Regulation of cardiac ion channels by transcription factors: Looking for new opportunities of druggable targets for the treatment of arrhythmias.

Cardiac electrical activity is governed by different ion channels that generate action potentials. Acquired or inherited abnormalities in the expression and/or function of ion channels usually result in electrophysiological changes that can cause cardiac arrhythmias. Transcription factors (TFs) control gene transcription by binding to specific DNA sequences adjacent to target genes. Linkage analysis, candidate-gene screening within families, and genome-wide association studies have linked rare and common genetic variants in the genes encoding TFs with genetically-determined cardiac arrhythmias. Besides its critical role in cardiac development, recent data demonstrated that they control cardiac electrical activity through the direct regulation of the expression and function of cardiac ion channels in adult hearts. This narrative review summarizes some studies showing functional data on regulation of the main human atrial and ventricular Na+, Ca2+, and K+ channels by cardiac TFs such as Pitx2c, Tbx20, Tbx5, Zfhx3, among others. The results have improved our understanding of the mechanisms regulating cardiac electrical activity and may open new avenues for therapeutic interventions in cardiac acquired or inherited arrhythmias through the identification of TFs as potential drug targets. Even though TFs have for a long time been considered as 'undruggable' targets, advances in structural biology have led to the identification of unique pockets in TFs amenable to be targeted with small-molecule drugs or peptides that are emerging as novel therapeutic drugs.

Dronedarone.

Atrial fibrillation (AF), the most common cardiac arrhythmia, is associated with substantial morbidity and mortality. Dronedarone is an amiodarone-like benzofuran which lacks the iodine moiety and presents a methane sulfonyl group that decreases its lipophilicity, thus shortening the half-life and decreasing tissue accumulation. Like amiodarone, dronedarone blocks multiple cardiac ion channels and ß-adrenoceptors, presenting electrophysiological characteristics of all four Vaughan Williams classes of antiarrhythmic drugs. In clinical trials, dronedarone has been found effective for both rhythm and rate control. Dronedarone was more effective than placebo in maintaining sinus rhythm in patients with paroxysmal and/or persistent AF and was also effective for ventricular rate control during AF recurrences, providing incremental rate control on top of standard drugs in permanent AF. Furthermore, in the ATHENA trial, dronedarone reduced the incidence of hospitalization due to cardiovascular events or death in patients with nonpermanent AF. Even when dronedarone was less effective than amiodarone in decreasing AF recurrence, it had a better safety profile, being devoid of thyroid, pulmonary and neurological toxicity. This review analyzes the electrophysiological and pharmacological properties, as well as the efficacy and safety of dronedarone in patients with atrial fibrillation.

New investigational drugs for the management of acute heart failure syndromes.

Acute heart failure syndromes (AHFS) enclose a broad spectrum of conditions with different clinical presentations, heart failure history, pathophysiology, prognosis and treatment. AHFS represent a major public health problem because of their high prevalence, high rates of mortality and readmissions and significant healthcare costs, and a therapeutic challenge for the clinicians because management strategies vary markedly. Traditionally used drugs for the treatment of AHFS, including diuretics, vasodilators and positive inotropics, improve clinical signs and symptoms as well as hemodynamics, but present important limitations, as they fail to reduce and may even increase in-hospital and postdischarge mortality, especially in patients with coronary artery disease. Thus, we need new pharmacological agents to not only improve signs and symptoms and cardiac performance, but also improve both short- and long-term outcomes (hospitalizations/survival). In the last decade, significant efforts have been made to identify new therapeutic targets involved in the genesis/progression of AHFS and to develop new therapeutic strategies that may safely improve outcomes. As a result, several new families of drugs have been developed and are currently studied in experimental models and in Phase II and III clinical trials, in an attempt to define their efficacy and safety profiles as well as their precise role in the treatment of AHFS patients. This review firstly analyzes the main clinical applications and limitations of conventional drugs, and then focuses on the mechanisms of action and effects of recently approved drugs and of new investigational agents on signs, symptoms, hemodynamics and outcomes in AHFS patients.

Effects of MiRP1 and DPP6 beta-subunits on the blockade induced by flecainide of Kv4.3/KChIP2 channels.

