Use-dependent Inhibition Research Articles

Development of Riluzole Analogs with Improved Use-Dependent Inhibition of Skeletal Muscle Sodium Channels.

Several commercially available and newly synthesized riluzole analogs were evaluated in vitro as voltage-gated skeletal muscle sodium-channel blockers. Data obtained from the patch-clamp technique demonstrated that potency is well correlated with lipophilicity and the introduction of a protonatable amino function in the benzothiazole 2-position enhances the use-dependent behavior. The most interesting compound, the 2-piperazine analog of riluzole (14), although slightly less potent than the parent compound in the patch-clamp assay as well as in an in vitro model of myotonia, showed greater use-dependent Nav1.4 blocking activity. Docking studies allowed the identification of the key interactions that 14 makes with the amino acids of the local anesthetic binding site within the pore of the channel. The reported results pave the way for the identification of novel compounds useful in the treatment of cell excitability disorders.

New aryl and acylsulfonamides as state-dependent inhibitors of Nav1.3 voltage-gated sodium channel

Voltage-gated sodium channels (Navs) play an essential role in neurotransmission, and their dysfunction is often a cause of various neurological disorders. The Nav1.3 isoform is found in the CNS and upregulated after injury in the periphery, but its role in human physiology has not yet been fully elucidated. Reports suggest that selective Nav1.3 inhibitors could be used as novel therapeutics to treat pain or neurodevelopmental disorders. Few selective inhibitors of this channel are known in the literature. In this work, we report the discovery of a new series of aryl and acylsulfonamides as state-dependent inhibitors of Nav1.3 channels. Using a ligand-based 3D similarity search and subsequent hit optimization, we identified and prepared a series of 47 novel compounds and tested them on Nav1.3, Nav1.5, and a selected subset also on Nav1.7 channels in a QPatch patch-clamp electrophysiology assay. Eight compounds had an IC50 value of less than 1 μM against the Nav1.3 channel inactivated state, with one compound displaying an IC50 value of 20 nM, whereas activity against the inactivated state of the Nav1.5 channel and Nav1.7 channel was approximately 20-fold weaker. None of the compounds showed use-dependent inhibition of the cardiac isoform Nav1.5 at a concentration of 30 μM. Further selectivity testing of the most promising hits was measured using the two-electrode voltage-clamp method against the closed state of the Nav1.1-Nav1.8 channels, and compound 15b displayed small, yet selective, effects against the Nav1.3 channel, with no activity against the other isoforms. Additional selectivity testing of promising hits against the inactivated state of the Nav1.3, Nav1.7, and Nav1.8 channels revealed several compounds with robust and selective activity against the inactivated state of the Nav1.3 channel among the three isoforms tested. Moreover, the compounds were not cytotoxic at a concentration of 50 μM, as demonstrated by the assay in human HepG2 cells (hepatocellular carcinoma cells). The novel state-dependent inhibitors of Nav1.3 discovered in this work provide a valuable tool to better evaluate this channel as a potential drug target.

Trichloroethanol, an active metabolite of chloral hydrate, modulates tetrodotoxin-resistant Na+ channels in rat nociceptive neurons

BackgroundChloral hydrate is a sedative-hypnotic drug widely used for relieving fear and anxiety in pediatric patients. However, mechanisms underlying the chloral hydrate-mediated analgesic action remain unexplored. Therefore, we investigated the effect of 2′,2′,2′-trichloroethanol (TCE), the active metabolite of chloral hydrate, on tetrodotoxin-resistant (TTX-R) Na+ channels expressed in nociceptive sensory neurons.MethodsThe TTX-R Na+ current (INa) was recorded from acutely isolated rat trigeminal ganglion neurons using the whole-cell patch-clamp technique.ResultsTrichloroethanol decreased the peak amplitude of transient TTX-R INa in a concentration-dependent manner and potently inhibited persistent components of transient TTX-R INa and slow voltage-ramp-induced INa at clinically relevant concentrations. Trichloroethanol exerted multiple effects on various properties of TTX-R Na+ channels; it (1) induced a hyperpolarizing shift on the steady-state fast inactivation relationship, (2) increased use-dependent inhibition, (3) accelerated the onset of inactivation, and (4) retarded the recovery of inactivated TTX-R Na+ channels. Under current-clamp conditions, TCE increased the threshold for the generation of action potentials, as well as decreased the number of action potentials elicited by depolarizing current stimuli.ConclusionsOur findings suggest that chloral hydrate, through its active metabolite TCE, inhibits TTX-R INa and modulates various properties of these channels, resulting in the decreased excitability of nociceptive neurons. These pharmacological characteristics provide novel insights into the analgesic efficacy exerted by chloral hydrate.

