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.     

A Ca²⁺-dependent chloride current and Ca²⁺ influx via Ca(v)1.2 ion channels play major roles in P2Y receptor-mediated pulmonary vasoconstriction

BACKGROUND AND PURPOSE

ATP, UTP and UDP act at smooth muscle P2X and P2Y receptors to constrict rat intrapulmonary arteries, but the underlying signalling pathways are poorly understood. Here, we determined the roles of the Ca²⁺-dependent chloride ion current (I_Cl,Ca), Ca_v1.2 ion channels and Ca²⁺ influx.

EXPERIMENTAL APPROACH

Isometric tension was recorded from endothelium-denuded rat intrapulmonary artery rings (i.d. 200–500 µm) mounted on a wire myograph.

KEY RESULTS

The I_Cl,Ca blockers, niflumic acid and 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid and the Ca_v1.2 channel blocker, nifedipine, reduced peak amplitude of contractions evoked by UTP and UDP by ∼45–50% and in a non-additive manner. Ca²⁺-free buffer inhibited responses by ∼70%. Niflumic acid and nifedipine similarly depressed contractions to ATP, but Ca²⁺-free buffer almost abolished the response. After peaking, contractions to UTP and UDP decayed slowly by 50–70% to a sustained plateau, which was rapidly inhibited by niflumic acid and nifedipine. Contractions to ATP, however, reversed rapidly and fully. Tannic acid contracted tissues per se and potentiated nucleotide-evoked contractions.

CONCLUSIONS AND IMPLICATIONS

I_Cl,Ca and Ca²⁺ influx via Ca_v1.2 ion channels contribute substantially and equally to contractions of rat intrapulmonary arteries evoked by UTP and UDP, via P2Y receptors. ATP also activates these mechanisms via P2Y receptors, but the greater dependence on extracellular Ca²⁺ most likely reflects additional influx through the P2X1 receptor pore. The lack of a sustained response to ATP is probably due to it acting at P2 receptor subtypes that desensitize rapidly. Thus multiple signalling mechanisms contribute to pulmonary artery vasoconstriction mediated by P2 receptors.     

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.     

Inhibition of vascular calcium-gated chloride currents by blockers of KCa1.1, but not by modulators of KCa2.1 or KCa2.3 channels

Background and purpose:

Recent pharmacological studies have proposed there is a high degree of similarity between calcium-activated Cl⁻ channels (CaCCs) and large conductance, calcium-gated K⁺ channels (K_Ca1.1). The goal of the present study was to ascertain whether blockers of K_Ca1.1 inhibited calcium-activated Cl⁻ currents (I_ClCa) and if the pharmacological overlap between K_Ca1.1 and CaCCs extends to intermediate and small conductance, calcium-activated K⁺ channels.

Experimental approaches:

Whole-cell Cl⁻ and K⁺ currents were recorded from murine portal vein myocytes using the whole-cell variant of the patch clamp technique. CaCC currents were evoked by pipette solutions containing 500 nM free [Ca²⁺].

Key results:

The selective K_Ca1.1 blocker paxilline (1 µM) inhibited I_ClCa by ∼90%, whereas penitrem A (1 µM) and iberiotoxin (100 and 300 nM) reduced the amplitude of I_ClCa by ∼20%, as well as slowing channel deactivation. Paxilline also abolished the stimulatory effect of niflumic acid on the CaCC. In contrast, an antibody against the Ca²⁺-binding domain of murine K_Ca1.1 had no effect on I_ClCa while inhibiting spontaneous K_Ca1.1 currents. Structurally different modulators of small and intermediate conductance calcium-activated K⁺ channels (K_Ca2.1 and K_Ca2.3), namely 1-EBIO, (100 µM); NS309, (1 µM); TRAM-34, (10 µM); UCL 1684, (1 µM) had no effect on I_ClCa.

