Inhibition of voltage-gated Na(+) currents in sensory neurones by the sea anemone toxin APETx2

BACKGROUND AND PURPOSE

APETx2, a toxin from the sea anemone Anthropleura elegantissima, inhibits acid-sensing ion channel 3 (ASIC3)-containing homo- and heterotrimeric channels with IC₅₀ values < 100 nM and 0.1–2 µM respectively. ASIC3 channels mediate acute acid-induced and inflammatory pain response and APETx2 has been used as a selective pharmacological tool in animal studies. Toxins from sea anemones also modulate voltage-gated Na⁺ channel (Na_v) function. Here we tested the effects of APETx2 on Na_v function in sensory neurones.

EXPERIMENTAL APPROACH

Effects of APETx2 on Na_v function were studied in rat dorsal root ganglion (DRG) neurones by whole-cell patch clamp.

KEY RESULTS

APETx2 inhibited the tetrodotoxin (TTX)-resistant Na_v 1.8 currents of DRG neurones (IC₅₀, 2.6 µM). TTX-sensitive currents were less inhibited. The inhibition of Na_v 1.8 currents was due to a rightward shift in the voltage dependence of activation and a reduction of the maximal macroscopic conductance. The inhibition of Na_v 1.8 currents by APETx2 was confirmed with cloned channels expressed in Xenopus oocytes. In current-clamp experiments in DRG neurones, the number of action potentials induced by injection of a current ramp was reduced by APETx2.

CONCLUSIONS AND IMPLICATIONS

APETx2 inhibited Na_v 1.8 channels, in addition to ASIC3 channels, at concentrations used in in vivo studies. The limited specificity of this toxin should be taken into account when using APETx2 as a pharmacological tool. Its dual action will be an advantage for the use of APETx2 or its derivatives as analgesic drugs.     

Biophysical properties of Na(v) 1.8/Na(v) 1.2 chimeras and inhibition by µO-conotoxin MrVIB

BACKGROUND AND PURPOSE

Voltage-gated sodium channels are expressed primarily in excitable cells and play a pivotal role in the initiation and propagation of action potentials. Nine subtypes of the pore-forming α-subunit have been identified, each with a distinct tissue distribution, biophysical properties and sensitivity to tetrodotoxin (TTX). Na_v1.8, a TTX-resistant (TTX-R) subtype, is selectively expressed in sensory neurons and plays a pathophysiological role in neuropathic pain. In comparison with TTX-sensitive (TTX-S) Na_vα-subtypes in neurons, Na_v1.8 is most strongly inhibited by the µO-conotoxin MrVIB from Conus marmoreus. To determine which domain confers Na_v1.8 α-subunit its biophysical properties and MrVIB binding, we constructed various chimeric channels incorporating sequence from Na_v1.8 and the TTX-S Na_v1.2 using a domain exchange strategy.

EXPERIMENTAL APPROACH

Wild-type and chimeric Na_v channels were expressed in Xenopus oocytes, and depolarization-activated Na⁺ currents were recorded using the two-electrode voltage clamp technique.

KEY RESULTS

MrVIB (1 µM) reduced Na_v1.2 current amplitude to 69 ± 12%, whereas Na_v1.8 current was reduced to 31 ± 3%, confirming that MrVIB has a binding preference for Na_v1.8. A similar reduction in Na⁺ current amplitude was observed when MrVIB was applied to chimeras containing the region extending from S6 segment of domain I through the S5-S6 linker of domain II of Na_v1.8. In contrast, MrVIB had only a small effect on Na⁺ current for chimeras containing the corresponding region of Na_v1.2.

CONCLUSIONS AND IMPLICATIONS

Taken together, these results suggest that domain II of Na_v1.8 is an important determinant of MrVIB affinity, highlighting a region of the α-subunit that may allow further nociceptor-specific ligand targeting.     

