Neurosteroids Allopregnanolone Sulfate and Pregnanolone Sulfate Have Diverse Effect on the α Subunit of the Neuronal Voltage-gated Sodium Channels Na_v1.2, Na_v1.6, Na_v1.7, and Na_v1.8 Expressed in Xenopus Oocytes

NEUROSTEROIDS are neuroactive steroids synthesized from cholesterol in both central and peripheral nervous systems, and they accumulate in the nervous system.¹  They rapidly alter neuronal excitability by mediating actions through ion-gated neurotransmitter receptors, but not through classic steroid hormone nuclear receptors.²  Many of them are converted to sulfated metabolites by hydroxysteroid sulfotransferases, and neurosteroid sulfates are also known to regulate physiological processes. They are thought to be potentially therapeutic because of their many pharmacological properties.^3,4 

Two 3α-hydroxylated metabolites of progesterone, allopregnanolone (3α-hydroxy-5α-pregnane-20-one) and pregnanolone (3α-hydroxy-5β-pregnane-20-one), are known to be positive modulators at γ-aminobutyric acid type A (GABA_A) receptors with high potency.⁵  These neurosteroids have been shown to have greater anesthetic potencies than those of other intravenous anesthetics that are clinically used, and not to cause acute tolerance that are observed in other anesthetics, suggesting usefulness of these neurosteroids as general anesthetics.^6,7  On the contrary, allopregnanolone was shown to have the most potent analgesic effects among all neurosteroids in pain models.⁸  Recent studies demonstrated its analgesic effects in neuropathic pain models. Allopregnanolone alleviates thermal and mechanical hyperalgesia by ligation of the sciatic nerve in rats,⁹  produces analgesic effects on formalin-induced pain in rats,¹⁰  and prevents anticancer drug oxaliplatin-induced cold and mechanical allodynia and hyperalgesia.¹¹  In addition, it was suggested that stimulation of allopregnanolone synthesis might be involved in the antinociceptive effects of several analgesic drugs in neuropathic pain models.^12–14  Its effect on GABA_A receptors may be important for its antinociceptive properties because GABA is involved in pain pathways in the nervous systems, and drugs targeting subtypes of GABA receptors have analgesic effects in chronic pain.¹⁵  However, these two neurosteroids, allopregnanolone and pregnanolone, also act on other ion channels in pain signaling pathways, including T-type calcium channels¹⁶  and N-methyl-d-aspartate receptors.¹⁷ 

Voltage-gated sodium channels (Na_v) have an important role in action potential initiation and propagation in excitable nerve and muscle cells. Nine α subunits (Na_v1.1 to Na_v1.9) and four auxiliary β subunits have been identified in mammals.^18,19  Each pore-forming α subunit has a different pattern of development and localization and has distinct physiological and pathophysiological roles. Sodium channel α subunits expressed in the dorsal root ganglion are considered possible targets for analgesics for inflammatory and neuropathic pain.^20–22  However, there has been little investigation on the effects of allopregnanolone on sodium channel function. It is important to examine these effects because they may be useful in clarifying the mechanisms of the analgesic effects of allopregnanolone and developing natural and safe neurosteroid-based analgesics for refractory pain. In addition, our recent report demonstrated the importance of neurosteroid sulfonation for regulation of ion channels because of more potent effects of sulfated steroid than those of nonsulfated steroids.²³  Here, we investigate the effects of two sulfated neurosteroids, allopregnanolone sulfate (APAS) and pregnanolone sulfate (PAS) (fig. 1), on several sodium channel α subunits, including Na_v1.2, which is expressed in the central nervous system; Na_v1.6, which is expressed in the central nervous system and dorsal root ganglion neurons; and Na_v1.7 and Na_v1.8, which are expressed in dorsal root ganglion neurons.

This study was approved by the Animal Research Committee of the University of Occupational and Environmental Health, Kitakyushu, Japan.

Allopregnanolone sulfate and PAS were purchased from Steraloids, Inc. (Newport, RI).

