Permanently charged chiral 1,4-dihydropyridines: molecular probes of L-type calcium channels. Synthesis and pharmacological characterization of methyl(omega-trimethylalkylammonium) 1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridinedicarboxylate iodide, calcium channel antagonists

We report the synthesis of the single enantiomers of permanently charged dihydropyridine derivatives (DHPs with alkyl linker lengths of two and eight carbon atoms) and their activities on cardiac and neuronal L-type calcium channels. Permanently charged chiral 1,4-dihydropyridines and methyl (omega)-trimethylalkylammonium) 1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridinedicarboxylate iodides were synthesized in high optical purities from (R)-(-) and (S)-(+)-1,4-dihydro-2,6-dimethyl-5-methoxycarbonyl-4-(3-nitrophenyl)-3-+ ++pyridinecarboxylic acid, obtained by resolution of racemic 1,4-dihydro-2,6-dimethyl-5-methoxycarbonyl-4-(3-nitrophenyl)-3-pyridi necarboxylic acid. Competition binding experiments with radioligand [3H]-(+)-PN200-110 and the block of whole cell barium currents through L-type calcium channels in GH4C1 cells show that the compounds with the eight-carbon alkyl linker optimally block the L-type Ca2+ channels, and that the S-enantiomer is more potent than the R-enantiomer.     

Actions of mibefradil, efonidipine and nifedipine block of recombinant T- and L-type Ca channels with distinct inhibitory mechanisms

We compared detailed efficacy of efonidipine and nifedipine, dihydropyridine analogues, and mibefradil using recombinant T- and L-type Ca2+ channels expressed separately in mammalian cells. All these Ca2+ channel antagonists blocked T-type Ca2+ channel currents (I(Ca(T))) with distinct blocking manners: I(Ca(T)) was blocked mainly by a tonic manner by nifedipine, by a use-dependent manner by mibefradil, and by a combination of both manners by efonidipine. IC50s of these Ca2+ channel antagonists to I(Ca(T)) and L-type Ca2+ channel current (I(Ca(L))) were 1.2 micromol/l and 0.14 nmol/l for nifedipine; 0.87 and 1.4 micromol/l for mibefradil, and 0.35 micromol/l and 1.8 nmol/l for efonidipine, respectively. Efonidipine, a dihydropyridine analogue, showed high affinity to T-type Ca2+ channel.     

Towards selective antagonists of T-type calcium channels: design, characterization and potential applications of NNC 55-0396

NNC 55-0396 is a structural analog of mibefradil (Ro 40-5967) that inhibits both T-type and high-voltage-activated (HVA) Ca2+ channels with a higher selectivity for T-type Ca2+ channels. The inhibitory effect of mibefradil on HVA Ca2+ channels can be attributed to a hydrolyzed metabolite of the drug: the methoxy acetate side chain of mibefradil is removed by intracellular enzymes, thus it forms (1S,2S)-2-(2-(N-[(3-benzoimidazol-2-yl)propyl]-N-methylamino)ethyl)-6-fluoro-1,2,3,4-tetrahydro-1-isopropyl-2-naphtyl hydroxy dihydrochloride (dm-mibefradil), which causes potent inhibition of HVA Ca2+ currents. By replacing the methoxy acetate chain of mibefradil with cyclopropanecarboxylate, a more stable analog was developed (NNC 55-0396). The acute IC50 of NNC 55-0396 to block recombinant Cav3.1 T-type channels expressed in HEK293 cells is approximately 7 muM, whereas 100 microM NNC 55-0396 has no detectable effect on high voltage-activated currents in INS-1 cells. Block of T-type Ca2+ current was partially reduced by membrane hyperpolarization and was enhanced at high stimulus frequency. Washing NNC 55-0396 out of the recording chamber did not reverse the T-type Ca2+ current activity, suggesting that the compound dissolves in or passes through the plasma membrane to exert its effect; however, intracellular perfusion of the compound did not block T-type Ca2+ currents, arguing against a cytoplasmic route of action. We conclude that NNC 55-0396, by virtue of its modified structure, does not produce the metabolite that causes inhibition of L-type Ca2+ channel channels, thus rendering it more selective to T-type Ca2+ channels.     