BACKGROUND AND PURPOSE: The human cardiac transient outward potassium current (Ito) is believed to be composed of the pore-forming Kv4.3 alpha-subunit, coassembled with modulatory beta-subunits as KChIP2, MiRP1 and DPP6 proteins. beta-Subunits can alter the pharmacological response of Ito; therefore, we analysed the effects of flecainide on Kv4.3/KChIP2 channels coassembled with MiRP1 and/or DPP6 beta-subunits. EXPERIMENTAL APPROACH: Currents were recorded in Chinese hamster ovary cells stably expressing K(V)4.3/KChIP2 channels, and transiently transfected with either MiRP1, DPP6 or both, using the whole-cell patch-clamp technique. KEY RESULTS: In control conditions, Kv4.3/KChIP2/MiRP1 channels exhibited the slowest activation and inactivation kinetics and showed an 'overshoot' in the time course of recovery from inactivation. The midpoint values (Vh) of the activation and inactivation curves for Kv4.3/KChIP2/DPP6 and Kv4.3/KChIP2/MiRP1/DPP6 channels were approximately 10 mV more negative than Vh values for Kv4.3/KChIP2 and Kv4.3/KChIP2/MiRP1 channels. Flecainide (0.1-100 microM) produced a similar concentration-dependent blockade of total integrated current flow (IC50 approximately 10 microM) in all the channel complexes. However, the IC50 values for peak current amplitude and inactivated channel block were significantly different. Flecainide shifted the Vh values of both the activation and inactivation curves to more negative potentials and apparently accelerated inactivation kinetics in all channels. Moreover, flecainide slowed recovery from inactivation in all the channel complexes and suppressed the 'overshoot' in Kv4.3/KChIP2/MiRP1 channels. CONCLUSIONS AND IMPLICATIONS: Flecainide directly binds to the Kv4.3 alpha-subunit when the channels are in the open and inactivated state and the presence of the beta-subunits modulates the blockade by altering the gating function.

Effects of atorvastatin and simvastatin on atrial plateau currents.

Recent evidence has shown that the inhibitors of the 3-hydroxy-3-methylglutaryl coenzyme A reductase (statins) might exert antiarrhythmic effects both in experimental models and in humans. In this study we analyzed the effects of atorvastatin and simvastatin acid (SVA) on the currents responsible for the duration of the plateau of human atrial action potentials: hKv1.5, Kv4.3, and L-type Ca(2+) (I(Ca,L)). hKv1.5 and Kv4.3 currents were recorded in transfected Ltk(-) and Chinese hamster ovary cells, respectively, and I(Ca,L) in mouse ventricular myocytes, using whole-cell patch-clamp. Atorvastatin and SVA produced a concentration-dependent block of hKv1.5 channels (IC(50)=4.5+/-1.7 microM and 5.7+/-0.03 microM, respectively) and shifted the midpoint of the activation and inactivation curves to more negative potentials. Importantly, atorvastatin- and SVA-induced block was added to that produced by quinidine, a drug that blocks hKv1.5 channels by binding to their pore cavity. Atorvastatin and SVA blocked Kv4.3 channels in a concentration-dependent manner (IC(50)=13.9+/-3.6 nM and 7.0+/-0.8 microM, respectively). Both drugs accelerated the inactivation kinetics and shifted the inactivation curve to more negative potentials. SVA (10 nM), but not atorvastatin, also blocked I(Ca,L) producing a frequency-dependent block that, at 2 Hz, reached a 50.2+/-1.5%. As a consequence of these effects, at nanomolar concentrations, atorvastatin lengthened, whereas SVA shortened, the duration of mouse atrial action potentials. The results suggest that atorvastatin and SVA alter Kv1.5 and Kv4.3 channel activity following a complex mechanism that does not imply the binding of the drug to the channel pore.

Bupivacaine effects on hKv1.5 channels are dependent on extracellular pH.

