Distinct properties of amlodipine and nicardipine block of the voltage-dependent Ca2+ channels Ca v 1.2 and Ca v 2.1 and the mutant channels Ca v 1.2/Dihydropyridine insensitive and Ca v 2.1/Dihydropyridine sensitive

The binding site within the L-type Ca(2+) channel Ca(v)1.2 for neutral dihydropyridines is well characterized. However, the contributions of the alkylamino side chains of charged dihydropyridines such as amlodipine and nicardipine to channel block are not clear. We tested the hypothesis that the distinct locations of the charged side chains on amlodipine and nicardipine would confer distinct properties of channel block by these two drugs. Using whole-cell voltage clamp, we investigated block of wild type Ca(v) 2.1, wild type Ca(v)1.2, and Ca(v)1.2/Dihydropyridine insensitive, a mutant channel insensitive to neutral DHPs, by amlodipine and nicardipine. The potency of nicardipine and amlodipine for block of closed (stimulation frequency of 0.05 Hz) Ca(v)1.2 channels was not different (IC(50) values of 60 nM and 57 nM, respectively), but only nicardipine block was enhanced by increasing the stimulation frequency to 1 Hz. The frequency-dependent block of Ca(v)1.2 by nicardipine is the result of a strong interaction of nicardipine with the inactivated state of Ca(v)1.2. However, nicardipine block of Ca(v)1.2/Dihydropyridine insensitive was much more potent than block by amlodipine (IC(50) values of 2.0 μM and 26 μM, respectively). A mutant Ca(v)2.1 channel containing the neutral DHP binding site (Ca(v)2.1/Dihydropyridine sensitive) was more potently blocked by amlodipine (IC(50)=41 nM) and nicardipine (IC(50)=175 nM) than the parent Ca(v)2.1 channel. These data suggest that the alkylamino group of nicardipine and amlodipine project into distinct regions of Ca(v)1.2 such that the side chain of nicardipine, but not amlodipine, contributes to the potency of closed-channel block, and confers frequency-dependent block.     

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.     

A-887826 is a structurally novel, potent and voltage-dependent Nav1.8 sodium channel blocker that attenuates neuropathic tactile allodynia in rats

Activation of sodium channels is essential to action potential generation and propagation. Recent genetic and pharmacological evidence indicates that activation of Na_v1.8 channels contributes to chronic pain. Herein, we describe the identification of a novel series of structurally related pyridine derivatives as potent Na_v1.8 channel blockers. A-887826 exemplifies this series and potently (IC₅₀ = 11nM) blocked recombinant human Na_v1.8 channels. A-887826 was ∼3 fold less potent to block Na_v1.2, ∼10 fold less potent to block tetrodotoxin-sensitive sodium (TTX-S Na⁺) currents and was >30 fold less potent to block Na_V1.5 channels. A-887826 potently blocked tetrodotoxin-resistant sodium (TTX-R Na⁺) currents (IC₅₀ = 8nM) from small diameter rat dorsal root ganglion (DRG) neurons in a voltage-dependent fashion. A-887826 effectively suppressed evoked action potential firing when DRG neurons were held at depolarized potentials and reversibly suppressed spontaneous firing in small diameter DRG neurons from complete Freund’s adjuvant inflamed rats. Following oral administration, A-887826 significantly attenuated tactile allodynia in a rat neuropathic pain model. Further characterization of TTX-R current block in rat DRG neurons demonstrated that A-887826 (100 nM) shifted the mid-point of voltage-dependent inactivation of TTX-R currents by ∼4 mV without affecting voltage-dependent activation and did not exhibit frequency-dependent inhibition. The present data demonstrate that A-887826 is a structurally novel and potent Na_v1.8 blocker that inhibits rat DRG TTX-R currents in a voltage-, but not frequency-dependent fashion. The ability of this structurally novel Na_v1.8 blocker to effectively reduce tactile allodynia in neuropathic rats further supports the role of Na_v1.8 sodium channels in pathological pain states.     