Tramadol as a Voltage-Gated Sodium Channel Blocker of Peripheral Sodium Channels Nav1.7 and Nav1.5

Tramadol is an opioid analog used to treat chronic and acute pain. Intradermal injections of tramadol at hundreds of millimoles have been shown to produce a local anesthetic effect. We used the whole-cell patch-clamp technique in this study to investigate whether tramadol blocks the sodium current in HEK293 cells, which stably express the pain threshold sodium channel Nav1.7 or the cardiac sodium channel Nav1.5. The half-maximal inhibitory concentration of tramadol was 0.73 mM for Nav1.7 and 0.43 mM for Nav1.5 at a holding potential of -100 mV. The blocking effects of tramadol were completely reversible. Tramadol shifted the steady-state inactivation curves of Nav1.7 and Nav1.5 toward hyperpolarization. Tramadol also slowed the recovery rate from the inactivation of Nav1.7 and Nav1.5 and induced stronger use-dependent inhibition. Because the mean plasma concentration of tramadol upon oral administration is lower than its mean blocking concentration of sodium channels in this study, it is unlikely that tramadol in plasma will have an analgesic effect by blocking Nav1.7 or show cardiotoxicity by blocking Nav1.5. However, tramadol could act as a local anesthetic when used at a concentration of several hundred millimoles by intradermal injection and as an antiarrhythmic when injected intravenously at a similar dose, as does lidocaine.

Use-Dependent Relief of Inhibition of Nav1.8 Channels by A-887826.

Sodium channel inhibitors used as local anesthetics, antiarrhythmics, or antiepileptics typically have the property of use-dependent inhibition, whereby inhibition is enhanced by repetitive channel activation. For targeting pain, Nav1.8 channels are an attractive target because they are prominent in primary pain-sensing neurons, with little or no expression in most other kinds of neurons, and a number of Nav1.8-targeted compounds have been developed. We examined the characteristics of Nav1.8 inhibition by one of the most potent Nav1.8 inhibitors so far described, A-887826, and found that when studied with physiologic resting potentials and physiologic temperatures, inhibition had strong "reverse use dependence", whereby inhibition was relieved by repetitive short depolarizations. This effect was much stronger with A-887826 than with A-803467, another Nav1.8 inhibitor. The use-dependent relief from inhibition was seen in both human Nav1.8 channels studied in a cell line and in native Nav1.8 channels in mouse dorsal root ganglion (DRG) neurons. In native Nav1.8 channels, substantial relief of inhibition occurred during repetitive stimulation by action potential waveforms at 5 Hz, suggesting that the phenomenon is likely important under physiologic conditions. SIGNIFICANCE STATEMENT: Nav1.8 sodium channels are expressed in primary pain-sensing neurons and are a prime current target for new drugs for pain. This work shows that one of the most potent Nav1.8 inhibitors, A-887826, has the unusual property that inhibition is relieved by repeated short depolarizations. This "reverse use dependence" may reduce inhibition during physiological firing and should be selected against in drug development.

Polysorbate 80 blocked a peripheral sodium channel, Nav1.7, and reduced neuronal excitability.