1. Bupivacaine-induced cardiotoxicity increases in hypoxic and acidotic conditions. We have analysed the effects of R(+)bupivacaine on hKv1.5 channels stably expressed in Ltk(-) cells using the whole-cell patch-clamp technique, at three different extracellular pH (pH(o)), 6.5, 7.4 and 10.0. 2. Acidification of the pH(o) from 7.4 to 6.5 decreased 4 fold the potency of R(+)bupivacaine to block hKv1.5 channels. At pH(o) 10.0, the potency of the drug increased approximately 2.5 fold. 3. Block induced by R(+)bupivacaine at pH(o) 6.5, 7.4 and 10.0, was voltage- and time-dependent in a manner consistent with an open state block of hKv1.5 channels. 4. At pH(o) 6.5, but not at pH(o) 7.4 or 10.0, R(+)bupivacaine increased by 95+/-3 % (n=6; P<0.05) the hKv1.5 current recorded at -10 mV, likely due to a drug-induced shift of the midpoint of activation (DeltaV=-8.5+/-1.4 mV; n=7). 5. R(+)bupivacaine development of block exhibited an 'instantaneous' component of block at the beginning of the depolarizing pulse, which averaged 12.5+/-1.8% (n=5) and 4.6+/-1.6% (n=6), at pH(o) 6.5 and 7.4, respectively, and that was not observed at pH(o) 10.0. 6. It is concluded that: (a) alkalinization of the pH(o) increases the potency of block of R(+)bupivacaine, and (b) at pH(o) 6.5, R(+)bupivacaine induces an 'agonist effect' of hKv1.5 current when recorded at negative membrane potentials.

Direct effects of candesartan and eprosartan on human cloned potassium channels involved in cardiac repolarization.

In the present study, we analyzed the effects of two angiotensin II type 1 receptor antagonists, candesartan (0.1 microM) and eprosartan (1 microM), on hKv1.5, HERG, KvLQT1+minK, and Kv4.3 channels expressed on Ltk(-) or Chinese hamster ovary cells using the patch-clamp technique. Candesartan and eprosartan produced a voltage-dependent block of hKv1.5 channels decreasing the current at +60 mV by 20.9 +/- 2.3% and 14.3 +/- 1.5%, respectively. The blockade was frequency-dependent, suggesting an open-channel interaction. Eprosartan inhibited the tail amplitude of HERG currents elicited on repolarization after pulses to +60 mV from 239 +/- 78 to 179 +/- 72 pA. Candesartan shifted the activation curve of HERG channels in the hyperpolarizing direction, thus increasing the current amplitude elicited by depolarizations to potentials between -50 and 0 mV. Candesartan reduced the KvLQT1+minK currents elicited by 2-s pulses to +60 mV (38.7 +/- 6.3%). In contrast, eprosartan transiently increased (8.8 +/- 2.7%) and thereafter reduced the KvLQT1+minK current amplitude by 17.7 +/- 3.0%. Eprosartan, but not candesartan, blocked Kv4.3 channels in a voltage-dependent manner (22.2 +/- 3.5% at +50 mV) without modifying the voltage-dependence of Kv4.3 channel inactivation. Candesartan slightly prolonged the action potential duration recorded in guinea pig papillary muscles at all driving rates. Eprosartan prolonged the action potential duration in muscles driven at 0.1 to 1 Hz, but it shortened this parameter at faster rates (2--3 Hz). All these results demonstrated that candesartan and eprosartan exert direct effects on Kv1.5, HERG, KvLQT1+minK, and Kv4.3 currents involved in human cardiac repolarization.

Effects of bupivacaine and a novel local anesthetic, IQB-9302, on human cardiac K+ channels.