Conclusions and implications:

These data show that the selective K_Ca1.1 blockers also reduce I_ClCa considerably. However, the pharmacological overlap that exists between CaCCs and K_Ca1.1 does not extend to the calcium-binding domain or to other calcium-gated K⁺ channels.     

Modulation of drug block of the cardiac potassium channel KCNA5 by the drug transporters OCTN1 and MDR1

BACKGROUND AND PURPOSE

A common site for drug binding on voltage-gated ion channels is at the interior face of the channel pore. In this study, we tested the hypothesis that the extent of drug block of the human cardiac KCNA5 (K_v1.5) channel underlying the atrial-specific, ultra-rapidly activating, delayed K⁺ current (I_Kur) is modulated by the drug uptake and efflux transporters encoded by organic cation transporter 1 (OCTN1) and multiple drug-resistant gene 1 (MDR1) and expressed in human heart.

EXPERIMENTAL APPROACH

Drug block of KCNA5 was assessed in Chinese hamster ovary cells transiently transfected with KCNA5 alone or in combination with the OCTN1 or MDR1 transporter construct, as well as in an MDR1 stably expressed cell line.

KEY RESUTLS

Co-expression of OCTN1 significantly facilitated block by quinidine (10 µM), verapamil (20 µM), propafenone (5 µM) and clofilium (30 µM). Further evidence of drug transport modulating drug block was the finding that with OCTN1, block developed faster and only partially washed-out, and that block potentiation was prevented by cimetidine, an inhibitor of OCTN1. MDR1 expression attenuated KCNA5 block by erythromycin (an MDR1 substrate). Block was restored by reversin-205 (10 µM, an MDR1 inhibitor). MDR1 did not affect KCNA5 inhibition by KN-93 (1 µM), a blocker acting on the outer mouth of the channel pore.

CONCLUSIONS AND IMPLICATIONS

The extent of drug block of KCNA5 can be modulated by drug uptake and efflux transporters. These data provide further support for the idea that modifying intracellular drug concentrations could modulate the effects of blocking ion channels in patients.     

Pentobarbital inhibition of human recombinant ��1A P/Q-type voltage-gated calcium channels involves slow, open channel block

BACKGROUND AND PURPOSE

Pre-synaptic neurotransmitter release is largely dependent on Ca²⁺ entry through P/Q-type (Ca_V2.1) voltage-gated Ca²⁺ channels (PQCCs) at most mammalian, central, fast synapses. Barbiturates are clinical depressants and inhibit pre-synaptic Ca²⁺ entry. PQCC barbiturate pharmacology is generally unclear, specifically in man. The pharmacology of the barbiturate pentobarbital (PB) in human recombinant α_1A PQCCs has been characterized.

EXPERIMENTAL APPROACH

PB effects on macroscopic Ca²⁺(I_Ca) and Ba²⁺(I_Ba) currents were studied using whole-cell patch clamp recording in HEK-293 cells heterologously expressing (α_1A)_human(β_2aα₂δ-1)_rabbit PQCCs.

KEY RESULTS

PB reversibly depressed peak current (I_peak) and enhanced apparent inactivation (fractional current at 800 ms, r₈₀₀) in a concentration-dependent fashion irrespective of charge carrier (50% inhibitory concentration: I_peak, 656 µM; r₈₀₀, 104 µM). Rate of mono-exponential I_Ba decay was linearly dependent on PB concentration. PB reduced channel availability by deepening non-steady-state inactivation curves without altering voltage dependence, slowed recovery from activity-induced unavailable states and produced use-dependent block. PB (100 µM) induced use-dependent block during physiological, high frequency pulse trains and overall depressed PQCC activity by two-fold.

CONCLUSION AND IMPLICATIONS

The results support a PB pharmacological mechanism involving a modulated receptor with preferential slow, bimolecular, open channel block (K_d = 15 µM). Clinical PB concentrations (<200 µM) inhibit PQCC during high frequency activation that reduces computed neurotransmitter release by 16-fold and is comparable to the magnitude of Ca²⁺-dependent facilitation, G-protein modulation and intrinsic inactivation that play critical roles in PQCC modulation underlying synaptic plasticity. The results are consistent with the hypothesis that PB inhibition of PQCCs contributes to central nervous system depression underlying anticonvulsant therapy and general anaesthesia.     