Polysorbate 80 is a non-ionic detergent derived from polyethoxylated sorbitan and oleic acid. It is widely used in pharmaceuticals, foods, and cosmetics as an emulsifier. Nav1.7 is a peripheral sodium channel that is highly expressed in sympathetic and sensory neurons, and it plays a critical role in determining the threshold of action potentials (APs). We found that 10 μg/mL polysorbate 80 either abolished APs or increased the threshold of the APs of dorsal root ganglions. We thus investigated whether polysorbate 80 inhibits Nav1.7 sodium current using a whole-cell patch-clamp recording technique. Polysorbate 80 decreased the Nav1.7 current in a concentration-dependent manner with a half-maximal inhibitory concentration (IC50) of 250.4 μg/mL at a holding potential of -120 mV. However, the IC50 was 1.1 μg/mL at a holding potential of -90 mV and was estimated to be 0.9 μg/mL at the resting potentials of neurons, where most channels are inactivated. The activation rate and the voltage dependency of activation of Nav1.7 were not changed by polysorbate 80. However, polysorbate 80 caused hyperpolarizing shifts in the voltage dependency of the steady-state fast inactivation curve. The blocking of Nav1.7 currents by polysorbate 80 was not reversible at a holding potential of -90 mV but was completely reversible at -120 mV, where the channels were mostly in the closed state. Polysorbate 80 also slowed recovery from inactivation and induced robust use-dependent inhibition, indicating that it is likely to bind to and stabilize the inactivated state. Our results indicate that polysorbate 80 inhibits Nav1.7 current in concentration-, state-, and use-dependent manners when used even below commercial concentrations. This suggests that polysorbate 80 may be helpful in pain medicine as an excipient. In addition, in vitro experiments using polysorbate 80 with neurons should be conducted with caution.

Contribution of tetrodotoxin-resistant persistent Na+ currents to the excitability of C-type dural afferent neurons in rats

BackgroundGrowing evidence supports the important role of persistent sodium currents (INaP) in the neuronal excitability of various central neurons. However, the role of tetrodotoxin-resistant (TTX-R) Na+ channel-mediated INaP in the neuronal excitability of nociceptive neurons remains poorly understood.MethodsWe investigated the functional role of TTX-R INaP in the excitability of C-type nociceptive dural afferent neurons, which was identified using a fluorescent dye, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchloride (DiI), and a whole-cell patch-clamp technique.ResultsTTX-R INaP were found in most DiI-positive neurons, but their density was proportional to neuronal size. Although the voltage dependence of TTX-R Na+ channels did not differ among DiI-positive neurons, the extent of the onset of slow inactivation, recovery from inactivation, and use-dependent inhibition of these channels was highly correlated with neuronal size and, to a great extent, the density of TTX-R INaP. In the presence of TTX, treatment with a specific INaP inhibitor, riluzole, substantially decreased the number of action potentials generated by depolarizing current injection, suggesting that TTX-R INaP are related to the excitability of dural afferent neurons. In animals treated chronically with inflammatory mediators, the density of TTX-R INaP was significantly increased, and it was difficult to inactivate TTX-R Na+ channels.ConclusionsTTX-R INaP apparently contributes to the differential properties of TTX-R Na+ channels and neuronal excitability. Consequently, the selective modulation of TTX-R INaP could be, at least in part, a new approach for the treatment of migraine headaches.

Sodium Channels and Local Anesthetics—Old Friends With New Perspectives

The long history of local anesthetics (LAs) starts out in the late 19th century when the content of coca plant leaves was discovered to alleviate pain. Soon after, cocaine was established and headed off to an infamous career as a substance causing addiction. Today, LAs and related substances—in modified form—are indispensable in our clinical everyday life for pain relief during and after minor and major surgery, and dental practices. In this review, we elucidate on the interaction of modern LAs with their main target, the voltage-gated sodium channel (Navs), in the light of the recently published channel structures. Knowledge of the 3D interaction sites of the drug with the protein will allow to mechanistically substantiate the comprehensive data available on LA gating modification. In the 1970s it was suggested that LAs can enter the channel pore from the lipid phase, which was quite prospective at that time. Today we know from cryo-electron microscopy structures and mutagenesis experiments, that indeed Navs have side fenestrations facing the membrane, which are likely the entrance for LAs to induce tonic block. In this review, we will focus on the effects of LA binding on fast inactivation and use-dependent inhibition in the light of the proposed new allosteric mechanism of fast inactivation. We will elaborate on subtype and species specificity and provide insights into modelling approaches that will help identify the exact molecular binding orientation, access pathways and pharmacokinetics. With this comprehensive overview, we will provide new perspectives in the use of the drug, both clinically and as a tool for basic ion channel research.