Optimization of Cav1.2 screening with an automated planar patch clamp platform

Introduction: Ca_v1.2 channels play an important role in shaping the cardiac action potential. Screening pharmaceutical compounds for Ca_v1.2 block is very important in developing drugs without cardiac liability. Ca_v1.2 screening has been traditionally done using fluorescence assays, but these assays have some limitations. Patch clamping is considered the gold standard for ion channel studies, but is very labor intensive. The purpose of this study was to develop a robust medium throughput Ca_v1.2 screening assay in PatchXpress^® 7000A by optimizing cell isolation conditions, recording solutions and experimental parameters. Under the conditions established, structurally different standard Ca_v1.2 antagonists and an agonist were tested. Methods: HEK-293 cells stably transfected with hCa_v1.2 L-type Ca channel were used. For experiments, cells were isolated using 0.05% Trypsin. Currents were recorded in the presence of 30 mM extracellular Ba²⁺ and low magnesium intracellular recording solution to minimize rundown. Ca_v1.2 currents were elicited from a holding potential of ? 60 mV at 0.05 Hz to increase pharmacological sensitivity and minimize rundown. Test compounds were applied at increasing concentrations for 5 min followed by a brief washout. Results: Averaged peak Ca_v1.2 current amplitudes were increased from 10 pA/pF to 15 pA/pF by shortening cell incubation and trypsin exposure time from 2.5 min at 37 °C to 1 min at room temperature and adding 0.2 mM cAMP to the intracellular solution. Rundown was minimized from 2%/min to 0.5%/min by reducing the intracellular free Mg²⁺ from 2.7 mM to 0.2 mM and adding 100 nM Ca²⁺. Under the established conditions, we tested 8 structurally different antagonists and an agonist. The IC₅₀ values obtained ranked well against published values and results obtained using traditional clamp experiments performed in parallel using the expressed cell line and native myocytes. Discussion: This assay can be used as a reliable pharmacological screening tool for Ca_v1.2 block to assess compounds for cardiac liability during lead optimization.     

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.     

Voltage and pH dependent block of cloned N-type Ca2+ channels by amlodipine

1. Two types of Ca²⁺ channel α₁-subunits were co-expressed in Xenopus oocytes with the Ca²⁺ channel α₂- and β₁-subunits. The Ba²⁺ current through the α_1Cα₂β and the α_1Bα₂β channels had electrophysiological and pharmacological properties of L- and N-type Ca²⁺ channels, respectively.
2. Amlodipine had a strong blocking action on both the L-type and N-type Ca²⁺ channels expressed in the oocyte. The potency of the amlodipine block on the N-type Ca²⁺ channel was comparable to that on the L-type Ca²⁺ channel. At −100 mV holding potential, the IC₅₀ values for amlodipine block on the L-type and N-type Ca²⁺ channel were 2.4 and 5.8 μM, respectively.
3. The blocking action of amlodipine on the N-type Ca²⁺ channel was dependent on holding potential and extracellular pH, as has been observed with amlodipine block on the L-type Ca²⁺ channel. A depolarized holding potential and high pH enhanced the blocking action of amlodipine.
4. The time course of block development by amlodipine was similar for L-type and N-type Ca²⁺ channels. However, it was slower than the time course of block development by nifedipine for the L-type Ca²⁺ channel.

     

Comparison of electrophysiological effects of calcium channel blockers on cardiac repolarization

Dihydropyridine (DHP) calcium channel blockers (CCBs) have been widely used to treat of several cardiovascular diseases. An excessive shortening of action potential duration (APD) due to the reduction of Ca²⁺ channel current (I_Ca) might increase the risk of arrhythmia. In this study we investigated the electrophysiological effects of nicardipine (NIC), isradipine (ISR), and amlodipine (AML) on the cardiac APD in rabbit Purkinje fibers, voltage-gated K⁺ channel currents (I_Kr, I_Ks) and voltage-gated Na⁺ channel current (I_Na). The concentration-dependent inhibition of Ca²⁺ channel currents (I_Ca) was examined in rat cardiomyocytes; these CCBs have similar potency on I_Ca channel blocking with IC₅₀ (the half-maximum inhibiting concentration) values of 0.142, 0.229, and 0.227 nM on NIC, ISR, and AML, respectively. However, ISR shortened both APD₅₀ and APD₉₀ already at 1 µM whereas NIC and AML shortened APD₅₀ but not APD₉₀ up to 30 µM. According to ion channel studies, NIC and AML concentration-dependently inhibited I_Kr and I_Ks while ISR had only partial inhibitory effects (<50% at 30 µM). Inhibition of I_Na was similarly observed in the three CCBs. Since the I_Kr and I_Ks mainly contribute to cardiac repolarization, their inhibition by NIC and AML could compensate for the AP shortening effects due to the block of I_Ca.     