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     

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.     

Quercetin and its principal metabolites, but not myricetin, oppose lipopolysaccharide-induced hyporesponsiveness of the porcine isolated coronary artery

BACKGROUND AND PURPOSE

Quercetin is anti-inflammatory in macrophages by inhibiting lipopolysaccharide (LPS)-mediated increases in cytokine and nitric oxide production but there is little information regarding the corresponding effect on the vasculature. We have examined the effect of quercetin, and its principal human metabolites, on inflammatory changes in the porcine isolated coronary artery.

EXPERIMENTAL APPROACH

Porcine coronary artery segments were incubated overnight at 37°C in modified Krebs-Henseleit solution with or without 1 µg·mL⁻¹ LPS. Some segments were also co-incubated with quercetin-related flavonoids or Bay 11-7082, an inhibitor of NFκB. Changes in isometric tension of segments to vasoconstrictor and vasodilator agents were recorded. Nitrite content of the incubation solution was estimated using the Griess reaction, while inducible nitric oxide synthase was identified immunohistochemically.

KEY RESULTS

Lipopolysaccharide reduced, by 35–50%, maximal contractions to KCl and U46619, thromboxane A₂ receptor agonist, and impaired endothelium-dependent relaxations to substance P. Nitrite content of the incubation medium increased 3- to 10-fold following exposure to LPS and inducible nitric oxide synthase was detected in the adventitia. Quercetin (0.1–10 µM) opposed LPS-induced changes in vascular responses, nitrite production and expression of inducible nitric oxide synthase. Similarly, 10 µM Bay 11-7082, 10 µM quercetin 3′-sulphate and 10 µM quercetin 3-glucuronide prevented LPS-induced changes, while myricetin (10 µM) was inactive. Myricetin (10 µM) prevented quercetin-induced modulation of LPS-mediated nitrite production.

CONCLUSION AND IMPLICATIONS

Quercetin, quercetin 3′-suphate and quercetin 3-glucuronide, exerted anti-inflammatory effects on the vasculature, possibly through a mechanism involving inhibition of NFκB. Myricetin-induced antagonism of the effect of anti-inflammatory action of quercetin merits further investigation.     

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.     

Interaction of diltiazem with an intracellularly accessible binding site on Ca(V)1.2

BACKGROUND AND PURPOSE

Diltiazem inhibits Ca_V1.2 channels and is widely used in clinical practice to treat cardiovascular diseases. Binding determinants for diltiazem are located on segments IIIS6, IVS6 and the selectivity filter of the pore forming α₁ subunit of Ca_V1.2. The aim of the present study was to clarify the location of the diltiazem binding site making use of its membrane-impermeable quaternary derivative d-cis-diltiazem (qDil) and mutant α₁ subunits.

EXPERIMENTAL APPROACH

Ca_V1.2 composed of α1, α2-δ and β2a subunits were expressed in tsA-201 cells and barium currents through Ca_V1.2 channels were recorded using the patch clamp method in the whole cell configuration. qDil was synthesized and applied to the intracellular side (via the patch pipette) or to the extracellular side of the membrane (by bath perfusion).

KEY RESULTS

Quaternary derivative d-cis-diltiazem inhibited Ca_V1.2 when applied to the intracellular side of the membrane in a use-dependent manner (59 ± 4% at 300 µM) and induced only a low level of tonic (non-use-dependent) block (16 ± 2% at 300 µM) when applied to the extracellular side of the membrane. Mutations in IIIS6 and IVS6 that have previously been shown to reduce the sensitivity of Ca_V1.2 to tertiary diltiazem also had reduced sensitivity to intracellularly applied qDil.