Rat Na_v1.2 α subunit complementary DNA (cDNA) was a gift from Dr. William A. Catterall, Ph.D. (Professor, Department of Pharmacology, University of Washington, Seattle, Washington). Rat Na_v1.6 α subunit cDNA was a gift from Dr. Alan L. Goldin, M.D., Ph.D. (Professor, Department of Anatomy and Neurobiology, University of California, Irvine, California). Rat Na_v1.7 α subunit cDNA was a gift from Gail Mandel, Ph.D. (Professor, Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, Oregon). Rat Na_v1.8 α subunit cDNA was a gift from Dr. Armen N. Akopian, Ph.D. (Assistant Professor, University of Texas Health Science Center, San Antonio, Texas), and human β₁ subunit cDNA was a gift from Dr. Alfred L. George, Jr., M.D. (Professor, Department of Pharmacology, Vanderbilt University, Nashville, Tennessee). The percentages of homology between rat and human protein of Na_v1.2, Na_v1.6, Na_v1.7, and Na_v1.8 are 98, 99, 93, and 83%, respectively, suggesting the possible limitations imposed by using rat α subunit for only Na_v1.8 to make conclusions in humans.

After linearization of cDNA with ClaI (Na_v1.2 α subunit), NotI (Na_v1.6, 1.7 α subunits), XbaI (Na_v1.8 α subunit), and EcoRI (β₁ subunit), cRNAs were transcribed using SP6 (Na_v 1.8 α, β₁ subunits) or T7 (Na_v1.2, 1.6, and 1.7 α subunits) RNA polymerase from the mMESSAGE mMACHINE kit (Ambion, Austin, TX). Adult female Xenopus laevis frogs were obtained from Kyudo Co., Ltd. (Saga, Japan). X. laevis oocytes and cRNA microinjection were prepared as described previously.²⁴  Na_v α subunit cRNAs were coinjected with β₁ subunit cRNA at a ratio of 1:10 (total volume was 20 to 40 ng/50 nl) into Xenopus oocytes (all α subunits were coinjected with the β₁ subunit) that were randomly assigned to four α subunit groups for injection. Injected oocytes were incubated at 19°C in incubation medium, and 2 to 6 days after injection, the cells were used for electrophysiological recordings.

All electrical recordings were performed at room temperature (23°C). Oocytes were placed in a 100-μl recording chamber and perfused at 2 ml/min with Frog Ringer’s solution containing 115 mmol/l NaCl, 2.5 mmol/l KCl, 10 mmol/l HEPES, 1.8 mmol/l CaCl₂, pH 7.2, using a peristaltic pump (World Precision Instruments Inc., Sarasota, FL). Recording electrodes were prepared, and the whole-cell voltage clamp and recordings were achieved as described previously.²⁴  Transients and leak currents were subtracted using the P/N procedure, in which N subsweeps each 1/Nth of the amplitude of the main stimulus waveform (P) are applied. APAS and PAS stocks were prepared in dimethylsulfoxide and diluted in Frog Ringer’s solution to a final dimethylsulfoxide concentration not exceeding 0.05%. APAS and PAS were perfused for 3 min to reach equilibrium. All recordings were performed by the experimenters who were blind to the type of compound.