Chronic exercise normalizes changes in Cav1.2 and KCa1.1 channels in mesenteric arteries from spontaneously hypertensive rats

Background and Purpose

Regular physical activity is an effective non-pharmacological therapy for prevention and control of hypertension. However, the underlying mechanisms are not fully understood. Accumulating evidence shows that the elevated vascular tone in hypertension is a consequence of the ‘ion channel remodelling’ that occurs during sustained high BP. The present study investigated the effects of aerobic exercise on the electrical remodelling of L-type Ca²⁺ (Ca_v1.2) and large-conductance Ca²⁺-activated K⁺ (K_Ca1.1) channels in mesenteric arteries (MAs) from spontaneously hypertensive rats (SHRs).

Experimental Approach

SHRs and normotensive (Wistar-Kyoto) rats were subjected to aerobic training or kept sedentary, and vascular mechanical and functional properties were evaluated.

Key Results

Exercise did not affect the heart weight, but reduced the heart rate and body weight in SHR. In mesenteric arterial myocytes, exercise normalized the increased Ca_v1.2 and K_Ca1.1 current density in SHRs. Exercise also ameliorated the increased open probability and mean open time of the single K_Ca1.1 channel in hypertension. The isometric contraction study revealed that both nifedipine (Ca_v1.2 channel blocker) and NS11021 (K_Ca1.1 channel activator) induced concentration-dependent vasorelaxation in MAs precontracted with noradrenaline. Exercise normalized the increased sensitivity of tissues to nifedipine and NS11021 in SHR. Furthermore, protein expression of the Ca_v1.2 α_1C-subunit together with the K_Ca1.1 α- and β1-subunit was significantly increased in SHRs; and exercise ameliorated these molecular alterations in hypertension.

Conclusions and Implications

Chronic exercise reduces BP and restores vascular function in MAs from SHR, which might be related to the correction of the Ca_v1.2 and K_Ca1.1 channel remodelling during hypertension.     

Antidepressants inhibit Nav1.3, Nav1.7, and Nav1.8 neuronal voltage-gated sodium channels more potently than Nav1.2 and Nav1.6 channels expressed in Xenopus oocytes

Tricyclic antidepressants (TCAs) and duloxetine are used to treat neuropathic pain. However, the mechanisms underlying their analgesic effects remain unclear. Although many investigators have shown inhibitory effects of antidepressants on voltage-gated sodium channels (Na_v) as a possible mechanism of analgesia, to our knowledge, no one has compared effects on the diverse variety of sodium channel α subunits. We investigated the effects of antidepressants on sodium currents in Xenopus oocytes expressing Na_v1.2, Na_v1.3, Na_v1.6, Na_v1.7, and Na_v1.8 with a β₁ subunit by using whole-cell, two-electrode, voltage clamp techniques. We also studied the role of the β₃ subunit on the effect of antidepressants on Na_v1.3. All antidepressants inhibited sodium currents in an inactivated state induced by all five α subunits with β₁. The inhibitory effects were more potent for Na_v1.3, Na_v1.7, and Na_v1.8, which are distributed in dorsal root ganglia, than Na_v1.2 and Na_v1.6, which are distributed primarily in the central nervous system. The effect of amitriptyline on Na_v1.7 with β₁ was most potent with a half-maximal inhibitory concentration (IC₅₀) 4.6 μmol/L. IC₅₀ for amitriptyline on Na_v1.3 coexpressed with β₁ was lowered from 8.4 to 4.5 μmol/L by coexpression with β₃. Antidepressants predominantly inhibited the sodium channels expressed in dorsal root ganglia, and amitriptyline has the most potent inhibitory effect. This is the first evidence, to our knowledge, showing the diverse effects of antidepressants on various α subunits. Moreover, the β₃ subunit appears important for inhibition of Na_v1.3. These findings may aid better understanding of the mechanisms underlying the pain relieving effects of antidepressants.