CONCLUSION AND IMPLICATIONS

The data show that use-dependent block of in Ca_V1.2 by diltiazem occurs by interaction with a binding site accessible via a hydrophilic route from the intracellular side of the membrane.     

Effect of the Ito activator NS5806 on cloned Kv4 channels depends on the accessory protein KChIP2

BACKGROUND AND PURPOSE

The compound NS5806 increases the transient outward current (I_to) in canine ventricular cardiomyocytes and slows current decay. In human and canine ventricle, I_to is thought to be mediated by K_V4.3 and various ancillary proteins, yet, the exact subunit composition of I_to channels is still debated. Here we characterize the effect of NS5806 on heterologously expressed putative I_to channel subunits and other potassium channels.

EXPERIMENTAL APPROACH

Cloned K_V4 channels were co-expressed with KChIP2, DPP6, DPP10, KCNE2, KCNE3 and K_V1.4 in Xenopus laevis oocytes or CHO-K1 cells.

KEY RESULTS

NS5806 increased K_V4.3/KChIP2 peak current amplitudes with an EC₅₀ of 5.3 ± 1.5µM and significantly slowed current decay. KCNE2, KCNE3, DPP6 and DPP10 modulated K_V4.3 currents and the response to NS5806, but current decay was slowed only in complexes containing KChIP2. The effect of NS5806 on K_V4.2 was similar to that on K_V4.3, and current decay was only slowed in presence of KChIP2. However, for K_V4.1, the slowing of current decay by NS5806 was independent of KChIP2. K_V1.4 was strongly inhibited by 10 µM NS5806 and K_V1.5 was inhibited to a smaller extent. Effects of NS5806 on kinetics of currents generated by K_V4.3/KChIP2/DPP6 with K_V1.4 in oocytes could reproduce those on cardiac I_to in canine ventricular myocytes. K_V7.1, K_V11.1 and K_ir2 currents were unaffected by NS5806.

CONCLUSION AND IMPLICATIONS

NS5806 modulated K_V4 channel gating depending on the presence of KChIP2, suggesting that NS5806 can potentially be used to address the molecular composition as well as the physiological role of cardiac I_to.     

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.     

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.     

Alternative splicing modulates diltiazem sensitivity of cardiac and vascular smooth muscle Cav1.2 calcium channels

Background and purpose:

As a calcium channel blocker, diltiazem acts mainly on the voltage-gated calcium channels, Ca_v1.2, for its beneficial effects in cardiovascular diseases such as hypertension, angina and/or supraventricular arrhythmias. However, the effects of diltiazem on different isoforms of Ca_v1.2 channels expressed in heart and vascular smooth muscles remain to be investigated. Here, we characterized the effects of diltiazem on the splice variants of Ca_v1.2 channels, predominant in cardiac and vascular smooth muscles.

Experimental approach:

Cardiac and smooth muscle isoforms of Ca_v1.2 channels were expressed in human embryonic kidney cells and their electrophysiological properties were characterized using whole-cell patch-clamp techniques.

Key results:

Under closed-channel and use-dependent block (0.03 Hz), cardiac splice variant Ca_v1.2CM was less sensitive to diltiazem than two major smooth muscle splice variants, Ca_v1.2SM and Ca_v1.2b. Ca_v1.2CM has a more positive half-inactivation potential than the smooth muscle channels, and diltiazem shifted it less to negative potential. Additionally, the current decay was slower in Ca_v1.2CM channels. When we modified alternatively spliced exons of cardiac Ca_v1.2CM channels into smooth muscle exons, we found that all three loci contribute to the different diltiazem sensitivity between cardiac and smooth muscle splice isoforms.

Conclusions and implications:

Alternative splicing of Ca_v1.2 channels modifies diltiazem sensitivity in the heart and blood vessels. Gating properties altered by diltiazem are different in the three channels.     