Sevoflurane modulation of tetrodotoxin-resistant Na+ channels in small-sized dorsal root ganglion neurons of rats.

Volatile anesthetics are widely used for general anesthesia during surgical operations. Voltage-gated Na+ channels expressed in central neurons are major targets for volatile anesthetics; but it is unclear whether these drugs modulate native tetrodotoxin-resistant (TTX-R) Na+ channels, which are involved in the development and maintenance of inflammatory pain. In this study, we examined the effects of sevoflurane on TTX-R Na+ currents (INa) in acutely isolated rat dorsal root ganglion neurons, using a whole-cell patch-clamp technique. Sevoflurane slightly potentiated the peak amplitude of transient TTX-R INa but more potently inhibited slow voltage-ramp-induced persistent INa in a concentration-dependent manner. Sevoflurane (0.86 ± 0.02 mM) (1) slightly shifted the steady-state fast inactivation relationship to hyperpolarizing ranges without affecting the voltage-activation relationship, (2) reduced the extent of use-dependent inhibition of Na+ channels, (3) accelerated the onset of inactivation and (4) delayed the recovery from inactivation of TTX-R Na+ channels. Thus, sevoflurane has diverse effects on TTX-R Na+ channels expressed in nociceptive neurons. The present results suggest that the inhibition of persistent INa and the modulation of the voltage dependence and inactivation might be, at least in part, responsible for the analgesic effects elicited by sevoflurane.

Propranolol modulation of tetrodotoxin-resistant Na+ channels in dural afferent neurons

Propranolol, a representative adrenergic β-receptor antagonist, is widely used to prevent migraine attacks. Although propranolol is well known to inhibit tetrodotoxin-resistant (TTX-R) Na+ channels in cardiac myocytes, it is unclear whether the drug modulates these channels expressed in dural afferent neurons. In this study, we examined the effects of propranolol on TTX-R Na+ channels in medium-sized dural afferent neurons identified by the fluorescent dye DiI. The TTX-R Na+ currents (INa) were recorded from acutely isolated DiI-positive neurons using a whole-cell patch clamp technique under voltage-clamp conditions. Propranolol inhibited the noninactivating steady-state component more potently than the peak component of transient TTX-R INa. Propranolol also potently inhibited the slow voltage ramp-induced TTX-R INa in a concentration-dependent manner, suggesting that it preferentially inhibited the noninactivating or persistent INa in DiI-positive neurons. Propranolol had little effect on voltage dependence, but it increased the extent of the use-dependent inhibition of TTX-R Na+ channels. Propranolol also accelerated the onset of inactivation and retarded recovery from inactivation in these channels. Under current-clamp conditions, propranolol decreased the number of action potentials elicited by depolarizing current stimuli. In conclusion, the propranolol-mediated preferential inhibition of persistent INa and modulation of the inactivation kinetics of TTX-R Na+ channels might represent additional mechanisms for migraine prophylaxis.

State-dependent inhibition of BK channels by the opioid agonist loperamide.