We have studied and compared the effects of bupivacaine with those induced by a new local anesthetic, IQB-9302, on human cardiac K+ channels hKv1.5, Kv2.1, Kv4.3, and HERG. Both drugs have a close chemical structure, only differing in their N-substituent (n-butyl and cyclopropylmethyl, for bupivacaine and IQB-9302, respectively). Both drugs blocked Kv2.1, Kv4.3, and HERG channels similarly. Bupivacaine inhibited these channels by 48.6 +/- 3.4, 45.4 +/- 12.4, and 43.1 +/- 9.1%, respectively, and IQB-9302 by 48.1 +/- 3.3, 36.1 +/- 3.7, and 50.3 +/- 6.6%, respectively. However, bupivacaine was 2.5 times more potent than IQB-9302 to block hKv1.5 channels (EC(50) = 8.9 +/- 1.4 versus 21.5 +/- 4.7 microM). Both drugs induced a time- and voltage-dependent block of hKv1.5 and Kv2.1 channels. Block of Kv4.3 channels induced by either drug was time- and voltage-dependent at membrane potentials coinciding with the activation of the channels. IQB-9302 produced an instantaneous block of Kv4.3 and hKv1.5 channels at the beginning of the depolarizing pulse that can be interpreted as a drug interaction with a nonconducting state. Bupivacaine and IQB-9302 induced a similar degree of block of HERG channels and induced a steep voltage-dependent decrease of the relative current. These results suggest that 1) bupivacaine and IQB-9302 block the open state of hKv1.5, Kv2.1, Kv4.3, and HERG channels; and 2) small differences at the N-substituent of these drugs do not affect the drug-induced block of Kv2.1, Kv4.3, or HERG, but specifically modify block of hKv1.5 channels.

Stereoselective effects of the enantiomers of a new local anaesthetic, IQB-9302, on a human cardiac potassium channel (Kv1.5).

1. The N-substituent of IQB-9302 has the same number of carbons as bupivacaine, but it exhibits a different spatial localization (n-butyl vs cyclopropylmethyl). Thus, the study of the effects of IQB-9302 enantiomers on hKv1.5 channels will lead to a better knowledge of the determinants of stereoselective block. 2. The effects of the IQB-9302 enantiomers were studied on hKv1.5 channels stably expressed in LTK:(-) cells using the whole-cell configuration of the patch-clamp technique. Drug molecular modelling was performed using Hyperchem software. 3. Block induced by IQB-9302 was stereoselective with the R(+) enantiomer being 3.2-fold more potent than the S(-) one (K(D) of 17.8+/-0.5 microM vs 58.6+/-4.0 microM). 4. S(-)- and R(+)IQB-9302 induced-block was time- and voltage-dependent consistent with an electrical distance from the cytoplasmic side of 0.173+/-0.022 (n=12) and 0.181+/-0.018 (n=10), respectively. 5. Potency of block of pipecoloxylidide local anaesthetics was linearly related to the length between the cationic tertiary amine and the end of the substituent. 6. Molecular modelling shows that only when S(-) and R(+) enantiomers are superimposed by their aromatic ring, their N-substituents are in opposite directions, which can explain the stereospecific block induced by bupivacaine and IQB-9302 with hKv1.5 channels. 7. These results suggest that: (a) IQB-9302 enantiomers block the open state of hKv1.5 channels, and (b) the length of the N-substituent in these local anaesthetics and not its volume determines the potency and degree of their stereoselective hKv1.5 channel block.

[Clinical pharmacology of the new antiarrhythmia agents]. / Farmacología clínica de los nuevos medicamentos antiarrítmicos.

Antiarrhythmic drugs (ADs) are the mainstay in the treatment of cardiac arrhythmias. Arrhythmias merit treatment for the relief of symptoms and for the prolongation of survival for decreasing arrhythmic deaths. Since class I ADs can increase arrhythmia mortality in patients with coronary artery disease the interest has shifted to class III ADs, particularly those with greater effect at fast heart rates or with greater selectivity for atrial tissue. The recognition that some cardiac arrhythmias can be attributed to variable expression of specific genes or variability in the function of their protein products offers new perspectives for developing new ADs Moreover, the finding that heart disease alters electrophysiological properties of cardiac tissue suggests that we must target the arrhythmogenic substrate rather than its final electrical product. Advances in molecular genetics and electrophysiology will provide an opportunity to identify new targets and to design new ADs that are more effective and with lower risk of complications than those presently prescribed.

Effects of a quaternary bupivacaine derivative on delayed rectifier K(+) currents.