Openers of small conductance calcium-activated potassium channels selectively enhance NO-mediated bradykinin vasodilatation in porcine retinal arterioles

Background and purpose:

Small (SK_Ca or K_Ca2) and intermediate (IK_Ca or K_Ca3.1) conductance calcium-activated potassium channels are involved in regulation of vascular tone and blood pressure. The present study investigated whether NS309 (6,7-dichloro-1H-indole-2,3-dione 3-oxime) and CyPPA (cyclohexyl-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-pyrimidin-4-yl]-amine), which are selective openers of SK_Ca and IK_Ca channels and of SK_Ca2 and SK_Ca3 channels, respectively, enhance endothelium-dependent vasodilatation in porcine retinal arterioles.

Experimental approach:

In porcine retinal arterioles, SK_Ca3 and IK_Ca protein localization was examined by immunolabelling. Endothelial cell calcium was measured by fluorescence imaging. For functional studies, arterioles with internal diameters of 116 ± 2 µm (n = 276) were mounted in microvascular myographs for isometric tension recordings.

Key results:

SK_Ca3 and IK_Ca protein was localized in the endothelium. Bradykinin, but not NS309 or CyPPA increased endothelial cell calcium. Pre-incubation with NS309 or CyPPA enhanced bradykinin relaxation without changing endothelial cell calcium. This enhanced relaxation was abolished by blocking SK_Ca channels with apamin. In the presence of NS309 or CyPPA, mainly inhibition of NO synthase with asymmetric dimethylarginine, but also inhibition of cyclooxygenase with indomethacin, reduced bradykinin relaxation. Bradykinin relaxation was completely abolished by NO synthase and cyclooxygenase inhibition together with a NO scavenger, oxyhaemoglobin.

Conclusions and implications:

In porcine retinal arterioles, bradykinin increases endothelial cell calcium leading to activation of SK_Ca and IK_Ca channels. Without altering endothelial cell calcium, NS309 and CyPPA open SK_Ca channels that enhance NO-mediated bradykinin relaxations. These results imply that opening SK_Ca channels improves endothelium-dependent relaxation and makes this channel a potential target for treatments aimed at restoring retinal blood flow.     

Phosphorylation alters the pharmacology of Ca2+-activated Cl? channels in rabbit pulmonary arterial smooth muscle cells

Background and purpose:

Ca²⁺-activated Cl⁻ currents (I_Cl(Ca)) in arterial smooth muscle cells are inhibited by phosphorylation. The Ca²⁺-activated Cl⁻ channel (Cl_Ca) blocker niflumic acid (NFA) produces a paradoxical dual effect on I_Cl(Ca), causing stimulation or inhibition at potentials below or above 0 mV respectively. We tested whether the effects of NFA on I_Cl(Ca) were modulated by phosphorylation.

Experimental approach:

I_Cl(Ca) was elicited with 500 nM free internal Ca²⁺ in rabbit pulmonary artery myocytes. The state of global phosphorylation was altered by cell dialysis with either 5 mM ATP or 0 mM ATP with or without an inhibitor of calmodulin-dependent protein kinase type II, KN-93 (10 µM).

Key results:

Dephosphorylation enhanced the ability of 100 µM NFA to inhibit I_Cl(Ca). This effect was attributed to a large negative shift in the voltage-dependence of block, which was converted to stimulation at potentials <−50 mV, ∼70 mV more negative than cells dialysed with 5 mM ATP. NFA dose-dependently blocked I_Cl(Ca) in the range of 0.1–250 µM in cells dialysed with 0 mM ATP and KN-93, which contrasted with the stimulation induced by 0.1 µM, which converted to block at concentrations >1 µM when cells were dialysed with 5 mM ATP.

Conclusions and implications:

Our data indicate that the presumed state of phosphorylation of the pore-forming or regulatory subunit of Cl_Ca channels influenced the interaction of NFA in a manner that obstructs interaction of the drug with an inhibitory binding site.     