     

Interactions between calcium channel blockers and α-adrenoceptors in the human coronary and mammary arteries: a radioligand binding study

1 The study was designed to assess whether Ca²⁺ channel blockers of the dihydropyridine family (felodipine, nicardipine, nifedipine, nimodipine, nitrendepine and nisoldipine), and of the phenylalkylamine family (verapamil) have any effect on α-adrenoceptor binding to sections of the human right coronary and mammary arteries, measured using [³H]-dihydroergocryptine (DHT) as a ligand. 2 Increasing concentrations of nicardipine, verapamil and nitrendepine competed with [³H]-DHT binding to sections of the human coronary and mammary arteries. The other compounds tested were without effect. Among the competitors of [³H]-DHT binding, nicardipine was the most powerful, with a 50% inhibition (IC₅₀) value of approximately 10 nM. 3 The pharmacological profile of competition with [³H]-DHT binding by nicardipine, in the presence or in the absence of guanosine triphosphate and NaCl, is consistent with antagonist activity of the dihydropyridine derivative at the α-adrenoceptor. 4 This property may account for the lower sympathetic stimulatory activity elicited by nicardipine, in comparison with other Ca²⁺ channel blockers used in cardiovascular therapy.     

Histamine H3 receptor activation inhibits glutamate release from rat striatal synaptosomes

The release of glutamate from striatal synaptosomes induced by depolarisation with 4-aminopyridine (4-AP) was studied by a method based on the fluorescent properties of the NAPDH formed by the metabolism of the neurotransmitter by glutamate dehydrogenase.Ca²⁺-dependent, depolarisation-induced glutamate release was inhibited in a concentration-dependent manner by the selective histamine H₃ agonist immepip. Best-fit estimates were: maximum inhibition 60±10% and IC₅₀ 68±10 nM. The effect of 300 nM immepip on depolarisation-evoked glutamate release was reversed by the selective H₃ antagonist thioperamide in a concentration-dependent manner (EC₅₀ 23 nM, K_i 4 nM).In fura-2-loaded synaptosomes, the increase in the intracellular concentration of Ca²⁺ ([Ca²⁺]_i) evoked by 4-AP-induced depolarisation (resting level 167±14 nM; Δ[Ca²⁺]_i 88±15 nM) was modestly, but significantly reduced (29±5% inhibition) by 300 nM immepip. The action of the H₃ agonist on depolarisation-induced changes in [Ca²⁺]_i was reversed by 100 nM thioperamide.Taken together, our results indicate that histamine modulates the release of glutamate from corticostriatal nerve terminals. Inhibition of depolarisation-induced Ca²⁺ entry through voltage-dependent Ca²⁺ channels appears to account for the effect of H₃ receptor activation on neurotransmitter release. Modulation of glutamatergic transmission in rat striatum may have important consequences for the function of basal ganglia and therefore for the control of motor behaviour.     

Synthesis of a New Series of 4-Aryl-1,4-Dihydropyridines with Calcium Channel Blocking and Vasodilatory Activity

Several new amide derivatives of 4-aryl-1,4-dihydropyridine carboxylic congeners have been synthesized in this study to obtain therapeutically useful compounds. The changes in pharmacological properties of dihydropyridines by the presence of polar groups at different positions of 4-phenyl substituent and also by introduction of unsymmetrical ester groups in the synthesized symmetrical analogs have been studied. In vitro calcium channel blocking activity has been evaluated in cultures of neonatal rat cortical neurons by measuring the inhibitory response at L-type calcium channels activated by veratridine. The newly synthesized dihydropyridines displayed moderate calcium channel blockade with IC₅₀ values ranging from 2 to 10 μM in comparison to nifedipine (IC₅₀ = 57.7 nM). The vasodilatory activity was evaluated on isolated rat thoracic aortic rings precontracted by phenylephrine/KCl (30 mM). The symmetrically substituted dihydropyridine 8a exhibited maximum activity with IC₅₀ = 0.64 μM but was found to be approximately 24 times less active in comparison to standard drug nifedipine with IC₅₀ = 27.5 nM.     

Levetiracetam-mediated emotional behavior in heterozygous rolling Nagoya CaV2.1 channel mutant mice