Ca2+ channel inhibition in a rat osteoblast-like cell line, UMR 106, by a new dihydropyridine derivative, S11568.

UMR 106 rat osteogenic sarcoma cells were studied with the whole cell patch clamp technique to investigate the presence of voltage-gated inward currents. In barium (Ba2+)-containing medium, depolarizing jumps revealed both transient (T-type) and sustained (L-type) Ba2+ currents. The L-type component was dihydropyridine-sensitive: the agonist Bay K 8644 increased the amplitude of the L-type Ba2+ current. A new dihydropyridine calcium channel blocker, S 11568 ((+/-)-2(2-[2-(aminoethoxy)ethoxyl]methyl)4-(2',3'- dichlorophenyl)3-ethoxycarbonyl-5-methoxycarbonyl-6-methyl-1,4- dihydropyridine, and its enantiomers, S 12967 ((+)-S 11568) and S 12968 ((-)-S 11568), inhibited the L-type Ba2+ current. IC50 values at a holding potential (VH) of -50 mV were 90 nM for S 11568, 800 nM for S 12967 and 45 nM for S 12968. At VH = -80 mV, S 12968 was less potent (IC50 near 500 nM). In contrast, S 12968 was without appreciable effect on the T-type component of the inward current through Ca2+ channels. Our results indicate that UMR 106 cells express both T-type and L-type Ca2+ channels and could be used to study the modulation by Ca2+ channel blocking agents, such as S 12968, of the hormonal regulation of Ca2+ fluxes across the osteoblast membrane.     

Differential blocking action of dihydropyridine Ca2+ antagonists on a T-type Ca2+ channel (alpha1G) expressed in Xenopus oocytes

Recent reports show that efonidipine, a dihydropyridine Ca2+ antagonist, has blocking action on T-type Ca2+ channels, which may produce favorable actions on cardiovascular systems. However, the effects of other dihydropyridine Ca2+ antagonists on T-type Ca2+ channels have not been investigated yet. Therefore, in this study, we examined the effects of dihydropyridine compounds clinically used for treatment of hypertension on a T-type Ca2+ channel subtype, alpha1G, expressed in Xenopus oocytes. These effects were compared with those on T-type Ca2+ channel. Rabbit L-type (alpha1Calpha2/deltabeta1a) or rat T-type (alpha1G) Ca2+ channel was expressed in Xenopus oocytes by injection of cRNA for each subunit. The Ba currents through expressed channels were measured by conventional 2-microelectrode voltage-clamp methods. Twelve DHPs (amlodipine, barnidipine, benidipine, cilnidipine, efonidipine, felodipine, manidipine, nicardipine, nifedipine, nilvadipine, nimodipine, nitrendipine) and mibefradil were tested. Cilnidipine, felodipine, nifedipine, nilvadipine, minodipine, and nitrendipine had little effect on the T-type channel. The blocks by drugs at 10 microM were less than 10% at a holding potential of -100 mV. The remaining 6 drugs had blocking action on the T-type channel comparable to that on the L-type channel. The blocking actions were also comparable to that by mibefradil. These results show that many dihydropyridine Ca2+ antagonists have blocking action on the alpha1G channel subtype. The action of dihydropyridine Ca2+ antagonists in clinical treatment should be evaluated on the basis of subtype selectivity.     

Blockade of T-type voltage-dependent Ca2+ channels by benidipine, a dihydropyridine calcium channel blocker, inhibits aldosterone production in human adrenocortical cell line NCI-H295R