Large-conductance Ca2+-activated K+ (BK) channels control a range of physiological functions, and their dysfunction is linked to human disease. We have found that the widely used drug loperamide (LOP) can inhibit activity of BK channels composed of either α-subunits (BKα channels) or α-subunits plus the auxiliary γ1-subunit (BKα/γ1 channels), and here we analyze the molecular mechanism of LOP action. LOP applied at the cytosolic side of the membrane rapidly and reversibly inhibited BK current, an effect that appeared as a decay in voltage-activated BK currents. The apparent affinity for LOP decreased with hyperpolarization in a manner consistent with LOP behaving as an inhibitor of open, activated channels. Increasing LOP concentration reduced the half-maximal activation voltage, consistent with relative stabilization of the LOP-inhibited open state. Single-channel recordings revealed that LOP did not reduce unitary BK channel current, but instead decreased BK channel open probability and mean open times. LOP elicited use-dependent inhibition, in which trains of brief depolarizing steps lead to accumulated reduction of BK current, whereas single brief depolarizing steps do not. The principal effects of LOP on BK channel gating are described by a mechanism in which LOP acts as a state-dependent pore blocker. Our results suggest that therapeutic doses of LOP may act in part by inhibiting K+ efflux through intestinal BK channels.

Increased Sympathetic Tone in Congestive Heart Failure and Comorbid PainAcute Friend Turns Chronic Foe and a Novel Pharmacologic Corrective Approach

Acute pain and congestive heart failure (CHF) both increase sympathetic (S) tone, decreasing pain and improving cardiac output. However, chronically high S increases pain and worsens CHF. Mechanisms will be discussed, and off-label ranolazine is a novel potential pharmacologic remedy in chronic CHF: (1) Fifty-four CHF patients were randomized to adjunctive RAN (RANCHF, 1000mg bid) vs. NORANCHF. Autonomic measurements (ANX 3.0 Autonomic Monitor) were taken at baseline and 1 yr. Fifty-nine % of patients in both groups were initially abnormal, including high sympathovagal balance (SB) that normalized in 10/12 (83%) RANCHF patients vs. 2/11 (18%) NORANCHF patients. High SB developed in 5/11 (45%) NORANCHF vs. 1/11(9%) RANCHF patients; (2) Matched CHF patients were given adjunctive RAN (1000 mg po-bid) (RANCHF, 41 systolic, 13 diastolic) vs. NORANCHF (43 systolic, 12 diastolic). Echocardiographic LVEF and autonomic measures were obtained at baseline and follow-up (mean 23.7 months). LVEF increased in 70% of RANCHF patients, an average of 11.3 units. Mean LVEF remained unchanged in NORANCHF patients. At baseline, 28% of patients had high SB. RAN normalized SB in >50%; the NORANCHF group had a 20% increase in patients with high SB. RAN reduced (composite endpoint) CHF admissions, cardiac death, ventricular tachycardia/fibrillation[vt/vf] by 40 % In conclusion, RAN substantially corrects the maladaptive SNS CHF response. Since 1 mechanism of action of RAN is a strongly use-dependent inhibition of the Nav1.7 in S ganglia, RAN should provide pain relief in chronically S-mediated pain syndromes.

Norquetiapine blocks the human cardiac sodium channel Nav1.5 in a state-dependent manner

Quetiapine, an atypical antipsychotic drug, is used for the treatment of schizophrenia and acute mania. Although a previous report showed that quetiapine blocked hERG potassium current, quetiapine has been considered relatively safe in terms of cardiovascular side effects. In the present study, we used the whole-cell patch-clamp technique to investigate the effect that quetiapine and its major metabolite norquetiapine can exert on human cardiac sodium channels (hNav1.5). The half-maximal inhibitory concentrations of quetiapine and norquetiapine at a holding potential of −90 mV near the resting potential of cardiomyocytes were 30 and 6 μM, respectively. Norquetiapine as well as quetiapine was preferentially bound in the inactivated state of the hNav1.5 channel. Norquetiapine slowed the recovery from inactivation of hNav1.5 and consequently induced strong use-dependent inhibition. Our results indicate that norquetiapine blocks hNav1.5 current in concentration-, state- and use-dependent manners, suggesting that the blockade of hNav1.5 current by norquetiapine may shorten the cardiac action potential duration and reduce the risk of QT interval prolongation induced by the inhibition of hERG potassium currents.

Screening Assay Protocols Targeting the Nav1.7 Channel Using Qube High-Throughput Automated Patch-Clamp System.