Natural and synthetic modulators of SK (K(ca)2) potassium channels inhibit magnesium-dependent activity of the kinase-coupled cation channel TRPM7

BACKGROUND AND PURPOSE

Transient receptor potential cation channel subfamily M member 7 (TRPM7) is a bifunctional protein comprising a TRP ion channel segment linked to an α-type protein kinase domain. TRPM7 is essential for proliferation and cell growth. Up-regulation of TRPM7 function is involved in anoxic neuronal death, cardiac fibrosis and tumour cell proliferation. The goal of this work was to identify non-toxic inhibitors of the TRPM7 channel and to assess the effect of blocking endogenous TRPM7 currents on the phenotype of living cells.

EXPERIMENTAL APPROACH

We developed an aequorin bioluminescence-based assay of TRPM7 channel activity and performed a hypothesis-driven screen for inhibitors of the channel. The candidates identified were further assessed electrophysiologically and in cell biological experiments.

KEY RESULTS

TRPM7 currents were inhibited by modulators of small conductance Ca²⁺-activated K⁺ channels (K_Ca2.1–2.3; SK) channels, including the antimalarial plant alkaloid quinine, CyPPA, dequalinium, NS8593, SKA31 and UCL 1684. The most potent compound NS8593 (IC₅₀ 1.6 µM) specifically targeted TRPM7 as compared with other TRP channels, interfered with Mg²⁺-dependent regulation of TRPM7 channel and inhibited the motility of cultured cells. NS8593 exhibited full and reversible block of native TRPM7-like currents in HEK 293 cells, freshly isolated smooth muscle cells, primary podocytes and ventricular myocytes.

CONCLUSIONS AND IMPLICATIONS

This study reveals a tight overlap in the pharmacological profiles of TRPM7 and K_Ca2.1–2.3 channels. NS8593 acts as a negative gating modulator of TRPM7 and is well-suited to study functional features and cellular roles of endogenous TRPM7.     

Blockade of I(Ca) suppresses early afterdepolarizations and reduces transmural dispersion of repolarization in a whole heart model of chronic heart failure

BACKGROUND AND PURPOSE

Chronic heart failure (CHF) is associated with action potential prolongation and Ca²⁺ overload, increasing risk of ventricular tachyarrhythmias (VT). We therefore investigated whether I_Ca blockade was anti-arrhythmic in an intact perfused heart model of CHF.

EXPERIMENTAL APPROACH

CHF was induced in rabbits after 4 weeks of rapid ventricular pacing. Hearts from CHF and sham-operated rabbits were isolated and perfused (Langendorff preparation), with ablation of the AV node. VT was induced by erythromycin and low [K⁺] (1.5mM). Electrophysiology of cardiac myocytes, with block of cation currents, was simulated by a mathematical model.

KEY RESULTS

Repolarization was prolonged in CHF hearts compared with sham-operated hearts. Action potential duration (APD) and overall dispersion of repolarization were further increased by erythromycin (300 µM) to block I_Kr in CHF hearts. After lowering [K⁺] to 1.5mM, CHF and sham hearts showed spontaneous episodes of polymorphic non-sustained VT. Additional infusion of verapamil (0.75 µM) suppressed early afterdepolarizations (EAD) and VT in 75% of sham and CHF hearts. Verapamil shortened APD and dispersion of repolarization, mainly by reducing transmural dispersion of repolarization via shortening of endocardial action potentials. Mathematical simulations showed that EADs were more effectively reduced by verapamil assuming a state-dependent block than a simple block of I_Ca.

CONCLUSIONS AND IMPLICATIONS

Blockade of I_Ca was highly effective in suppressing VT via reduction of transmural dispersion of repolarization and suppression of EAD. Such blockade might represent a novel therapeutic option to reduce risk of VT in structurally normal hearts and also in heart failure.

LINKED ARTICLE

This article is commented on by Stams et al., pp. 554–556 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2011.01818.x