Benidipine, a long-lasting dihydropyridine calcium channel blocker, is used for treatment of hypertension and angina. Benidipine exerts pleiotropic pharmacological features, such as renoprotective and cardioprotective effects. In pathophysiological conditions, the antidiuretic hormone aldosterone causes development of renal and cardiovascular diseases. In adrenal glomerulosa cells, aldosterone is produced in response to extracellular potassium, which is mainly mediated by T-type voltage-dependent Ca2+ channels. More recently, it has been demonstrated that benidipine inhibits T-type Ca2+ channels in addition to L-type Ca2+ channels. Therefore, effect of calcium channel blockers, including benidipine, on aldosterone production and T-type Ca2+ channels using human adrenocortical cell line NCI-H295R was investigated. Benidipine efficiently inhibited KCl-induced aldosterone production at low concentration (3 and 10 nM), with inhibitory activity more potent than other calcium channel blockers. Patch clamp analysis indicated that benidipine concentration-dependently inhibited T-type Ca2+ currents at 10, 100 and 1000 nM. As for examined calcium channel blockers, inhibitory activity for T-type Ca2+ currents was well correlated with aldosterone production. L-type specific calcium channel blockers calciseptine and nifedipine showed no effect in both assays. These results indicate that inhibition of T-type Ca2+ channels is responsible for inhibition of aldosterone production in NCI-H295R cells. Benidipine efficiently inhibited KCl-induced upregulation of 11-beta-hydroxylase mRNA and aldosterone synthase mRNA as well as KCl-induced Ca2+ influx, indicating it as the most likely inhibition mechanism. Benidipine partially inhibited angiotensin II-induced aldosterone production, plus showed additive effects when used in combination with the angiotensin II type I receptor blocker valsartan. Benidipine also partially inhibited angiotensin II-induced upregulation of the above mRNAs and Ca2+ influx inhibitory activities of benidipine for aldosterone production. T-type Ca2+ channels may contribute to additional benefits of this drug for treating renal and cardiovascular diseases, beyond its primary anti-hypertensive effects from blocking L-type Ca2+ channels.     

Pharmacological properties of Cav3.2, a low voltage-activated Ca2+ channel cloned from human heart

Three genes encoding T-type Ca2+ channels have been described but their correspondence to the various native T-type Ca2+ currents remains uncertain. In particular, Ca(V)3.2 (or alpha1H) was cloned from a human heart library, its message was found abundantly in cardiac tissue, and expressed Ca(V)3.2 was shown to conduct low voltage-activated currents, which inactivate rapidly and are sensitive to Ni2+ and mibefradil. These observations suggested that Ca(V)3.2 might encode native cardiac T-type Ca2+ channels but more information on the pharmacology of Ca(V)3.2 was needed to confirm this hypothesis. In the present study, we compare the pharmacology of Ca(V)3.2 expressed in HEK293 cells and of native T-type Ca2+ channels in guinea pig atrial myocytes ("native-T"). (1) Ca(V)3.2 and native-T are insensitive to TTX and to toxins selective for N-, P-, or Q-type Ca2+ channels (omega-CTx-GVIA, omega-Aga-IVA, omega-CTx-MVIIC). (2) The half-maximal blocking concentration (IC50) of mibefradil on Ca(V)3.2 is near that on native-T and the block is similarly voltage-dependent. (3) Ca(V)3.2 is five- to sixfold less sensitive than native-T to the 1,4-dihydropyridine (DHP) amlodipine, suggesting a difference in the DHP binding site. (4) Both channels display similar (but not identical) sensitivities to the inorganic blockers Ni2+ and Cd2+ and the IC50s are in the range of values found for T-type Ca2+ currents in other cell types. (5) Ni2+ shifts the voltage dependence of Ca(V)3.2 activation but not that of native-T. The many similarities between the two channels support the contention that Ca(V)3.2 encodes cardiac T-type Ca2+ channels. The slight differences may be due to species variations and/or to the choice of splice variant.     

Inhibition of L-type but not T-type calcium channel current by a new dihydropyridine derivative, S11568.

S11568, (+-)[((amino-2-ethoxy)-2-ethoxy]-methyl)-2-(dichloro-2', 3'-phenyl)-4-ethoxycarbonyl-3-methoxycarbonyl-5-methyl-6-dihydro-1,4-pyr idine HCl, is a new dihydropyridine derivative that is water soluble and relatively insensitive to light. The Ca2+ channel inhibitory activity of S11568 was tested in whole-cell patch clamp recordings from cultured embryonic chick cardiomyocytes in 40 mM Ba2(+)-containing medium that revealed T-type and L-type components of inward current through calcium channels. S11568 inhibited L-type Ca2+ current with an IC50 value near 1 microM but was without effect on the T-type barium current.     