The voltage dependence of activation was determined using 50-ms depolarizing pulses from a holding potential causing maximal current (V_max) (−90 mV for Na_v1.2 and Na_v1.6, −100 mV for Na_v1.7 and Na_v1.8) and from a holding potential causing half-maximal current (V_1/2) (from approximately −40 mV to −70 mV) to 60 mV in 10-mV increments. V_max and V_1/2 holding potentials induce resting and inactivated states of sodium channels. Because the effects of many analgesics in the inactivated state are known to be important for analgesic action,²⁵  we used these two different holding potentials to compare the effects of compounds in the resting and inactivated states. Normalized activation curves were fitted to the Boltzmann equation as described previously²⁴ : briefly, G/G_max = 1/(1 + exp(V_1/2 − V)/k), where G is the voltage-dependent sodium conductance, G_max is the maximal sodium conductance, G/G_max is the normalized fractional conductance, V_1/2 is the potential at which activation is half maximal, and k is the slope factor. To measure steady-state inactivation, currents were elicited by a 50-ms test pulse to −20 mV for Na_v1.2 and Na_v1.6, −10 mV for Na_v1.7, and +10 mV for Na_v1.8 after 200 ms (500 ms for only Na_v1.8) prepulses ranging from −140 to 0 mV in 10-mV increments from a holding potential of V_max. Steady-state inactivation curves were fitted to the Boltzmann equation: I/I_max = 1/(1 + exp(V_1/2 − V)/k), where I_max is the maximal sodium current, I/I_max is the normalized current, V_1/2 is the voltage of half-maximal inactivation, and k is the slope factor. To investigate a use-dependent sodium channel block, currents were elicited at 10 Hz by a 20-ms depolarizing pulse of −20 mV for Na_v1.2 and Na_v1.6, −10 mV for Na_v1.7, and +10 mV for Na_v1.8 from a V_1/2 holding potential in both the absence and presence of 100 μmol/l APAS and PAS. Peak currents were measured and normalized to the first pulse and plotted against the pulse number. Data were fitted to the monoexponential equation I_Na = exp(−τ_use·n) + C, where n is pulse number, C is the plateau I_Na, and τ_use is the time constant of use-dependent decay.

The GraphPad Prism software (GraphPad Software, Inc., San Diego, CA) was used to perform the statistical analysis, and a statistical power analysis was performed using G*Power software. All values are presented as means ± SEM. The n values refer to the number of oocytes examined. Each experiment was performed with oocytes taken from at least two frogs. Data were statistically evaluated by paired t test (two-tailed). We assessed the inhibitory effects at different APAS concentrations in the concentration–response curve, using one-way ANOVA followed by Dunnet post hoc test for multiple comparisons. Hill slope, half-maximal inhibitory concentration (IC₅₀), and half-maximal effective concentration (EC₅₀) values were also calculated. P value less than 0.05 was considered to indicate a significant difference.

Currents were elicited using a 50-ms depolarizing pulse to −20 mV for Na_v1.2 and Na_v1.6, −10 mV for Na_v1.7, and +10 mV for Na_v1.8 applied every 10 s from a V_max or V_1/2 holding potential in both the absence and presence of 100 μmol/l APAS and PAS (fig. 2). The amplitude of expressed sodium currents was typically 2 to 15 μA, and oocytes that showed a maximal current greater than 20 μA were not included in the data collection in all the following experiments. APAS had dual effects on sodium currents depending on the holding potential and α subunit (figs. 2 and 3). At V_1/2, APAS reduced the peak I_Na (sodium current) induced by Na_v1.2, Na_v1.6, and Na_v1.7 by 79 ± 1%, 71 ± 2%, and 49 ± 3%, respectively. At V_max, APAS also reduced I_Na induced by Na_v1.2 by 60 ± 4%, whereas it enhanced I_Na induced by Na_v1.6 and Na_v1.7 by 15 ± 6% and 14 ± 1%, respectively, although these effects were small. In contrast, APAS greatly enhanced I_Na induced by Na_v1.8 at both V_1/2 and V_max by 112 ± 34% and 202 ± 14%, respectively (fig. 3A). PAS reduced I_Na induced by Na_v1.2, Na_v1.6, and Na_v1.7 at V_1/2 by 54 ± 4%, 71 ± 1%, and 48 ± 2%, respectively. Effects of PAS on I_Na at V_max were smaller than those at V_1/2, and the magnitudes of inhibitory effects on Na_v1.2, Na_v1.6, and Na_v1.7 were 31 ± 5%, 10 ± 1%, and 6 ± 1%, respectively. While PAS enhanced I_Na induced by Na_v1.8 at V_max by 39 ± 6%, it did not affect I_Na induced by Na_v1.8 at V_1/2 (fig. 3B). In summary, PAS inhibited I_Na induced by Na_v1.2, Na_v1.6, and Na_v1.7 at both V_/1/2 and V_max holding potentials. APAS had inverse effects on Na_v1.6 and Na_v1.7 according to the different holding potentials, whereas it suppressed I_Na induced by Na_v1.2 at both V_/1/2 and V_max. Moreover, APAS markedly enhanced I_Na induced by Na_v1.8 at both V_/1/2 and V_max.