This article describes the basic procedure for setting up the screening protocol and recording data for Nav1.7 on a Qube automated patch-clamp system. Three protocols along with step-by-step details are provided. First, we describe a protocol to estimate Vhalf , the voltage at which half of the channels are inactivated, using traditional steady-state inactivation measurement as well as a new adaptive online estimation. Second, we establish a state-dependent protocol using adaptive online Vhalf measurement to obtain a concentration response curve (CRC) on known reference blockers. Last, we introduce a use-dependent protocol. In our hands, the sample reference demonstrated good state- and use-dependent inhibition of Nav1.7. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Comparing Vhalf measurements using offline calculation and a new adaptive online estimation Basic Protocol 2: Concentration response experiments using a state-dependent protocol Basic Protocol 3: Concentration response experiments using a use-dependent protocol Support Protocol: Cell culture protocol.

Sodium channel blockers in the treatment of neuropathic pain

Chronic pain is a global unmet medical need because of complex and incompletely understood underlying pathophysiological mechanisms and because treatment is mostly ineffective and can be addictive. Genetic and functional studies have linked mutations in voltage-gated sodium channels NaV1.7, NaV1.8 and Nav1.9 and pain syndromes. Studies of several families with Nav1.7 mutations uncovered different pain profiles in affected members of the same family. Using patient-specific iPSC-based differentiated sensory neurons, modulatory genetic factors that explain inter-individual variability of pain have begun to emerge. The robust genetic validation of Nav1.7 as a key factor in human pain disorders has led to intensive efforts for the development of novel isoform-specific drugs which are being tested in clinical studies. Importantly, a subset of mutations in Nav1.7 have been shown to respond to existing drugs which act in a novel mode of action, shifting channel activation in a deploarized direction, in addition to the classical state- and use-dependent inhibition. This new mode of action has now been shown to extend to a corresponding mutation in another sodium channel, Nav1.8. These studies show how monogenic pain disorders have advanced our understanding of the pathophysiology of chronic pain and show proof-of-principle studies implementing a precision medicine approach to treat pain.

THAR SHE BLOWS! The Search for the Great Spermine Whale of Carbamazepine Resistance.

Polyamine Modulation of Anticonvulsant Drug Response: A Potential Mechanism Contributing to Pharmacoresistance in Chronic Epilepsy Beckonert NM, Opitz T, Pitsch J, Soares da Silva P, Beck H. J Neurosci. 2018;38(24):5596-5605. doi:10.1523/JNEUROSCI.0640-18.2018.Despite the development of numerous novel anticonvulsant drugs, ∼30% of patients with epilepsy remain refractory to antiepileptic drugs (AEDs). Many established and novel AEDs reduce hyperexcitability via voltage- and use-dependent inhibition of voltage-gated Na+ channels. For the widely used anticonvulsant carbamazepine (CBZ), use-dependent block of Na+ channels is significantly reduced both in experimental and human epilepsies. However, the molecular underpinnings of this potential cellular mechanism for pharmacoresistance have remained enigmatic. Here, we describe the mechanism that leads to the emergence of CBZ-resistant Na+ channels. We focused on the endogenous polyamine system, which powerfully modulates Na+ channels in a use-dependent manner. We had shown previously that the intracellular polyamine spermine is reduced in chronic epilepsy, resulting in increased persistent Na+ currents. Because both spermine and CBZ bind use-dependently in spatial proximity within the Na+ channel pore, we hypothesized that spermine loss might also be related to diminished CBZ response. Using the pilocarpine model of refractory epilepsy in male rats and whole-cell patch-clamp recordings, we first replicated the reduction of use-dependent block by CBZ in chronically epileptic animals. We then substituted intracellular spermine via the patch pipette in different concentrations. Under these conditions, we found that exogenous spermine significantly rescues use-dependent block of Na+ channels by CBZ. These findings indicate that an unexpected modulatory mechanism, depletion of intracellular polyamines, leads both to increased persistent Na+ currents and to diminished CBZ sensitivity of Na+ channels. These findings could lead to novel strategies for overcoming pharmacoresistant epilepsy that target the polyamine system. Significance Statement: Pharmacoresistant epilepsy affects ∼18 million people worldwide, and intense efforts have therefore been undertaken to uncover the underlying molecular and cellular mechanisms. One of the key known candidate mechanisms of pharmacoresistance has been a loss of use-dependent Na+ channel block by the anticonvulsant CBZ, both in human and experimental epilepsies. Despite intense scrutiny, the molecular mechanisms underlying this phenomenon have not been elucidated. We now show that a loss of intracellular spermine in chronic epilepsy is a major causative factor leading to the development of CBZ-resistant Na+ currents. This finding can be exploited both for the screening of anticonvulsants in expression systems and for novel strategies to overcome pharmacoresistance that target the polyamine system.