Sites of action of Ca2+ channel inhibitors

Ca2+ channel inhibitors are viewed as a subgroup of Ca2+ antagonists. Most of the currently used Ca2+ channel inhibitors are thought to act by reducing Ca2+ entry into the cell through Ca2+ channels. There is substantial electrophysiological evidence that the major site of action of verapamil, nifedipine and diltiazem in cardiac cells is a sarcolemmal Ca2+ channel. Cytosolic sites of action may contribute to their effects but probably only at higher than therapeutic concentrations. The recent ligand binding studies also tend to support the view that the sarcolemma is the site of action of Ca2+ channel inhibitors in smooth muscle. High affinity binding sites for 1,4-dihydropyridines without any established function are found in fast skeletal muscle and some neuronal membranes. The binding of [3H]nitrendipine to membranes from cardiac, skeletal and smooth muscle, and from brain is saturable, reversible and of high affinity; it is sensitive to cations and other drugs that interact with Ca2+ channels. Inhibition of [3H]nitrendipine binding and blockade of K+ responses in guinea pig ileum by 1,4-dihydropyridines are well correlated, supporting the view that the observed binding is to Ca2+ channel. In contrast, blockade of Ca2+ channels in cardiac and skeletal muscle and in brain synaptosomes occurs only at higher concentrations than needed to saturate the high affinity binding sites. The therapeutic success of Ca2+ channel inhibitors in the treatment of angina pectoris, hypertension, peripheral vascular diseases, and many other disease entities is based on selective inhibition of Ca2+ entry into smooth muscle cells. The specificity of some of these drugs for Ca2+ channels in different cell types, organs, or vascular beds is probably determined by receptor modulation and the effect of reflex mechanisms, which in turn determine the indications for their therapeutic use.     

Synthesis and voltage-clamp studies of methyl 1,4-dihydro-2, 6-dimethyl-5-nitro-4-(benzofurazanyl)pyridine-3-carboxylate racemates and enantiomers and of their benzofuroxanyl analogues

Racemic methyl 1,4-dihydro-2, 6-dimethyl-5-nitro-4-(benzofurazanyl)pyridine-3-carboxylates (+/-)-10 and (+/-)-11 and their benzofuroxanyl analogues (+/-)-12 and (+/-)-13 were prepared using a modified Hantzsch reaction that involved the condensation of nitroacetone with methyl 3-aminocrotonate and the appropriate aldehydes. The racemic mixtures were resolved into the corresponding enantiomers. Whole-cell voltage-clamp studies on L-type Ca2+ channels expressed in a rat insulinoma cell line (RINm5F) showed that all the dextrorotatory antipodes were effective agonists of L-type Ca2+ currents, while the levorotatory ones were weak Ca2+ entry blockers. The (+)-enantiomer of benzofurazan-5'-yl derivative 11 demonstrated unusual activity in that, in addition to producing a potentiation of L-type currents, it interfered with the voltage-dependent gating of L-type channels by producing a net delay of their activation at low voltages. This compound represents an interesting tool to probe L-type Ca2+ channel structure and function.     

Inhibition of T-type and L-type calcium channels by mibefradil: physiologic and pharmacologic bases of cardiovascular effects

Ca2+ channel antagonists of the dihydropyridine, benzothiazepine, and phenylalkylamine classes have selective effects on L-type versus T-type Ca2+ channels. In contrast, mibefradil was reported to be more selective for T-type channels. We used the whole-cell patch-clamp technique to investigate the effects of mibefradil on T-type and L-type Ca2+ currents (I(CaT) and I(CaL)) recorded at physiologic extracellular Ca2+ in different cardiac cell types. At a stimulation rate of 0.1 Hz, mibefradil blocked I(CaT) evoked from negative holding potentials (HPs) (-100 mV to -80 mV) with an IC50 of 0.1 microM in rat atrial cells. This concentration had no effect on I(CaL) in rat ventricular cells (IC50: approximately3 microM). However, block of I(CaL) was enhanced when the HP was depolarized to -50 mV (IC50: approximately 0.1 microM). Besides a resting block, mibefradil displayed voltage- and use-dependent effects on both I(CaT) and I(CaL). In addition, inhibition was enhanced by increasing the duration of the step-depolarizations. Similar effects were observed in human atrial and rabbit sinoatrial cells. In conclusion, mibefradil combines the voltage- and use-dependent effects of dihydropyridines and benzothiazepines on I(CaL). Inhibition of I(CaL), which has probably been underestimated before, may contribute to most of the cardiovascular effects of mibefradil.     