Block of hKv1.5 channels by R-bupivacaine has been attributed to the interaction of the charged form of the drug with an intracellular receptor. However, bupivacaine is present as a mixture of neutral and charged forms both extra- and intracellularly. We have studied the effects produced by the R(+) enantiomer of a quaternary bupivacaine derivative, N-methyl-bupivacaine, (RB(+)1C) on hKv1.5 channels stably expressed in Ltk(-) cells using the whole-cell configuration of the patch-clamp technique. When applied from the intracellular side of the membrane, RB(+)1C induced a time- and voltage-dependent block similar to that induced by R-bupivacaine. External application of 50 microM RB(+)1C reduced the current at +60 mV by 24+/-2% (n=10), but this block displayed neither time- nor voltage-dependence. External RB(+)1C partially relieved block induced by R-bupivacaine (61+/-2% vs 56+/-3%, n=4, P<0.05), but it did not relieve block induced by internal RB(+)1C. In addition, it did not induce use-dependent block, but when applied in combination with internal RB(+)1C a use-dependent block that increased with pulse duration was observed. These results indicate that RB(+)1C induces different effects on hKv1.5 channels when applied from the intra or the extracellular side of the membrane, suggesting that the actions of bupivacaine are the resulting of those induced on the external and the internal side of hKv1.5 channels.

Losartan and its metabolite E3174 modify cardiac delayed rectifier K(+) currents.

BACKGROUND: The effects of type 1 angiotensin II receptor antagonist losartan and its metabolite E3174 on transmembrane action potentials, hKv1.5, HERG, and I(Ks) currents were analyzed. METHODS AND RESULTS: Guinea pig ventricular action potentials were recorded with microelectrode techniques and hKv1.5 and HERG currents with the whole-cell patch-clamp technique. I(Ks) was recorded in guinea pig ventricular myocytes with the perforated-nystatin-patch configuration. Losartan and E3174 transiently increased the hKv1.5 current by 8.0+/-1.4% and 7.4+/-1.6%, respectively. Thereafter, they produced a voltage-dependent block, E3174 being more potent than losartan (P<0.05) for this effect. Losartan decreased HERG currents elicited at 0 mV (23.3+/-4.8%), whereas E3174 increased the current (30.5+/-6.2%). Both drugs shifted the midpoint of the activation curve of HERG channels to more negative potentials. In ventricular myocytes, losartan and E3174 inhibited the I(Ks) (18.4+/-3.2% and 6. 5+/-0.7%, respectively). Losartan-induced block was voltage-independent, whereas E3174 shifted the midpoint of the activation curve to more negative potentials. Losartan lengthened the duration of the action potentials at both 50% and 90% of repolarization, whereas E3174 slowed only the final phase of the repolarization process. CONCLUSIONS: These results demonstrated that at therapeutic concentrations, both losartan and E3174 modified the cardiac delayed rectifier hKv1.5, HERG, and Ks currents.

Blockade of cardiac potassium and other channels by antihistamines.

The use of terfenadine and astemizole, two long-acting nonsedating histamine H1 receptor antagonists, has been associated with prolongation of the QT interval, development of ventricular arrhythmias, particularly torsade de pointes, and sudden cardiac death. Both drugs block the rapidly activating component of the delayed rectifier channel, I(Kr). At much higher concentrations, they also block several other cardiac channels (Na+, Ca2+, K+). Since many other antihistamines can also block one or other of the cardiac ion currents (e.g. loratadine blocks the human cardiac K+ channel, hKv1.5, with the same potency as terfenadine), these results are also reviewed and their clinical relevance discussed. Because of the proarrhythmic risk, some antihistamines should be taken only at the recommended doses and avoided in patients with liver disease or in those taking medications that inhibit oxidative cytochrome P-450 enzymes. These drugs should also be avoided in those with the congenital long QT syndrome or with secondary forms of delayed repolarisation (hypokalaemia, bradycardia, drug-induced QT prolongation). Identification of predisposing factors could enable physicians to anticipate, and thereby avoid, this potentially lethal complication of antihistamine therapy.

Effects of rupatadine, a new dual antagonist of histamine and platelet-activating factor receptors, on human cardiac kv1.5 channels.