A gain-of-function sodium channel β2-subunit mutation in painful diabetic neuropathy.

Diabetes mellitus is a global challenge with many diverse health sequelae, of which diabetic peripheral neuropathy is one of the most common. A substantial number of patients with diabetic peripheral neuropathy develop chronic pain, but the genetic and epigenetic factors that predispose diabetic peripheral neuropathy patients to develop neuropathic pain are poorly understood. Recent targeted genetic studies have identified mutations in α-subunits of voltage-gated sodium channels (Navs) in patients with painful diabetic peripheral neuropathy. Mutations in proteins that regulate trafficking or functional properties of Navs could expand the spectrum of patients with Nav-related peripheral neuropathies. The auxiliary sodium channel β-subunits (β1–4) have been reported to increase current density, alter inactivation kinetics, and modulate subcellular localization of Nav. Mutations in β-subunits have been associated with several diseases, including epilepsy, cancer, and diseases of the cardiac conducting system. However, mutations in β-subunits have never been shown previously to contribute to neuropathic pain. We report here a patient with painful diabetic peripheral neuropathy and negative genetic screening for mutations in SCN9A, SCN10A, and SCN11A—genes encoding sodium channel α-subunit that have been previously linked to the development of neuropathic pain. Genetic analysis revealed an aspartic acid to asparagine mutation, D109N, in the β2-subunit. Functional analysis using current-clamp revealed that the β2-D109N rendered dorsal root ganglion neurons hyperexcitable, especially in response to repetitive stimulation. Underlying the hyperexcitability induced by the β2-subunit mutation, as evidenced by voltage-clamp analysis, we found a depolarizing shift in the voltage dependence of Nav1.7 fast inactivation and reduced use-dependent inhibition of the Nav1.7 channel.

Modulation of tetrodotoxin-resistant Na+ channels by amitriptyline in dural afferent neurons

Migraine is characterized by recurrent and disabling headaches; therefore, several drugs have been widely prescribed to prevent acute migraine attacks. Amitriptyline, a tricyclic antidepressant, is among the most commonly administered. It is poorly known, however, whether amitriptyline modulates the excitability of dural afferent neurons that transmit pain signals from the dura mater. In this study, the effects of amitriptyline on tetrodotoxin-resistant (TTX-R) Na+ channels were examined in acutely isolated rat dural afferent neurons, which were identified by the fluorescent dye DiI. The TTX-R Na+ currents (INa) were recorded from medium-sized DiI-positive neurons using a whole-cell patch clamp technique. Amitriptyline (3 μM) slightly reduced the peak component of transient INa and induced a marked decrease in the steady-state component of transient TTX-R INa, as well as in the slow ramp-induced TTX-R INa. Our findings suggest that amitriptyline specifically inhibits persistent Na+ currents mediated by TTX-R Na+ channels. While amitriptyline had minor effects on voltage-activation/inactivation, it increased the extent of the use-dependent inhibition of TTX-R Na+ channels. Amitriptyline also affected the inactivation kinetics of TTX-R Na+ channels by significantly accelerating the inactivation of TTX-R Na+ channels and slowing the subsequent recovery. Amitriptyline decreased the number of action potentials by increasing the threshold for their generation. In conclusion, the amitriptyline-mediated diverse modulation of TTX-R Na+ channels would be, at least in part, responsible for its prophylactic efficacy for migraine attacks.