Mibefradil (Ro 40-5967): the first selective T-type Ca2+ channel blocker

Mibefradil is a novel Ca2+ antagonist acting on both L- and T-type Ca2+ channels, with a ten-fold selectivity for T-type Ca2+ channels. It belongs to a chemical class different from other Ca2+ antagonists (tetralol derivative), and binds to a new receptor site on the L-type Ca2+ channel, where it does not affect dihydropyridine (DHP) binding but appears to overlap the verapamil and fantofarone sites. In vitro and in vivo studies indicate that mibefradil has a high selectivity for the coronary vasculature over the peripheral vasculature and the myocardium. It has no relevant negative inotropic effects in various animal models, in normotensive patients, and patients with hypertension or angina pectoris. Instead, treatment with mibefradil slightly decreases heart rate and improves cardiac function. Clinical studies confirm that mibefradil is an effective antihypertensive and anti-ischaemic drug, which may be beneficial in the treatment of heart failure. Its excellent pharmacological and safety profile combined with high bioavailability makes it a promising new drug. Many of the unique pharmacological properties of mibefradil may derive from its selective block of T-type Ca2+ channels.     

Nicergoline inhibits T-type Ca2+ channels in rat isolated hippocampal CA1 pyramidal neurones.

1. The effects of nicergoline on the T- and L-type Ca2+ currents in pyramidal cells freshly isolated from rat hippocampal CA1 region were investigated by use of a 'concentration-clamp' technique. The technique combines a suction-pipette technique, which allows intracellular perfusion under a single-electrode voltage-clamp, and rapid exchange of extracellular solution within 2 ms. 2. T-type Ca2+ currents were evoked by step depolarizations from a holding potential of -100 mV to potentials more positive than -70 to -60 mV, and reached a peak at about -30 mV in the current-voltage relationship. Activation and inactivation of T-type Ca2+ currents were highly potential-dependent. 3. Nicergoline and other Ca2+ antagonists dose-dependently blocked the T-type Ca2+ channel with an order of potency nicardipine greater than nicergoline greater than diltiazem. 4. The L-type Ca2+ channel was also blocked in the order nicardipine greater than nicergoline greater than diltiazem, although the T-type Ca2+ channel was more sensitive to nicergoline. 5. The inhibitory effects of nicergoline and nicardipine on the T-type Ca2+ current were voltage-, time-, and use-dependent, and the inhibition increased with a decrease in the external Ca2+ concentration. Diltiazem showed only a use-dependent block.     

High affinity interaction of mibefradil with voltage-gated calcium and sodium channels

Mibefradil is a novel Ca(2+) antagonist which blocks both high-voltage activated and low voltage-activated Ca(2+) channels. Although L-type Ca(2+) channel block was demonstrated in functional experiments its molecular interaction with the channel has not yet been studied. We therefore investigated the binding of [(3)H]-mibefradil and a series of mibefradil analogues to L-type Ca(2+) channels in different tissues. [(3)H]-Mibefradil labelled a single class of high affinity sites on skeletal muscle L-type Ca(2+) channels (K(D) of 2.5+/-0.4 nM, B(max)=56.4+/-2.3 pmol mg(-1) of protein). Mibefradil (and a series of analogues) partially inhibited (+)-[(3)H]-isradipine binding to skeletal muscle membranes but stimulated binding to brain L-type Ca(2+) channels and alpha1C-subunits expressed in tsA201 cells indicating a tissue-specific, non-competitive interaction between the dihydropyridine and mibefradil binding domain. [(3)H]-Mibefradil also labelled a heterogenous population of high affinity sites in rabbit brain which was inhibited by a series of nonspecific Ca(2+) and Na(+)-channel blockers. Mibefradil and its analogue RO40-6040 had high affinity for neuronal voltage-gated Na(+)-channels as confirmed in binding (apparent K(i) values of 17 and 1.0 nM, respectively) and functional experiments (40% use-dependent inhibition of Na(+)-channel current by 1 microM mibefradil in GH3 cells). Our data demonstrate that mibefradil binds to voltage-gated L-type Ca(2+) channels with very high affinity and is also a potent blocker of voltage-gated neuronal Na(+)-channels. More lipophilic mibefradil analogues may possess neuroprotective properties like other nonselective Ca(2+)-/Na(+)-channel blockers.     