HDAC inhibitors restore C-fibre sensitivity in experimental neuropathic pain model

Background and Purpose

Hypoesthesia is a clinical feature of neuropathic pain. The feature is partly explained by the evidence of epigenetic repression of Na_v1.8 sodium channel in the dorsal root ganglion (DRG).

Experimental Approach

We investigated the possibility of trichostatin A (TSA), valproic acid (VPA) and suberoylanilide hydroxamic acid (SAHA) to reverse the unique C-fibre sensitivity observed following partial ligation of sciatic nerve in mice.

Key Results

Nerve injury-induced down-regulation of DRG Na_v1.8 sodium channel and C-fibre-related hypoesthesia were reversed by TSA, VPA and SAHA treatments, which inhibit histone deacetylase (HDAC), and increase histone acetylation at the regulatory sequence of Na_v1.8.

Conclusions and Implications

Taken together, these studies provide the evidence that hypoesthesia and underlying down-regulation of Na_v1.8, negative symptoms observed in nerve injury-induced neuropathic pain models are regulated by an epigenetic chromatin remodelling through HDAC-related machineries.     

Rosiglitazone inhibits Kv4.3 potassium channels by open-channel block and acceleration of closed-state inactivation

BACKGROUND AND PURPOSE

Rosiglitazone is a widely used oral hypoglycaemic agent, which improves insulin resistance in type 2 diabetes. Chronic rosiglitazone treatment is associated with a number of adverse cardiac events. The present study was designed to characterize the effects of rosiglitazone on cloned K_v4.3 potassium channels.

EXPERIMENTAL APPROACH

The interaction of rosiglitazone with cloned K_v4.3 channels stably expressed in Chinese hamster ovary cells was investigated using whole-cell patch-clamp techniques.

KEY RESULTS

Rosiglitazone decreased the currents carried by K_v4.3 channels and accelerated the current inactivation, concentration-dependently, with an IC₅₀ of 24.5 µM. The association and dissociation rate constants for rosiglitazone were 1.22 µM⁻¹·s⁻¹ and 31.30 s⁻¹ respectively. Block by rosiglitazone was voltage-dependent, increasing in the voltage range for channel activation; however, no voltage dependence was found in the voltage range required for full activation. Rosiglitazone had no effect on either the deactivation kinetics or the steady-state activation of K_v4.3 channels. Rosiglitazone shifted the steady-state inactivation curves in the hyperpolarizing direction, concentration-dependently. The K_i for the interaction between rosiglitazone and the inactivated state of K_v4.3 channels was 1.49 µM, from the concentration-dependent shift in the steady-state inactivation curves. Rosiglitazone also accelerated the kinetics of the closed-state inactivation of K_v4.3 channels. Rosiglitazone did not affect either use dependence or recovery from inactivation of K_v4.3 currents.

CONCLUSION AND IMPLICATIONS

Our results indicate that rosiglitazone potently inhibits currents carried by K_v4.3 channels by interacting with these channels in the open state and by accelerating the closed-state inactivation of K_v4.3 channels.

LINKED ARTICLE

This article is commented on by Hancox, pp. 496–498 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2011.01281.x     

Hydrogen sulfide-induced mechanical hyperalgesia and allodynia require activation of both Cav3.2 and TRPA1 channels in mice

BACKGROUND AND PURPOSE

Hydrogen sulfide, a gasotransmitter, facilitates somatic pain signals via activation of Ca_v3.2 T-type calcium channels in rats. Given evidence for the activation of transient receptor potential ankyrin-1 (TRPA1) channels by H₂S, we asked whether TRPA1 channels, in addition to Ca_v3.2 channels, contribute to the H₂S-induced mechanical hyperalgesia and allodynia in mice.

EXPERIMENTAL APPROACH

Mechanical hyperalgesia and allodynia were evaluated by the von Frey test in mice. Ca_v3.2 or TRPA1 channels in the sensory neurons were silenced by repeated intrathecal administration of antisense oligodeoxynucleotides in mice.