Ca_V2.1, which is highly expressed in the nervous system, plays an essential role in presynaptic neurotransmitter release. Although recent data suggest that the antiepileptic drug levetiracetam (LEV) inhibits presynaptic Ca_V2.1 activity, the precise physiological role of Ca_V2.1/LEV-regulated emotional performance has not been elucidated. We examined whether Ca_V2.1/LEV mediates emotional behavior using a combined pharmacologic and genetic approach. Heterozygous rolling Nagoya (rol/+) mice carrying the Ca_V2.1α₁ mutation demonstrated normal emotional behavior. Exposure to 75 mg/kg LEV, which had no effect in wild-type controls, reduced anxiety in elevated plus maze and light-dark exploration tests and reduced depression in forced swimming and tail suspension behavioral tests in rol/+ mice. Similar behavioral patterns in motor activity were noted in wild-type and rol/+ mice injected with 0-150 mg/kg LEV. The phosphorylation of tryptophan hydroxylase at serine-58 and serotonin concentration were increased in the brainstems of rol/+ mice injected with 75 mg/kg LEV but not in those of wild-type controls. These results indicate that Ca_V2.1/LEV mediates serotonin signaling leading to alterations in emotion. Our results also indicate that a combination of subthreshold pharmacologic and genetic approaches can be used to study functional signaling pathways in neuronal circuits.     

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.     

A peripherally acting,selective T-type calcium channel blocker,ABT-639, effectively reduces nociceptive and neuropathic pain in rats

Activation of T-type Ca²⁺ channels contributes to nociceptive signaling by facilitating action potential bursting and modulation of membrane potentials during periods of neuronal hyperexcitability. The role of T-type Ca²⁺ channels in chronic pain is supported by gene knockdown studies showing that decreased Ca_v3.2 channel expression results in the loss of low voltage-activated (LVA) currents in dorsal root ganglion (DRG) neurons and attenuation of neuropathic pain in the chronic constriction injury (CCI) model. ABT-639 is a novel, peripherally acting, selective T-type Ca²⁺ channel blocker. ABT-639 blocks recombinant human T-type (Ca_v3.2) Ca²⁺ channels in a voltage-dependent fashion (IC₅₀ = 2 μM) and attenuates LVA currents in rat DRG neurons (IC₅₀ = 8 μM). ABT-639 was significantly less active at other Ca²⁺ channels (e.g. Ca_v1.2 and Ca_v2.2) (IC₅₀ > 30 μM). ABT-639 has high oral bioavailability (%F = 73), low protein binding (88.9%) and a low brain:plasma ratio (0.05:1) in rodents. Following oral administration ABT-639 produced dose-dependent antinociception in a rat model of knee joint pain (ED₅₀ = 2 mg/kg, p.o.). ABT-639 (10–100 mg/kg, p.o.) also increased tactile allodynia thresholds in multiple models of neuropathic pain (e.g. spinal nerve ligation, CCI, and vincristine-induced, and capsaicin secondary hypersensitivity). ABT-639 did not attenuate hyperalgesia in inflammatory pain models induced by complete Freund's adjuvant or carrageenan. At higher doses (e.g. 100 - 300 mg/kg) ABT-639 did not significantly alter hemodynamic or psychomotor function. The antinociceptive profile of ABT-639 provides novel insights into the role of peripheral T-type (Ca_v3.2) channels in chronic pain states.     

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.     

In-vitro Negative Chronotropic and Inotropic Effects of a Novel Dihydropyridine Derivative,CD-832, in the Guinea-pig: Comparison with Calcium-channel Antagonists

The effects of CD-832 (4R-(-)-2-(nicotinoylamino)ethyl-3-nitroxypropyl-1,4-dihydro-2,6-dimethyl-4,3-nitrophenyl, 3,5-pyridine dicarboxylate), a novel dihydropyridine derivative, on guinea-pig isolated myocardial preparations have been compared with those of Ca²⁺-channel antagonists. All ten compounds induced concentration-dependent negative chronotropic effects on preparations of isolated right atria and negative inotropic effects on isolated right ventricular papillary muscles. The order of potency for the negative chronotropic effect was CD-832 > nicardipine = gallopamil > clentiazem > nifedipine = efonidipine > amlodipine = semotiadil > verapamil > diltiazem; that for the negative inotropic effect was nicardipine = gallopamil > nifedipine > verapamil > CD-832 > diltiazem > clentiazem > efonidipine = semotiadil > amlodipine. The ratio of the EC50 (the concentration of Ca²⁺ antagonist having 50% of the maximum effect) for the negative inotropic effect divided by the EC50 for the negative chronotropic effect, considered to be an index of selectivity for negative chronotropic effect, was higher for CD-832, amlodipine, efonidipine and semotiadil than for the other Ca²⁺ antagonists. The ratio for CD-832, nifedipine, nicardipine, efonidipine, amlodipine, verapamil, gallopamil, diltiazem, clentiazem and semotiadil was 11·4, 0·29, 0·87, 35·4, 37·1, 0·65, 0·87, 0·92, 7·11 and 30·0, respectively. These findings indicate that CD-832 and the newly developed Ca²⁺ antagonists including amlodipine, efonidipine, semotiadil and clentiazem were selective for a negative chronotropic effect rather than for a negative inotropic effect. This ‘chrono-selective’ effect of these drugs might be of benefit in the treatment of cardiovascular disorders.     