Inhibition of T-type and L-type Ca(2+) currents by aranidipine, a novel dihydropyridine Ca(2+) antagonist

The effects of aranidipine, a novel dihydropyridine Ca(2+) channel antagonist, on membrane currents in guinea pig ventricular myocytes and on action potentials in rabbit sinoatrial node tissue were examined. In myocytes, aranidipine (10 nmol/l to 1 micromol/l) concentration-dependently decreased T-type and L-type Ca(2+) currents. Aranidipine (1 micromol/l) had little effect on K(+) currents. In the sinoatrial node, 0.1 micromol/l aranidipine increased cycle length, and decreased +V(max) and the slope of the phase 4 depolarization. Thus, inhibition of both T-type and L-type Ca(2+) currents by aranidipine may partly explain its potent negative chronotropic activity.     

Preferential block of T-type calcium channels by neuroleptics in neural crest-derived rat and human C cell lines.

We have used the whole-cell version of the patch-clamp technique to analyze the inhibition of Ca2+ currents by antipsychotic agents in neural crest-derived rat and human thyroid C cell lines. Diphenylbutylpiperidine (DPBP) antipsychotics, including penfluridol and fluspirilene, potently and preferentially block T-type Ca2+ current in the rat medullary thyroid carcinoma 6-23 (clone 6) cell line. When step depolarizations were applied at 0.1 Hz from a holding potential of -80 mV, with 10 mM Ca2+ as the charge carrier, the DPBP penfluridol inhibited T-type current with an IC50 of 224 nM. High voltage-activated L and N currents were less potently blocked. At a concentration of 500 nM, penfluridol inhibited 78.0 +/- 2.3% (n = 29) of inactivating T-type Ca2+ current, whereas the sustained high voltage-activated current was reduced by 25.6 +/- 3.5% (n = 28). Block of T-type current by penfluridol was enhanced by depolarizing test pulses applied at frequencies above 0.03 Hz. The use-dependent component of block was largely reversed by pulse-free periods at -80 mV. T-type Ca2+ channels in the human TT C cell line were blocked by penfluridol, and the potency was enhanced by reduction of extracellular Ca2+. Non-DPBP antipsychotics, including haloperidol, clozapine, and thioridazine, also blocked T-type channels, but these were 20-100 times less potent than the DPBPs. These results identify the DPBPs as a new class of organic Ca2+ channel antagonists, which are distinctive in their ability to preferentially block T-type channels. These agents will be useful in defining the function of T channels in various excitable cells. Their potent block of T-type Ca2+ channels, which would be enhanced in rapidly firing cells, suggests that this action may be relevant to the therapeutic or toxic effects of these drugs when used in clinical pharmacology.     

Verapamil block of T-type calcium channels

Verapamil is a prototypical phenylalkylamine (PAA), and it was the first calcium channel blocker to be used clinically. It tonically blocks L-type channels in the inner pore with micromolar affinity, and its affinity increases at depolarized membrane potentials. In T-type calcium channels, verapamil blocks with micromolar affinity and has modestly increased affinity at depolarized potentials. We found that a related PAA, 4-desmethoxyverapamil (D888), is comparable with verapamil both in affinity and in state-dependence. Permanently charged verapamil was more effective intracellularly than neutral verapamil. Charged PAAs were able to access their binding site from both inside and outside the cell. Furthermore, membrane-impermeant [2-(trimethylammonium)ethyl]methanethiosulfonate was able to access the inner pore from outside of the cell. We examined a homology model of the T-type calcium channel to look for possible routes of drug entry. Mutation of L1825W produced a channel that was blocked significantly more slowly by charged verapamil from the outside, with an increase in apparent affinity when the drug was applied from the inside. Data suggest that T-type channels have a back pathway through which charged drugs can access the inner pore of the channel without passing through the plasma membrane.     