Next, we examined the concentration–response relationship for suppression of the peak I_Na induced through Na_v1.2, Na_v1.6, and Na_v1.7 by APAS and PAS at V_1/2 holding potential because suppression by both neurosteroids of these α subunits at V_1/2 was more potent than that at V_max (fig. 4, A and B). In addition, we investigated the concentration–response relationship for potentiation of the peak I_Na of Na_v1.8 by APAS and PAS at V_max, because both neurosteroids showed potent enhancement of I_Na at V_max compared with that at V_1/2 (fig. 4C). IC₅₀ values, EC₅₀ values, and Hill slopes calculated from nonlinear regression analyses of the dose–response curves are shown in table 1. From these analyses, the effect of APAS on Na_v1.2 was the most potent among the two neurosteroids and four α subunits.

We examined the effects of APAS and PAS on four α subunits in sodium current activation. Voltage dependence of activation was determined using 50-ms depolarizing pulses from a holding potential of V_max to 50 mV in 10-mV increments or from a holding potential of V_1/2 to 60 mV in 10-mV increments for Na_v1.2, Na_v1.6, Na_v1.7, and Na_v1.8 in both the absence and presence of 100 μmol/l APAS and PAS (fig. 5). Activation curves were derived from the I–V curves (see Electrophysiological Recordings under Materials and Methods). At V_max, APAS greatly reduced the peak I_Na induced by Na_v1.2, whereas it greatly enhanced the peak I_Na induced by Na_v1.8 in the depolarizing region where channel opening begins. It also enhanced the peak I_Na induced by Na_v1.6 and Na_v1.7, similar to its effects on Na_v1.8, although both effects were small. At V_1/2, APAS greatly suppressed the peak I_Na induced by Na_v1.2, Na_v1.6, and Na_v1.7, but it enhanced the peak I_Na induced by Na_v1.8, similar to its effects on Na_v1.8 at V_max. PAS reduced I_Na induced by Na_v1.2, Na_v1.6, and Na_v1.7 at both V_/1/2 and V_max, whereas it enhanced I_Na induced by Na_v1.8 in the depolarizing region at V_max, but had no effect at V_1/2.

At V_max holding potential, APAS significantly shifted the midpoint of the steady-state activation (V_1/2) in a depolarizing direction for Na_v1.2, but it significantly shifted V_1/2 in a hyperpolarizing direction for Na_v1.6, Na_v1.7, and Na_v1.8. At V_1/2, APAS also shifted V_1/2 in a similar direction as the shift at V_max, although the shift was small and not significant, except for Na_v1.8. The shifts of V_1/2 by PAS were smaller than those by APAS. PAS significantly shifted V_1/2 in a depolarizing direction for Na_v1.2 and Na_v1.6 at V_1/2, but it had no or slight effects on all α subunits at V_max, and on Na_v1.7 and Na_v1.8 at V_1/2 (fig. 6 and tables 2 and 3).