KEY RESULTS

Intraplantar administration of NaHS evoked hyperalgesia and allodynia in mice, an effect attenuated or abolished by NNC 55–0396 or mibefradil, T-type calcium channel blockers, and by ascorbic acid or zinc chloride, known to selectively inhibit Ca_v3.2 channels, out of the three isoforms of T-type calcium channels. Silencing of Ca_v3.2 channels in the sensory neurons also prevented the NaHS-induced hyperalgesia and allodynia in mice. The NaHS-induced hyperalgesia and allodynia in mice were significantly suppressed by AP18, a TRPA1 channel blocker, and by silencing of TRPA1 channels in the sensory neurons.

CONCLUSIONS AND IMPLICATIONS

Mechanical hyperalgesia and allodynia induced by NaHS/H₂S required activation of both Ca_v3.2 and TRPA1 channels in mice.     

On the relationship between block of the cardiac Na⁺ channel and drug-induced prolongation of the QRS complex

BACKGROUND AND PURPOSE

Inhibition of the human cardiac Na⁺ channel (hNa_v1.5) can prolong the QRS complex and has been associated with increased mortality in patients with underlying cardiovascular disease. The safety implications of blocking hNa_v1.5 channels suggest the need to test for this activity early in drug discovery in order to design out any potential liability. However, interpretation of hNa_v1.5 blocking potency requires knowledge of how hNa_v1.5 block translates into prolongation of the QRS complex.

EXPERIMENTAL APPROACH

We tested Class I anti-arrhythmics, other known QRS prolonging drugs and drugs not reported to prolong the QRS complex. Their block of hNa_v1.5 channels (as IC₅₀ values) was measured in an automated electrophysiology-based assay. These IC₅₀ values were compared with published reports of the corresponding unbound (free) plasma concentrations attained during clinical use (fC_max) to provide an IC₅₀ : fC_max ratio.

KEY RESULTS

For 42 Class I anti-arrhythmics and other QRS prolonging drugs, 67% had IC₅₀ : fC_max ratios <30. For 55 non-QRS prolonging drugs tested, 72% had ratios >100. Finally, we determined the relationship between the IC₅₀ value and the free drug concentration associated with prolongation of the QRS complex in humans. For 37 drugs, QRS complex prolongation was observed at free plasma concentrations that were about 15-fold lower than the corresponding IC₅₀ at hNa_v1.5 channels.

CONCLUSIONS AND IMPLICATIONS

A margin of 30- to 100-fold between hNa_v1.5 IC₅₀ and fC_max appears to confer an acceptable degree of safety from QRS prolongation. QRS prolongation occurs on average at free plasma levels 15-fold below the IC₅₀ at hNa_v1.5 channels.

LINKED ARTICLE

This article is commented on by Gintant et al., pp. 254–259 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2011.01433.x     

F 15845, a new blocker of the persistent sodium current prevents consequences of hypoxia in rat femoral artery

Background and purpose:

Selective cyclooxygenase-2 (COX-2) inhibitors such as rofecoxib (Vioxx) and celecoxib (Celebrex) were developed as NSAIDs with reduced gastric side effects. Celecoxib has now been shown to affect cellular physiology via an unexpected, COX-independent, pathway – by inhibiting K_v2.1 and other ion channels. In this study, we investigated the mechanism of the action of celecoxib on K_v2.1 channels.

Experimental approach:

The mode of action of celecoxib on rat K_v2.1 channels was studied by whole-cell patch-clamping to record currents from channels expressed in HEK-293 cells.

Key results:

Celecoxib reduced current through K_v2.1 channels when applied from the extracellular side. At low concentrations (≤3 µM), celecoxib accelerated kinetics of activation, deactivation and inactivation. Recovery of rat K_v2.1 channels from inactivation could be characterized by two components, with celecoxib selectively accelerating the slow component of recovery at ≤10 µM. At >3 µM, celecoxib led to closed-channel block with relative slowing of activation. At 30 µM, it additionally induced open-channel block that manifested in use-dependent inhibition and slower recovery from inactivation.