1. The effects of rupatadine, a new dual antagonist of both histamine H1 and platelet-activating factor receptors, were studied on human cloned hKv1.5 channels expressed in Ltk- cells using the whole-cell patch-clamp technique. 2. Rupatadine produced a use- and concentration-dependent block of hKv1.5 channels (KD=2.4+/-0.7 micronM) and slowed the deactivation of the tail currents, thus inducing the 'crossover' phenomenon. 3. Rupatadine-induced block was voltage-dependent increasing in the voltage range for channel opening suggesting an open channel interaction. At potentials positive to +10 mV the blockade decreased with a shallow voltage-dependence. Moreover, rupatadine also modified the voltage-dependence of hKv1.5 channel activation, which exhibited two components, the midpoint of the steeper component averaging -25. 2+/-2.7 mV. 4. When the intracellular K+ concentration ([K+]i) was lowered to 25% the voltage-dependent unblock observed at positive potentials was suppressed and the activation curve in the presence of rupatadine did not exhibit two components even when the midpoint of the activation curve was shifted to more negative potentials (-30. 3+/-1.3 mV). 5. On channels mutated on the residue R485 (R485Y) which is located on the external entryway of the pore the rupatadine-induced block did not decrease at potentials positive to +10 mV. In contrast, on V512M channels rupatadine reproduced all the features of the blockade observed on wild type channels. 6. All these results suggest that rupatadine blocks hKv1.5 channels binding to an external and to an internal binding site but only at concentrations much higher than therapeutic plasma levels in man. Efflux of K+ promotes the unbinding from the external site. Furthermore, rupatadine binds to an internal site and dramatically modifies the voltage-dependence of channel opening.

Benzocaine enhances and inhibits the K+ current through a human cardiac cloned channel (Kv1.5).

OBJECTIVE: The aim of this study was to analyze the effects of a neutral local anaesthetic, benzocaine, on a cardiac K+ channel cloned from human ventricle. METHODS: Experiments were performed on hKv1.5 channels stably expressed on mouse cells using the whole-cell configuration of the patch clamp technique. RESULTS: At 10 nM, benzocaine increased the current amplitude ("agonist effect") by shifting the activation curve 8.4 +/- 2.7 mV in the negative direction, and slowed the time course of tail current decline. In contrast, benzocaine (100-700 microM) inhibited hKv1.5 currents (KD = 901 +/- 81 microM), modified the voltage-dependence of channel activation, which became biphasic, and accelerated the channel deactivation. Extracellular K+ concentration ([K+]o) also affected the channel gating. At 140 mM [K+]o, the time course of tail currents deactivation was significantly accelerated, whereas at 0 mM [K+]o, it was slowed. At both [K+]o the activation curve became biphasic. Benzocaine accelerated the tail current decay at 0 mM but not at 140 mM [K+]o. The reduction in the permeation of K+ through the pore did not modify the blocking effects of micromolar concentrations of benzocaine, but suppressed the agonist effect observed at nanomolar concentrations. CONCLUSIONS: All these results suggest that benzocaine blocks and modifies the voltage- and time-dependent properties of hKv1.5 channels, binding to an extracellular and to an intracellular site at the channel level. Moreover, both sites are related to each other and can also interact with K+.

Effects of propafenone and 5-hydroxy-propafenone on hKv1.5 channels.

1. The goal of this study was to analyse the effects of propafenone and its major metabolite, 5-hydroxy-propafenone, on a human cardiac K+ channel (hKv1.5) stably expressed in Ltk- cells and using the whole-cell configuration of the patch-clamp technique. 2. Propafenone and 5-hydroxy-propafenone inhibited in a concentration-dependent manner the hKv1.5 current with K(D) values of 4.4+/-0.3 microM and 9.2+/-1.6 microM, respectively. 3. Block induced by both drugs was voltage-dependent consistent with a value of electrical distance (referenced to the cytoplasmic side) of 0.17+/-0.55 (n=10) and 0.16+/-0.81 (n=16). 4. The apparent association (k) and dissociation (l) rate constants for propafenone were (8.9+/-0.9) x 10(6) M(-1) s(-1) and 39.5+/-4.2 s(-1), respectively. For 5-hydroxy-propafenone these values averaged (2.3+/-0.3) x 10(6) M(-1) s(-1) and 21.4+/-3.1 s(-1), respectively. 5. Both drugs reduced the tail current amplitude recorded at -40 mV after 250 ms depolarizing pulses to +60 mV, and slowed the deactivation time course resulting in a 'crossover' phenomenon when the tail currents recorded under control conditions and in the presence of each drug were superimposed. 6. Both compounds induced a small but statistically significant use-dependent block when trains of depolarizations at frequencies between 0.5 and 3 Hz were applied. 7. These results indicate that propafenone and its metabolite block hKv1.5 channels in a concentration-, voltage-, time- and use-dependent manner and the concentrations needed to observe these effects are in the therapeutical range.

