as bupivacaine and amitriptyline, generally induce a very strong use-dependent block.23,33 It is possible that this property is predisposed for a high cardiac toxicity, and that the obvious lack of cardiotoxicity of AJA is due to its failure to induce use-dependent block. However, use-dependent inhibition may also be an important mechanism when sodium channel blockers are applied for treatment of neuropathic pain.34 Another remarkable property of AJA observed in this study was an impressive shift of the voltage dependency of steady-state fast inactivation. Even though this shift is also induced by local anesthetics and the opiod methadone,35 we are not aware of any substance inducing such a strong shift at low micromolar concentrations (approximately 20 mV by 3 μmol/L). For local anesthetics, this effect is due to an interaction with the local anesthetic site since it is abbreviated in local anesthetic channel mutants.32 However, the shift induced by AJA is perfectly preserved on the mutants Nav1.5—N406K and F1760A, and thus, this effect appears independent of the local anesthetic binding site.
Considering the lipophilic structure and chemical properties of AJA, interactions in the lipid bilayer of the cell membrane could be one possible site of action as suggested for halothane.36 AJA shares some features with volatile anesthetics. Both AJA and volatile anesthetics such as sevoflurane, desflurane, or isoflurane show a hyperpolarizing shift of the voltage-dependency of fast inactivation as well as a voltage-dependent block of sodium currents among the different sodium channel subunits.37–39 Unlike AJA, volatile anesthetics induce significant use-dependent inhibition, whose extent varies due to substance-specific differences in block among different volatile anesthetics.37
AJA’s LogP value (octanol:water partition coefficient) is approximately 7.55. Its lipophilicity might facilitate effects on binding sites in the lipid bilayer to shift the channel gating. But also an interaction with a hydrophobic pocket within the channel protein itself is hypothetically conceivable. Thus, further studies are needed to explore the detailed molecular mechanism of this drug’s action.
A comparable effect on steady-state fast inactivation observed for AJA in this study was recently demonstrated for the endogenous cannabinoid anandamide on Nav1.7.19 The authors of this study found that this property correlates with the ability of the drug to inhibit resurgent currents, and postulated that substances inducing a preferential block of resurgent currents might be useful therapeutic tools. It is interesting to note that this inhibitory property was suggested to be independent from the local anesthetic binding site.19 In our study, AJA inhibited Navβ4-peptide-mediated resurgent Nav1.5-currents more potently than transient currents generated by wild-type Nav1.5. Wild-type Nav1.5 was chosen because it was previously demonstrated to generate large Navβ4 peptide-mediated resurgent currents.40 In contrast, wild-type Nav1.7 hardly generates any detectable resurgent currents.19 As resurgent currents of different α-subunits have been suggested to drive different pain syndromes in humans,21,22 it is possible that the analgesic efficacy of AJA is in part due to inhibition of resurgent currents.
Limitations of Our Study
We have studied the effects of AJA in vitro in sodium channel α-subunits. Alpha-subunits of sodium channels show normal gating characteristics (with respect to experiments in native tissue) when expressed in a mammalian cell line in the absence of the β-subunit.41,42 In HEK293 cells mRNA encoding, the β1α splicing of the putative regulatory sodium channel subunit is abundantly expressed.43
Irrespective of the role that altered expression of β-subunits may have in the development of pain states in vivo,44 concerns have been raised that exogenous introduction of β1 in HEK cells in vitro may lead to an overexpression of β-subunits which in turn might have unpredictable effects on the pharmacologic interaction between the sodium channel α-subunit and channel blockers.43
The advantage of this experimental approach is that drug interaction with the sodium channel can be studied without confounding factors in this model since it is generally accepted that the receptor sites for pharmacological agents interfering with voltage-gated sodium channels are located on the α-subunits.45
In summary, our in vitro study identified AJA as an unselective inhibitor of voltage-dependent sodium channels. This inhibition is concentration- and state-dependent, properties that AJA shares with other cannabinoids. It is assumed that these inhibitory effects of AJA are mediated by mechanisms distinct from the local anesthetic binding site. This could be one of the mechanisms by which AJA exerts analgesic effects in patients with neuropathic pain. Thus, AJA is not only an interesting drug with implications for pain treatment but also as a tool to further explore a yet poorly defined route to inhibit voltage-dependent sodium channels.
Name: Nilufar Foadi, MD.
Contribution: Nilufar Foadi designed experiments, acquired, analyzed, interpreted data, and participated in writing the manuscript.
Attestation: Nilufar Foadi read and approved the final manuscript and is the archival author.
Name: Christian Berger, MD.
Contribution: Christian Berger performed experiments and acquired, analyzed, and interpreted data.
Attestation: Christian Berger read and approved the final manuscript.
Name: Igor Pilawski, VMD.
Contribution: Igor Pilawski performed experiments and acquired, analyzed, and interpreted data.
Attestation: Igor Pilawski read and approved the final manuscript.
Name: Carsten Stoetzer, MD.
Contribution: Carsten Stoetzer performed experiments and acquired, analyzed, and interpreted data.
Attestation: Carsten Stoetzer read and approved the final manuscript.
Name: Matthias Karst, MD.
Contribution: Matthias Karst participated in design of experiments, contributed to the methods and illustrations, and edited the manuscript.
Attestation: Matthias Karst read and approved the final manuscript.
Name: Gertrud Haeseler, MD.
Contribution: Gertrud Haeseler participated in design of experiments, contributed to the methods and illustrations, and edited the manuscript.
Attestation: Gertrud Haeseler read and approved the final manuscript.
Name: Florian Wegner, MD.
Contribution: Florian Wegner participated in design of experiments, contributed to the methods and illustrations, and edited the manuscript.
Attestation: Florian Wegner read and approved the final manuscript.
Name: Andreas Leffler, MD.
Contribution: Andreas Leffler mentored, conceived, designed, coordinated the study, and drafted the manuscript.
Attestation: Andreas Leffler read and approved the final manuscript.
Name: Jörg Ahrens, MD.
Contribution: Jörg Ahrens mentored, conceived, designed, coordinated the study, and drafted the manuscript.
Attestation: Jörg Ahrens read and approved the final manuscript.
This manuscript was handled by: Marcel E. Durieux, MD, PhD.
We are indebted to Prof. Frank Lehmann-Horn (Ulm, Germany) for providing us with transfected cells, Andreas Niesel (Hannover, Germany) for technical support and Prof. Sumner Burstein, (Boston) for his kind supply of AJA.
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