Conclusions and implications:

Celecoxib reduced current through K_v2.1 channels by modifying gating and inducing closed- and open-channel block, with the three effects manifesting at different concentrations. These data will help to elucidate the mechanisms of action of this widely prescribed drug on ion channels and those underlying its neurological, cardiovascular and other effects.     

Treatment with the Kv7 potassium channel activator flupirtine is beneficial in two independent mouse models of pulmonary hypertension

Background and purpose:

Voltage-gated potassium (K_v) channels contribute to resting membrane potential in pulmonary artery smooth muscle cells and are down regulated in patients with pulmonary arterial hypertension (PAH) and a contribution from K_v7 channels has been recently proposed. We investigated the effect of the K_v7 channel activator, flupirtine, on PAH in two independent mouse models: PAH induced by hypoxia and spontaneous PAH in mice over-expressing the 5-HT transporter (SERT⁺ mice).

Experimental approach:

Right ventricular pressure was assessed in vivo in mice chronically treated with flupirtine (30 mg·kg⁻¹·day⁻¹). In separate in vitro experiments, pulmonary arteries from untreated mice were mounted in a wire myograph. Relaxations to acute administration of flupirtine and contractions to K_v channel blocking drugs, including the K_v7 channel blocker linopirdine, were measured.

Key results:

In wild-type (WT) mice, hypoxia increased right ventricular pressure, pulmonary vascular remodelling and right ventricular hypertrophy. These effects were attenuated by flupirtine, which also attenuated these indices of PAH in SERT⁺ mice. In the in vitro experiments, flupirtine induced a potent relaxant response in arteries from untreated WT and SERT⁺ mice. The relaxation was fully reversed by linopirdine, which potently contracted mouse pulmonary arteries while other K_v channel blockers did not.

Conclusions and implications:

Flupirtine significantly attenuated development of chronic hypoxia-induced PAH in mice and reversed established PAH in SERT⁺ mice, apparently via K_v7 channel activation. These results provide the first direct evidence that drugs activating K_v7 channels may be of benefit in the treatment of PAH with different aetiologies.     

Addition of a single methyl group to a small molecule sodium channel inhibitor introduces a new mode of gating modulation

Background and Purpose

Aryl sulfonamide Na_v1.3 or Na_v1.7 voltage‐gated sodium (Na_v) channel inhibitors interact with the Domain 4 voltage sensor domain (D4 VSD). During studies to better understand the structure‐activity relationship of this interaction, an additional mode of channel modulation, specifically slowing of inactivation, was revealed by addition of a single methyl moiety. The objective of the current study was to determine if these different modulatory effects are mediated by the same or distinct interactions with the channel.

Experimental Approach

Electrophysiology and site‐directed mutation were used to compare the effects of PF‐06526290 and its desmethyl analogue PF‐05661014 on Na_v channel function.

Key Results

PF‐05661014 selectively inhibits Na_v1.3 versus Na_v1.7 currents by stabilizing inactivated channels via interaction with D4 VSD. In contrast, PF‐06526290, which differs from PF‐05661014 by a single methyl group, exhibits a dual effect. It greatly slows inactivation of Na_v channels in a subtype‐independent manner. However, upon prolonged depolarization to induce inactivation, PF‐06526290 becomes a Na_v subtype selective inhibitor similar to PF‐05661014. Mutation of the D4 VSD modulates inhibition of Na_v1.3 or Na_v1.7 by both PF‐05661014 and PF‐06526290, but has no effect on the inactivation slowing produced by PF‐06526290. This finding, along with the absence of functional inhibition of PF‐06526290‐induced inactivation slowing by PF‐05661014, suggests that distinct interactions underlie the two modes of Na_v channel modulation.