Structural determinants of potency and stereoselective block of hKv1.5 channels induced by local anesthetics.

Block of hKv1.5 channels by bupivacaine is stereoselective, with (R)-(+)-bupivacaine being 7-fold more potent than (S)-(-)-bupivacaine. The study of the effects of chemically related enantiomers on these channels may help to elucidate the structural determinants of stereoselective hKv1.5 channels block by local anesthetics. In this study, we analyzed the effects of (R)-(+)-ropivacaine, (R)-(+)-mepivacaine, and (S)-(-)-mepivacaine on hKv1.5 channels stably expressed in Ltk- cells. (R)-(+)-Ropivacaine inhibited hKv1.5 current and induced a fast initial decline superimposed to the slow inactivation during the application of depolarizing pulses, which reached steady state at the end of 250-msec depolarizing pulses. The concentration-dependence block induced by (R)-(+)-ropivacaine yielded a KD value of 32 +/- 1 microM [i.e., 2.5-fold more potent than (S)-(-)-ropivacaine]. (R)-(+)-Ropivacaine block also was voltage dependent, with a fractional electrical distance (delta) of 0.156 +/- 0.003 (n = 14) referred to the inner surface. Both (S)-(-)- and (R)-(+)-mepivacaine blocked hKv1.5 channels, with KD values of 286.8 +/- 34.1 and 379.0 +/- 56.0 microM, respectively [i.e., block was not stereoselective (p > 0.05)]. (S)-(-)-Mepivacaine and (R)-(+)-mepivacaine block displayed no apparent time-dependence due to a very fast dissociation rate constant. However, block by mepivacaine enantiomers was voltage dependent, with delta values of 0.154 +/- 0.015 and 0.160 +/- 0.008 for the (S)-(-)- and (R)-(+)-enantiomers, respectively. We conclude that (1) (R)-(+)-ropivacaine and mepivacaine enantiomers block the open state of hKv1.5 channels and (2) the length of their alkyl substituent at position 1 determines the potency and the degree of stereoselectivity.

Molecular determinants of stereoselective bupivacaine block of hKv1.5 channels.

Enantiomers of local anesthetics are useful probes of ion channel structure that can reveal three-dimensional relations for drug binding in the channel pore and may have important clinical consequences. Bupivacaine block of open hKv1.5 channels is stereoselective, with the R(+)-enantiomer being 7-fold more potent than the S(-)-enantiomer (Kd = 4.1 mumol/L versus 27.3 mumol/L). Using whole-cell voltage clamp of hKv1.5 channels and site-directed mutants stably expressed in Ltk- cells, we have identified a set of amino acids that determine the stereoselectivity of bupivacaine block. Replacement of threonine 505 by hydrophobic amino acids (isoleucine, valine, or alanine) abolished stereoselective block, whereas a serine substitution preserved it [Kd = 60 mumol/L and 7.4 mumol/L for S(-)- and R(+)-bupivacaine, respectively]. A similar substitution at the internal tetraethylammonium binding site (T477S) reduced the affinity for both enantiomers similarly, thus preserving the stereoselectivity [Kd = 45.5 mumol/L and 7.8 mumol/L for S(-)- and R(+)-bupivacaine, respectively]. Replacement of L508 or V512 by a methionine (L508M and V512M) abolished stereoselective block, whereas substitution of V512 by an alanine (V512A) preserved it. Block of Kv2.1 channels, which carry valine, leucine, and isoleucine residues at T505, L508, and V512 equivalent sites, respectively, was not stereoselective [Kd = 8.3 mumol/L and 13 mumol/L for S(-)- and R(+)-bupivacaine, respectively]. These results suggest that (1) the bupivacaine binding site is located in the inner mouth of the pore, (2) stereoselective block displays subfamily selectivity, and (3) a polar interaction with T505 combined with hydrophobic interactions with L508 and V512 are required for stereoselective block.