Synthesis and evaluation of a new class of nifedipine analogs with T-type calcium channel blocking activity

We have synthesized a novel series of 18 dialkyl 1,4-dihydro-4-(2'alkoxy-6'-pentadecylphenyl)-2,6-dimethyl-3,5 pyridine dicarboxylates from anacardic acid, a natural compound found in cashew nut shells, and investigated their blocking action on L- and T-type calcium channels transiently expressed in tSA-201 cells. The IC(50) values for L-type calcium channel block obtained with the series ranged from 1 to approximately 40 microM, with higher affinities being favored by increasing the size of the alkoxy group on the 4-phenyl ring and ester substituent in the 3,5 positions. A detailed analysis of the strongest L-type channel blocker of the series (PPK-12) revealed that block was poorly reversible and mediated an apparent speeding of the time course of inactivation. Moreover, in the presence of PPK-12, the midpoint of the steady state inactivation curve was shifted by 20 mV toward more hyperpolarized potentials, resulting in an increase in blocking efficacy at more depolarized holding potentials. Surprisingly, PPK-12 blocked T- and L-type calcium channels with similar affinities. One of the weakest L-type channel inhibitors (PPK-5) exhibited a T-type channel affinity that was similar to that seen with PPK-12, resulting in a 40-fold selectivity of PPK-5 for T- over L-type channels. Thus, dialkyl 1,4-dihydro-4-(2'alkoxy-6'-pentadecylphenyl)-2,6-dimethyl-3,5 pyridine dicarboxylates may serve as excellent candidates for the development of T-type calcium-channel specific blockers.     

钙通道在心房颤动时表达及功能变化的研究进展

细胞内钙超载在心房颤动(AF)的发生和维持中的作用被广泛关注。AF时快速心房率引起细胞内钙超载,细胞内钙通道的表达和功能改变,使AF维持。T型钙通道参与基础钙的调节,而心肌收缩时,L型钙通道在钙释放过程中起到触发作用。收缩期胞浆内90%的Ca2+由SR上的RyR2释放;舒张期胞浆内70%～75%的Ca2+由SR上的Ca2+-ATP酶摄取贮存。本文对AF时L型、T型钙通道以及SR上RyR2和Ca2+-ATP酶的表达和功能变化进行概述,为AF的防治提供更多的理论依据。     

State-dependent verapamil block of the cloned human Ca(v)3.1 T-type Ca(2+) channel

Verapamil is a potent phenylalkylamine antihypertensive believed to exert its therapeutic effect primarily by blocking high-voltage-activated L-type calcium channels. It was the first clinically used calcium channel blocker and remains in clinical use, although it has been eclipsed by other calcium channel blockers because of its short half-life and interactions with other channels. In addition to blocking L-type channels, it has been reported to block T-type (low-voltage activated) calcium channels. This type of cross-reactivity is likely to be beneficial in the effective control of blood pressure. Although the interactions of T channels with a number of drugs have been described, the mechanisms by which these agents modulate channel activity are largely unknown. Most calcium channel blockers exhibit state-dependence (i.e., preferential binding to certain channel conformations), but little is known about state-dependent verapamil block of T channels. We stably expressed human Ca(v)3.1 T-type channels in human embryonic kidney 293 cells and studied the state-dependence of the drug with macroscopic and gating currents. Verapamil blocked currents at micromolar concentrations at polarized potentials similar to those reported for L-type channels, although unlike for L-type currents, it did not affect current time course. The drug exhibited use-dependence and significantly slowed the apparent recovery from inactivation. Current inhibition was dependent on potential. This dependence was restricted to negative potentials, although all data were consistent with verapamil binding in the pore. Gating currents were unaffected by verapamil. We propose that verapamil achieves its inhibitory effect via occlusion of the channel pore associated with an open/inactivated conformation of the channel.