Conclusions and Implications

Addition of a methyl group to a Na_v channel inhibitor introduces an additional mode of gating modulation, implying that a single compound can affect sodium channel function in multiple ways.

Abbreviations

VSD

(voltage sensor domain)

PF‐05661014

(4‐(3‐benzylureido)‐N‐(thiazol‐2‐yl) benzenesulfonamide)

PF‐06526290

(4‐(3‐benzyl‐3‐methylureido)‐N‐(thiazol‐2‐yl) benzenesulfonamide)

DRG

(dorsal root ganglion)

     

Characteristics and molecular basis of celecoxib modulation on K(v)7 potassium channels

BACKGROUND AND PURPOSE

Celecoxib is a selective cyclooxygenase-2 (COX-2) inhibitor used for the treatment of pain and inflammation. Emerging and accumulating evidence suggests that celecoxib can affect cellular targets other than COX, such as ion channels. In this study, we characterized the effects of celecoxib on K_v7 K⁺ channels and compared its effects with the well-established K_v7 channel opener retigabine.

EXPERIMENTAL APPROACH

A perforated whole-cell patch technique was used to record K_v7currents expressed in HEK 293 cells and M-type currents from rat superior cervical ganglion neurons.

KEY RESULTS

Celecoxib enhanced K_v7.2–7.4, K_v7.2/7.3 and K_v7.3/7.5 currents but inhibited K_v7.1 and K_v7.1/KCNE1 currents and these effects were concentration dependent. The IC₅₀ value for inhibition of K_v7.1 channels was approximately 4 µM and the EC₅₀ values for activation of K_v7.2–7.4, K_v7.2/K_v7.3 and K_v7.3/K_v7.5 channels were approximately 2–5 µM. The effects of celecoxib were manifested by increasing current amplitudes, shifting the voltage-dependent activation curve in a more negative direction and slowing the deactivation of K_v7 currents. 2,5-Dimethyl-celecoxib, a celecoxib analogue devoid of COX inhibition activity, has similar but greater effects on K_v7currents. K_v7.2(A235T) and K_v7.2(W236L) mutant channels, which have greatly attenuated responses to retigabine, showed a reversed response to celecoxib, from activation to inhibition.

CONCLUSIONS AND IMPLICATIONS

These results suggest that K_v7 channels are targets of celecoxib action and provide new mechanistic evidence for understanding the effects of celecoxib. They also provide a new approach to developing K_v7 modulators and for studying the structure–function relationship of K_v7 channels.     

Protein kinase C-independent inhibition of arterial smooth muscle K(+) channels by a diacylglycerol analogue

BACKGROUND AND PURPOSE

Analogues of the endogenous diacylglycerols have been used extensively as pharmacological activators of protein kinase C (PKC). Several reports show that some of these compounds have additional effects that are independent of PKC activation, including direct block of K⁺ and Ca²⁺ channels. We investigated whether dioctanoyl-sn-glycerol (DiC8), a commonly used diacylglycerol analogue, blocks K⁺ currents of rat mesenteric arterial smooth muscle in a PKC-independent manner.

EXPERIMENTAL APPROACH

Conventional whole-cell and inside-out patch clamp was used to measure the inhibition of K⁺ currents of rat isolated mesenteric smooth muscle cells by DiC8 in the absence and presence of PKC inhibitor peptide.

KEY RESULTS

Mesenteric artery smooth muscle K_v currents inactivated very slowly with a time constant of about 2 s following pulses from −65 to +40 mV. Application of 1 µM DiC8 produced an approximate 40-fold increase in the apparent rate of inactivation. Pretreatment of the cells with PKC inhibitor peptide had a minimal effect on the action of DiC8, and substantial inactivation still occurred, indicating that this effect was mainly independent of PKC. We also found that DiC8 blocked BK and K_ATP currents, and again a significant proportion of these blocks occurred independently of PKC activation.