Block of human cardiac Kv1.5 channels by loratadine: voltage-, time- and use-dependent block at concentrations above therapeutic levels.

OBJECTIVE: The aim of this study was to analyze the effects of loratadine on a human cardiac K+ channel (hKv1.5) cloned from human ventricle and stably expressed in a mouse cell line. METHODS: Currents were studied using the whole-cell configuration of the patch-clamp technique in Ltk- cells transfected with the gene encoding hKv1.5 channels. RESULTS: Loratadine inhibited in a concentration-dependent manner the hKv1.5 current, the apparent affinity being 1.2 +/- 0.2 microM. The blockade increased steeply between -30 and 0 mV which corresponded with the voltage range for channel opening, thus suggesting that the drug binds preferentially to the open state of the channel. The apparent association and dissociation rate constants were (3.6 +/- 0.5) x 10(6).M-1.s-1 and 3.7 +/- 1.6.s-1, respectively. Loratadine, 1 microM, increased the time constant of deactivation of tail currents elicited on return to -40 mV after 500 ms depolarizing pulses to +60 mV from 36.2 +/- 3.4 to 64.9 +/- 3.6 ms (n = 6, P < 0.01), thus inducing a 'crossover' phenomenon. Application of trains of pulses at 1 Hz lead to a progressive increase in the blockade reaching a final value of 48.6 +/- 4.3%. Recovery from loratadine-induced block at -80 mV exhibited a time constant of 743.0 +/- 78.0 ms. Finally, the results of a mathematical stimulation of the effects of loratadine, based on an open-channel block model, reproduced fairly well the main effects of the drug. CONCLUSIONS: The present results demonstrated that loratadine blocked hKv1.5 channels in a concentration-, voltage-, time- and use-dependent manner but only at concentrations much higher than therapeutic plasma levels in man.

[Pharmacology of nitrates]. / Pharmacologie des dérivés nitrés.

Nitrates (nitroglycerin, isosorbide dinitrate and isosorbide 5-mononitrate) are drugs used in the treatment of angina pectoris and in the prevention of myocardial ischaemia. By providing nitric oxide (NO), which, in turn, increases the level of cGMP and decreases (Ca)i, they induce effects of vasodilatation and anti-platelet aggregation. In patients suffering from endothelial dysfunction, they therefore act like exogenous NO providers. At low doses, they act as venous vasodilators by decreasing the tension (preload) and volume of the ventricle, as well as myocardial oxygen requirements (MVO2). The reduction of ventricular tension and volume also indirectly increases subendocardial blood flow. In the coronary vessels, they produce epicardial vasodilatation (all the more marked the smaller the calibre of the vessels), increase collateral blood flow and, through stenoses, induce reduction of coronary tone and the excess pressure due to vasospasm. At higher doses, the vasodilator effect is exerted on arteries and, although it reduces peripheral vascular resistance (afterload) and blood pressure, it can also produce reflex tachycardia which annihilates the reduction of MVO2. Finally, nitrates exert anti-platelet aggregation effects. All these properties account for the beneficial effects of nitrates in acute or chronic coronary ischaemic syndromes, in patients suffering from left ventricular dysfunction, during the acute phase of myocardial infarction and in post-myocardial infarction ventricular remodelling. There are many pharmaceutical formulations allowing administration of nitrates via a variety of routes. However, the efficacy of repeated nitrate administration is limited by the appearance of tolerance, which can be prevented by observing short periods without nitrates (8-24 hours). The use of sustained-release formulations, with a single daily dose, ensures a maximum anti-ischaemic effect, reduces the risk of tolerance and facilitates the patient's compliance with treatment, which makes it the treatment of choice in angina patients.