We also investigated the effects of APAS and PAS on steady-state inactivation. Currents were elicited by a 50-ms test pulse to −20 mV for Na_v1.2 and Na_v1.6, −10 mV for Na_v1.7, and +10 mV for Na_v1.8 after 200 ms (500 ms for only Na_v1.8) prepulses ranging from −140 mV to 0 mV in 10-mV increments from V_max holding potential. Steady-state inactivation curves were fitted to the Boltzmann equation (see Electrophysiological Recordings under Materials and Methods). APAS and PAS significantly shifted the midpoint of steady-state inactivation (V_1/2) in the hyperpolarizing direction for all α subunits; APAS shifted by 8.0, 8.9, 6.7, and 8.9 mV and PAS shifted by 4.5, 8.0, 6.6, and 10.2 mV for Na_v1.2, Na_v1.6, Na_v1.7, and Na_v1.8, respectively (fig. 7 and tables 2 and 3). The effects of APAS and PAS in the hyperpolarizing range were consistent with the effects of these two neurosteroids on the peak I_Na at V_max and their effects on the I–V curves in the hyperpolarizing range at V_max.

The use-dependent block of sodium currents by APAS and PAS was also investigated. Currents were elicited at 10 Hz by a 20-ms depolarizing pulse of −20 mV for Na_v1.2 and Na_v1.6 and −10 mV for Na_v1.7 from a V_1/2 holding potential in both the absence and presence of 100 μmol/l APAS and PAS. Peak currents were measured and normalized to the first pulse and plotted against the pulse number (fig. 8, A–D). Data were fitted by the monoexponential equation (see Electrophysiological Recordings under Materials and Methods). APAS significantly reduced the plateau I_Na amplitude of Na_v1.2, Na_v1.6, and Na_v1.7 from 0.80 ± 0.03 to 0.57 ± 0.03, 0.89 ± 0.01 to 0.49 ± 0.07, and 0.89 ± 0.02 to 0.62 ± 0.06, respectively (fig. 8E). PAS also reduced the plateau I_Na amplitudes of Na_v1.2, Na_v1.6, and Na_v1.7 from 0.81 ± 0.2 to 0.70 ± 0.03, 0.94 ± 0.01 to 0.73 ± 0.02, and 0.91 ± 0.02 to 0.75 ± 0.01, respectively, and the reductions were significant except for Na_v1.2 (fig. 8F). These results demonstrated a use-dependent block of APAS and PAS on sodium channels, and the block by APAS was more potent than that by PAS.

In the current study, we demonstrated that APAS and PAS differentially affected I_Na induced by four α subunits at both V_max and V_1/2 holding potentials. Moreover, we found that both neurosteroids suppress Na_v1.2, Na_v1.6, and Na_v1.7 at V_1/2 in a concentration-dependent manner. IC₅₀ values indicated that the effect of APAS on Na_v1.2 was most potent among the two compounds and three α subunits. To the best of our knowledge, this is the first direct evidence of the various effects of these two neurosteroids on neuronal sodium channel α subunits. It is thought that APAS is synthesized from allopregnanolone by 3α-hydroxysteroid sulfotransferase in vivo, because 3α-hydroxysteroid sulfotransferase has been isolated in vivo.²⁶  Therefore, allopregnanolone likely exerts a portion of its effects through APAS, which is its metabolite.