CONCLUSIONS AND IMPLICATIONS

These results show that DiC8 has a direct effect on arterial smooth muscle K⁺ channels, and this precludes its use as a PKC activator when investigating PKC-mediated effects on vascular K⁺ channels.     

Expression and function of the K+ channel KCNQ genes in human arteries

BACKGROUND AND PURPOSE

KCNQ-encoded voltage-gated potassium channels (K_v7) have recently been identified as important anti-constrictor elements in rodent blood vessels but the role of these channels and the effects of their modulation in human arteries remain unknown. Here, we have assessed KCNQ gene expression and function in human arteries ex vivo.

EXPERIMENTAL APPROACH

Fifty arteries (41 from visceral adipose tissue, 9 mesenteric arteries) were obtained from subjects undergoing elective surgery. Quantitative RT-PCR experiments using primers specific for all known KCNQ genes and immunohistochemsitry were used to show K_v7 channel expression. Wire myography and single cell electrophysiology assessed the function of these channels.

KEY RESULTS

KCNQ4 was expressed in all arteries assessed, with variable contributions from KCNQ1, 3 and 5. KCNQ2 was not detected. K_v7 channel isoform-dependent staining was revealed in the smooth muscle layer. In functional studies, the K_v7 channel blockers, XE991 and linopirdine increased isometric tension and inhibited K⁺ currents. In contrast, the K_v7.1-specific blocker chromanol 293B did not affect vascular tone. Two K_v7 channel activators, retigabine and acrylamide S-1, relaxed preconstricted arteries, actions reversed by XE991. K_v7 channel activators also suppressed spontaneous contractile activity in seven arteries, reversible by XE991.

CONCLUSIONS AND IMPLICATIONS

This is the first study to demonstrate not only the presence of KCNQ gene products in human arteries but also their contribution to vascular tone ex vivo.

LINKED ARTICLE

This article is commented on by Mani and Byron, pp. 38–41 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2010.01065.x     

Effect of verapamil on the action of methanethiosulfonate reagents on human voltage-gated K(v)1.3 channels: implications for the C-type inactivated state

BACKGROUND AND PURPOSE

Voltage-gated K_v1.3 channels appear on T-lymphocytes and are characterized by their typical C-type inactivation. In order to develop drugs stabilizing the C-type inactivated state and thus potentially useful in treatment of autoimmune diseases, it is important to know more about the three-dimensional structure of this inactivated state of the channel.

EXPERIMENTAL APPROACH

The patch-clamp technique was used to study effects of methanethiosulphonate (MTS) compounds on currents through wild-type human K_v1.3 (hK_v1.3) and two mutant channels, hK_v1.3 V417C and hK_v1.3 H399T-V417C, in the closed, open and inactivated states.

KEY RESULTS

Extracellular application of 2-aminoethyl methanethiosulphonate (MTSEA) irreversibly reduced currents through hK_v1.3 V417C channels in the open and inactivated, but not in the closed state, indicating that a modification was possible. Co-application of verapamil prevented this reduction. Intracellular application of MTSEA and [2-(trimethylammonium)ethyl] methanethiosulphonate (MTSET) also modified the mutant channels, whereas extra- and intracellular application of sodium (2-sulfonatoethyl)methanethiosulphonate (MTSES) and intracellular application of MTSET did not.

CONCLUSIONS AND IMPLICATIONS

Our experiments showed that the binding site for MTS compounds was intracellular in the mutant channels and that the V417C mutant channels were modified in the open and the inactivated states, and this modification was prevented by verapamil. Therefore, the activation gate on the intracellular side of the selectivity filter must be open during inactivation. Furthermore, although the S6 segment is moving further apart during inactivation, this change does not include a movement of the side chain of the amino acid at position 417, away from lining the channel pore.     