Multiple structural elements contribute to voltage-dependent facilitation of neuronal α1C (CaV1.2) L-type calcium channels

Voltage- and frequency-dependent facilitation of calcium channel activity has been implicated in a number of key physiological processes. Various mechanisms have been proposed to mediate these regulations, including a switch between channel gating modes, voltage-dependent phosphorylation, and a voltage-dependent deinhibition of G-protein block. Studying such modulation on recombinant Ca channels expressed in oocytes, we previously reported that α_1C L-type calcium channel contrast with non-L type Ca channels by its ability to exhibit facilitation by pre-depolarization (Voltage-dependent facilitation of a neuronal α_IC L-type calcium channel, E. Bourinet et al., EMBO Journal, 1994; 13, 5032–5039). To further analyze this effect, we have investigated the molecular determinants which mediate the differences in voltage-dependent facilitation between «facilitable» α_1C and «non facilitable» α_1E calcium channels. We used a series of chimeras which combine the four transmembrane domains of the two channels. Results show that the four domains of α_1C contribute to facilitation, with domain I being most critical. This domain is required but not sufficient alone to generate facilitation. The minimal requirement to observe the effect is the presence of domain I plus one of the three others. We conclude that similarly to activation gating, voltage-dependent facilitation of α_1C is a complex process which involves multiple structural elements were domains I and III play the major role.     

Inhibition of human Na(v)1.5 sodium channels by strychnine and its analogs

Strychnine and brucine from the seeds of the plant Strychnos nux vomica have been shown to have interesting pharmacological effects on several neurotransmitter receptors. In this study, we have characterized the pharmacological properties of strychnine and its analogs on human Na_v1.5 channels to assess their potential therapeutic advantage in certain arrhythmias. Among the eight alkaloids, only strychnine and icajine exhibited inhibition potency on the Na_v1.5 channel with the half-maximum inhibition (IC₅₀) values of 83.1 μM and 104.6 μM, respectively. Structure–function analysis indicated that the increased bulky methoxy groups on the phenyl ring or the negatively charged oxygen atom may account for this lack of inhibition on the Na_v1.5 channel. Strychnine and icajine may bind to the channel by cation–π interactions. The substitution with a large side chain on the phenyl ring or the increased molecular volume may alter the optimized position for the compound close to the binding sites of the channel. Strychnine and icajine bind to the Na_v1.5 channel with a new mechanism that is different from TTX and local anesthetics. They bind to the outer vestibule of the channel pore with fast association and dissociation rates at resting state. Strychnine and icajine had little effect on steady-state fast inactivation but markedly shifted the slow inactivation of Na_v1.5 currents toward more hyperpolarized potentials. The property of icajine influencing slow-inactivated state of Na_v1.5 channel would be potential therapeutic advantages in certain arrhythmias.     

Effect of sulfur dioxide inhalation on the expression of KATP and L-Ca2+ channels in rat hearts

Epidemiological studies have revealed an association between sulfur dioxide (SO₂) exposure and cardiovascular diseases. This study is designed to investigate the SO₂ effect on the expression of ATP-sensitive K⁺ (K_ATP) channel and L-type calcium (L-Ca²⁺) channel in rat hearts. The results show that the mRNA and protein levels of the K_ATP channel subunits Kir6.2 and SUR2A of rat hearts in SO₂ groups were higher than those in control group. SO₂ at 14 mg/m³ significantly decreased the expression of the L-Ca²⁺ channel subunits Ca_v1.2 and Ca_v1.3. This suggests that SO₂ can activate the K_ATP channels by up-regulating the expression of Kir6.2 and SUR2A, while it inhibits the L-Ca²⁺ channels by down-regulating the expression of Ca_v1.2 and Ca_v1.3 in rat hearts. The molecular mechanism of SO₂-induced negative inotropic effect might be linked to the expression changes of these subunits, which may contribute to the pathogenesis of SO₂-associated cardiovascular diseases.