It was reported that the level of endogenous allopregnanolone changes in many physiological and pathological situations within a serum concentration range of 1 to 10 nmol/l.^27,28  However, it is not clear whether allopregnanolone has an analgesic effect in physiological concentrations. A recent study demonstrated that 1 and 10 μmol/l of allopregnanolone reduced mechanical allodynia and thermal heat hyperalgesia in normal and neuropathic pain models in rats after 10-μl intrathecal injection.²⁹  Another investigator reported that intrathecal administration of 10 μmol/l of allopregnanolone showed antihyperalgesic effects in hyperalgesic rats after spinal nerve ligation.³⁰  From these previous studies, concentrations approximately 1 μmol/l allopregnanolone at receptive fields are estimated to have an analgesic effect. In the current study, APAS tended to, albeit not significantly, suppress the I_Na of Na_v1.2 at 0.3 μmol/l by 8% and significantly (P < 0.01) inhibited it at 1 μmol/l by 19 ± 2%. The IC₅₀ value of Na_v1.2 inhibition by APAS was 12 μmol/l. It was reported that relatively small degrees of sodium channel inhibition could have profound effects on the neuronal firing rate because a 10% inhibition of sodium current reduces the number of action potentials to 10 from a control response of 21 in 750 ms.²⁴  Therefore, APAS may reduce neuronal firing for Na_v1.2 at a concentration exhibiting the antinociceptive effects of allopregnanolone in animal models, whereas the effects of APAS and PAS on another three α and four α subunits, respectively, may not be pharmacologically relevant because these effects were observed at concentrations over 10 μmol/l. In addition, the effects of highly hydrophobic compounds—such as neurosteroids—we used tend to be attenuated in the voltage-clamp techniques with Xenopus oocytes, compared with the whole-cell voltage-clamp methods using mammalian cells. Indeed, it was reported that the enhancing effect by allopregnanolone on GABA_A receptor combination (α₁β₂γ_2L) was more potent in the human embryonic kidney 293 cells system (EC₅₀; 41 ± 2 nmol/l)³¹  than that in the Xenopus oocyte system (EC₅₀; 177 ± 2 nmol/l).³²  This may be a limitation of experiments using the Xenopus oocyte expression system; this limitation indicates that APAS might inhibit function of Na_v1.2 more potently in a mammalian cell system than in the oocyte system, however, it also could potentiate Na_v1.8 function more potently in a mammalian cell. Therefore, further investigation is needed to consider the roles of these α subunits in humans.

Analysis of gating revealed common characteristics but also some differences in the effects of APAS and PAS on different α subunits. A common effect on all α subunits was enhancement of inactivation. Because of this enhancement effect, the inhibitions by two compounds at V_1/2 holding potentials could be interpreted as stronger effects because they shift inactivation curve to the hyperpolarizing direction, which makes the channel into further inactivated state. In contrast, APAS enhanced peak I_Na at V_max, shifted activation in the hyperpolarizing direction, and increased sodium currents in the hyperpolarizing range of the inactivation curves for Na_v1.6, Na_v1.7, and Na_v1.8. These changes indicate that APAS shifts channel gating equilibrium toward the open channel state and activates sodium channels. This action might attenuate the effects on the inactivated state and, especially, lead to enhancement of I_Na even in the inactivated state (V_1/2 holding potential) for Na_v1.8 in spite of the great enhancement of inactivation. However, for Na_v1.2, APAS profoundly suppressed peak I_Na at V_max, shifted activation in the depolarizing direction at V_max, and greatly decreased sodium currents in the hyperpolarizing range of the inactivation curve, indicating that resting channel block is an important mechanism of APAS inhibition for only Na_v1.2. Both compounds demonstrated use-dependency for inhibition of Na_v1.2, Na_v1.6, and Na_v1.7, suggesting the ability to slow the recovery time from inactivation.³³  Many investigators have shown that sodium channel blockers, including local anesthetics, tricyclic antidepressants, and volatile anesthetics, enhance steady-state inactivation with no effect on activation and exhibit use-dependent block.^34–36  We demonstrated that APAS enhances inactivation and shows use-dependent block similar to other sodium channel blockers, yet it also has diverse effects on activation according to differences in α subunits. These actions suggest that APAS may have different binding sites or allosteric conformational mechanisms to change sodium channel function, although further investigation with site-directed mutagenesis is needed to rule out nonspecific membrane effects. PAS may have common binding sites with APAS, because it shows similar effects, although these changes were small.

The α subunit consists of four homologous domains (I to IV) containing six transmembrane segments (S1 to S6), and one reentrant P-region connecting S5 to S6 (SS1/SS2). Tetrodotoxin-sensitive α subunits, Na_v1.2, Na_v1.6, and Na_v1.7, are phylogenetically related and show 70 to 80% amino acid sequence identity. In contrast, tetrodotoxin-resistant α subunits, Na_v1.8, are phylogenetically distant and show only 55 to 56% sequence identity to the other three α subunits. In addition, the lengths of amino acid sequences of four α subunits differed within the range of 1957 to 2005 residues. Therefore, these differences would result in the diversity in neurosteroid action, especially in the effects on channel activation. Indeed, the longest extracellular regions in the α subunit (IS5 to SS1) are 93, 77, 73, and 66 amino acid residues in Na_v1.2, Na_v1.6, Na_v1.7, and Na_v1.8, respectively. The diversity in sequence and differences in the effects on activation according to α subunit may be important for clarifying binding sites and the mechanism of Na_v1.2 inhibition by APAS in further investigations.