The flavonoid scaffold as a template for the design of modulators of the vascular Ca(v) 1.2 channels

BACKGROUND AND PURPOSE

Previous studies have pointed to the plant flavonoids myricetin and quercetin as two structurally related stimulators of vascular Ca_v1.2 channel current (I_Ca1.2). Here we have tested the proposition that the flavonoid structure confers the ability to modulate Ca_v1.2 channels.

EXPERIMENTAL APPROACH

Twenty-four flavonoids were analysed for their effects on I_Ca1.2 in rat tail artery myocytes, using the whole-cell patch-clamp method.

KEY RESULTS

Most of the flavonoids stimulated or inhibited I_Ca1.2 in a concentration- and voltage-dependent manner with EC₅₀ values ranging between 4.4 µM (kaempferol) and 16.0 µM (myricetin) for the stimulators and IC₅₀ values between 13.4 µM (galangin) and 100 µM [(±)-naringenin] for the inhibitors. Key structural requirements for I_Ca1.2 stimulatory activity were the double bond between C2 and C3 and the hydroxylation pattern on the flavonoid scaffold, the latter also determining the molecular charge, as shown by molecular modelling techniques. Absence of OH groups in the B ring was key in I_Ca1.2 inhibition. The functional interaction between quercetin and either the stimulator myricetin or the antagonists resokaempferol, crysin, genistein, and 5,7,2′-trihydroxyflavone revealed that quercetin expressed the highest apparent affinity, in the low µM range, for Ca_v1.2 channels. Neither protein tyrosine kinase nor protein kinase Cα were involved in quercetin-induced stimulation of I_Ca1.2.

CONCLUSIONS AND IMPLICATIONS

Quercetin-like plant flavonoids were active on vascular Ca_v1.2 channels. Thus, the flavonoid scaffold may be a template for the design of novel modulators of vascular smooth muscle Ca_v1.2 channels, valuable for the treatment of hypertension and stroke.     

Verapamil- and state-dependent effect of 2-aminoethylmethanethiosulphonate (MTSEA) on hKv1.3 channels

BACKGROUND AND PURPOSE

T-cells usually express voltage-gated K_v1.3 channels. These channels are distinguished by their typical C-type inactivation. Therefore, to be able to rationally design drugs specific for the C-type inactivation state that may have therapeutic value in autoimmune disease therapy, it is necessary to identify those amino acids that are accessible for drug binding in C-type inactivated channels.

EXPERIMENTAL APPROACH

The influence of 2-aminoethylmethanethiosulphonate (MTSEA) on currents through wild-type human K_v1.3 (hK_v1.3) and three mutant channels, hK_v1.3_L418C, hK_v1.3_T419C and hK_v1.3_I420C, in the closed, open and inactivated states was investigated by the patch-clamp technique.

KEY RESULTS

Currents through hK_v1.3_L418C and hK_v1.3_T419C channels were irreversibly reduced after the external application of MTSEA in the open state but not in the inactivated and closed states. Currents through hK_v1.3_I420C channels were irreversibly reduced in the open and inactivated states but not in the closed state. In the presence of verapamil, the MTSEA modification of hK_v1.3_T419C and hK_v1.3_I420C channels was prevented, while the MTSEA modification of hK_v1.3_L418C channels was unaffected.

CONCLUSION AND IMPLICATIONS

From our experiments, we conclude that the activation gate of all mutant channels must be open for modification by MTSEA and must also be open during inactivation. In addition, the relative movement of the S6 segments that occur during C-type inactivation includes a movement of the side chains of the amino acids at positions 418 and 419 away from the pore lining. Furthermore, the overlapping binding site for MTSEA and verapamil does not include position 418 in hK_v1.3 channels.     