Polyamine Modulation of Anticonvulsant Drug Response: A Potential Mechanism Contributing to Pharmacoresistance in Chronic Epilepsy.

Despite the development of numerous novel anticonvulsant drugs, ∼30% of epilepsy patients remain refractory to antiepileptic drugs (AEDs). Many established and novel AEDs reduce hyperexcitability via voltage- and use-dependent inhibition of voltage-gated Na+ channels. For the widely used anticonvulsant carbamazepine (CBZ), use-dependent block of Na+ channels is significantly reduced both in experimental and human epilepsy. However, the molecular underpinnings of this potential cellular mechanism for pharmacoresistance have remained enigmatic.Here, we describe the mechanism that leads to the emergence of CBZ-resistant Na+ channels. We focused on the endogenous polyamine system, which powerfully modulates Na+ channels in a use-dependent manner. We had shown previously that the intracellular polyamine spermine is reduced in chronic epilepsy, resulting in increased persistent Na+ currents. Because spermine and CBZ both bind use-dependently in spatial proximity within the Na+ channel pore, we hypothesized that spermine loss might also be related to diminished CBZ response. Using the pilocarpine model of refractory epilepsy in male rats and whole-cell patch-clamp recordings, we first replicated the reduction of use-dependent block by CBZ in chronically epileptic animals. We then substituted intracellular spermine via the patch pipette in different concentrations. Under these conditions, we found that exogenous spermine significantly rescues use-dependent block of Na+ channels by CBZ. These findings indicate that an unexpected modulatory mechanism, depletion of intracellular polyamines, leads both to increased persistent Na+ currents and to diminished CBZ sensitivity of Na+ channels. These findings could lead to novel strategies for overcoming pharmacoresistant epilepsy that target the polyamine system.SIGNIFICANCE STATEMENT Pharmacoresistant epilepsy affects ∼18 million people worldwide, and intense efforts have therefore been undertaken to uncover the underlying molecular and cellular mechanisms. One of the key known candidate mechanisms of pharmacoresistance has been a loss of use-dependent Na+ channel block by the anticonvulsant carbamazepine (CBZ), both in human and experimental epilepsies. Despite intense scrutiny, the molecular mechanisms underlying this phenomenon have not been elucidated. We now show that a loss of intracellular spermine in chronic epilepsy is a major causative factor leading to the development of CBZ-resistant Na+ currents. This finding can be exploited both for the screening of anticonvulsants in expression systems, and for novel strategies to overcome pharmacoresistance that target the polyamine system.

Effect of carbamazepine on tetrodotoxin-resistant Na+ channels in trigeminal ganglion neurons innervating to the dura.

Migraine is a neurological disorder characterized by recurrent and disabling severe headaches. Although several anticonvulsant drugs that block voltage-dependent Na+ channels are widely used for migraine, far less is known about the therapeutic actions of carbamazepine on migraine. In the present study, therefore, we characterized the effects of carbamazepine on tetrodotoxin-resistant (TTX-R) Na+ channels in acutely isolated rat dural afferent neurons, which were identified by the fluorescent dye DiI. The TTX-R Na+ currents were measured in medium-sized DiIpositive neurons using the whole-cell patch clamp technique in the voltage-clamp mode. While carbamazepine had little effect on the peak amplitude of transient Na+ currents, it strongly inhibited steady-state currents of transient as well as persistent Na+ currents in a concentration-dependent manner. Carbamazepine had only minor effects on the voltage-activation relationship, the voltage-inactivation relationship, and the use-dependent inhibition of TTX-R Na+ channels. However, carbamazepine changed the inactivation kinetics of TTX-R Na+ channels, significantly accelerating the development of inactivation and delaying the recovery from inactivation. In the current-clamp mode, carbamazepine decreased the number of action potentials without changing the action potential threshold. Given that the sensitization of dural afferent neurons by inflammatory mediators triggers acute migraine headaches and that inflammatory mediators potentiate TTX-R Na+ currents, the present results suggest that carbamazepine may be useful for the treatment of migraine headaches.