γ-Aminobutyric acid type A receptors have been considered to be important for the analgesic effects of allopregnanolone because it has high potency as a positive GABA_A modulator compared with other neurosteroids. Pregnanolone also affects GABA_A receptors in a manner similar to that of allopregnanolone; nevertheless, its analgesic effect is weak. In fact, pregnanolone was shown to reduce mechanical allodynia without reduction of thermal heat hyperalgesia in a neuropathic pain model in contrast to attenuation of both by allopregnanolone.²⁸  The investigators suggested that the partial analgesic effects of pregnanolone are caused by suppression of glycine receptors by demonstrating that pregnanolone had a significant analgesic effect only in animals displaying a strychnine-induced allodynia in two types of allodynia models induced by bicuculline and strychnine.²⁸  Moreover, a recent report demonstrated that allopregnanolone shows analgesic effects in rats through suppression of T-type Ca²⁺ currents and potentiation of GABA_A currents.¹⁶  These previous reports indicate several mechanisms underlying the analgesic effect of allopregnanolone likely exist, as well as potentiation of GABA_A receptors.

Sodium channel α subunits expressed in the dorsal root ganglion (Na_v1.7, Na_v1.8, and Na_v1.9) are thought to be involved in the pathogenesis of inflammatory and neuropathic pain. A recent study reported that Na_v1.2 also plays an important role in pain signaling. It was reported that Na_v1.2 and Na_v1.3 predominantly compose functional sodium channel currents within lamina I/II (dorsal horn) neurons, which mediate acute and chronic nociceptive signals from peripheral nociceptors to pain-processing regions in the brain.³⁷  Another recent report showed that mutations in Na_v1.2 are associated with seizures and pain characterized by headaches and back pain.³⁸  A disubstituted succinamide, a potent sodium channel blocker, was reported to attenuate nociceptive behavior in a rat model of tonic pain and was demonstrated to potently block Na_v1.2, as well as Na_v1.7 and Na_v1.8, with a potency two orders of magnitude higher than anticonvulsant and antiarrhythmic sodium channel blockers currently used to treat neuropathic pain.³⁹  Other investigators demonstrated that four sodium channel blockers, including lidocaine, mexiletine, benzocaine, and ambroxol, which are used clinically to treat pain, suppressed recombinant Na_v1.2 currents as well as tetrodotoxin-resistant Na⁺ channel currents in rat sensory neurons, which comprised mostly Na_v1.8 currents. The authors suggested that these sodium channel blockers would induce analgesia according to the amount of sodium channel blocking, including Na_v1.2 and Na_v1.8.⁴⁰  These recent reports support that suppression of Na_v1.2 function by APAS might be a mechanism underlying the analgesic effects of allopregnanolone.

In conclusion, APAS and PAS have diverse effects on Na_v1.2, Na_v1.6, Na_v1.7, and Na_v1.8 α subunits expressed in Xenopus oocytes, with differences in the effects on sodium channel gating. In particular, only APAS inhibited sodium currents of Na_v1.2 at pharmacologically relevant concentrations. These results raise the possibility that suppression of Na_v1.2 by APAS may be important for pain relief by allopregnanolone and provide a better understanding of the mechanisms underlying the analgesic effects of allopregnanolone. However, further studies are needed to clarify the relevance of sodium channel inhibition by APAS.

This study was supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science, Tokyo, Japan (grant no. 21791480 to Dr. Horishita).

The authors declare no competing interests.