Concomitant facilitation of GABAA receptors and KV7 channels by the non-opioid analgesic flupirtine

BACKGROUND AND PURPOSE

Flupirtine is a non-opioid analgesic that has been in clinical use for more than 20 years. It is characterized as a selective neuronal potassium channel opener (SNEPCO). Nevertheless, its mechanisms of action remain controversial and are the purpose of this study.

EXPERIMENTAL APPROACH

Effects of flupirtine on native and recombinant voltage- and ligand-gated ion channels were explored in patch-clamp experiments using the following experimental systems: recombinant K_IR3 and K_V7 channels and α3β4 nicotinic acetylcholine receptors expressed in tsA 201 cells; native voltage-gated Na⁺, Ca²⁺, inward rectifier K⁺, K_V7 K⁺, and TRPV1 channels, as well as GABA_A, glycine, and ionotropic glutamate receptors expressed in rat dorsal root ganglion, dorsal horn and hippocampal neurons.

KEY RESULTS

Therapeutic flupirtine concentrations (≤10 µM) did not affect voltage-gated Na⁺ or Ca²⁺ channels, inward rectifier K⁺ channels, nicotinic acetylcholine receptors, glycine or ionotropic glutamate receptors. Flupirtine shifted the gating of K_V7 K⁺ channels to more negative potentials and the gating of GABA_A receptors to lower GABA concentrations. These latter effects were more pronounced in dorsal root ganglion and dorsal horn neurons than in hippocampal neurons. In dorsal root ganglion and dorsal horn neurons, the facilitatory effect of therapeutic flupirtine concentrations on K_V7 channels and GABA_A receptors was comparable, whereas in hippocampal neurons the effects on K_V7 channels were more pronounced.

CONCLUSIONS AND IMPLICATIONS

These results indicate that flupirtine exerts its analgesic action by acting on both GABA_A receptors and K_V7 channels.     

Topical application of disodium isostearyl 2-O-L-ascorbyl phosphate, an amphiphilic ascorbic acid derivative, reduces neuropathic hyperalgesia in rats

BACKGROUND AND PURPOSE

Ca_v3.2 T-type calcium channels, targeted by H₂S, are involved in neuropathic hyperalgesia in rats and ascorbic acid inhibits Ca_v3.2 channels. Therefore, we evaluated the effects of intraplantar (i.pl.) administration of ascorbic acid or topical application of disodium isostearyl 2-O-L-ascorbyl phosphate (DI-VCP), a skin-permeable ascorbate derivative on hyperalgesia induced by NaHS, an H₂S donor, and on neuropathic hyperalgesia.

EXPERIMENTAL APPROACH

In rats mechanical hyperalgesia was evoked by i.pl. NaHS, and neuropathic hyperalgesia was induced by L5 spinal nerve cutting (L5SNC) or by repeated administration of paclitaxel, an anti-cancer drug. Dermal ascorbic acid levels were determined colorimetrically.

KEY RESULTS

The NaHS-evoked Ca_v3.2 channel-dependent hyperalgesia was inhibited by co-administered ascorbic acid. Topical application of DI-VCP, but not ascorbic acid, prevented the NaHS-evoked hyperalgesia, and also increased dermal ascorbic acid levels. Neuropathic hyperalgesia induced by L5SNC or paclitaxel was reversed by i.pl. NNC 55–0396, a selective T-type calcium channel blocker, ascorbic acid or DI-VCP, and by topical DI-VCP, but not by topical ascorbic acid. The effects of i.pl. ascorbic acid and topical DI-VCP in the paclitaxel-treated rats were characterized by the faster onset and greater magnitude, compared with their effects in the L5SNC rats. Dermal ascorbic acid levels in the hindpaw significantly decreased after paclitaxel treatment, but not L5SNC, which was reversed by topical DI-VCP.

CONCLUSIONS AND IMPLICATIONS

Ascorbic acid, known to inhibit Ca_v3.2 channels, suppressed neuropathic hyperalgesia. DI-VCP ointment for topical application may be of benefit in the treatment of neuropathic pain.