Skip Navigation

Cardiovascular Research 2004 64(3):457-466; doi:10.1016/j.cardiores.2004.07.022
© 2004 by European Society of Cardiology
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Caballero, R.
Right arrow Articles by Delpón, E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Caballero, R.
Right arrow Articles by Delpón, E.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 2004, European Society of Cardiology

Diltiazem inhibits hKv1.5 and Kv4.3 currents at therapeutic concentrations

Ricardo Caballero*,1, Ricardo Gómez1, Lucía Núñez, Ignacio Moreno, Juan Tamargo and Eva Delpón

Department of Pharmacology, School of Medicine, Universidad Complutense, 28040 Madrid, Spain

* Corresponding author. Tel.: +34 91 394 14 74; fax: +34 91 394 14 70. Email address: rcaballero{at}ift.csic.es

Received 26 April 2004; revised 27 July 2004; accepted 28 July 2004


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Objective: In the present study we examined the effects of diltiazem, an L-type Ca2+ channel blocker widely used for the control of the ventricular rate in patients with supraventricular arrhythmias, on hKv1.5 and Kv4.3 channels that generate the cardiac ultrarapid delayed rectifier (IKur) and the 4-aminopyridine sensitive transient outward (Ito) K+ currents, respectively.

Methods: hKv1.5 and Kv4.3 channels were stably and transiently expressed in mouse fibroblast and Chinese hamster ovary cells, respectively. Currents were recorded using the whole-cell patch clamp.

Results: Diltiazem (0.01 nM-500 µM) blocked hKv1.5 channels, in a frequency-dependent manner exhibiting a biphasic dose-response curve (IC50=4.8±1.5 nM and 42.3±3.6 µM). Diltiazem delayed the initial phase of the tail current decline and shifted the midpoint of the activation (Vh=–16.5±2.1 mV vs –20.4±2.6 mV, P<0.001) and inactivation (Vh=–22.4±0.7 mV vs. –28.2±1.9 mV, P<0.001) curves to more negative potentials. The analysis of the development of the diltiazem-induced block yielded apparent association (k) and dissociation (P) rate constants of (1.6±0.2)x106 M–1s–1 and 46.8±4.8 s–1, respectively. Diltiazem (0.1 nM-100 µM) also blocked Kv4.3 channels in a frequency-dependent manner exhibiting a biphasic dose-response curve (IC50=62.6±11.1 nM and 109.9±12.8 µM). Diltiazem decreased the peak current and, at concentrations ≤0.1 µM, accelerated the inactivation time course. The apparent association and dissociation rate constants resulted (1.7±0.2)x106 M–1s–1 and 258.6±38.1 s–1, respectively. Diltiazem, 10 nM, shifted to more negative potentials the voltage-dependence of Kv4.3 channel inactivation (Vh=–33.1±2.3 mV vs –38.2±3.5 mV, n=6, Plt;0.05) the blockade increasing at potentials at which the amount of inactivated channels increased.

Conclusion: The results demonstrated for the first time that diltiazem, at therapeutic concentrations, decreased hKv1.5 and Kv4.3 currents by binding to the open and the inactivated state of the channels.


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
L-type Ca2+ channel blockers are widely used for the treatment of hypertension or coronary artery disease. Verapamil and diltiazem slow heart rate and prolong atrioventricular conduction, effects that can control the rates of ventricular response in patients with atrial arrhythmias [1]. Ca2+ channel blockers also block several K+ channels. Verapamil is a potent blocker of HERG channels that generate the rapidly activating delayed rectifier current (IKr), whereas diltiazem only weakly suppresses HERG current, and nifedipine has no effect [2]. Verapamil and nifedipine also block hKv1.5 channels at the low micromolar range ({approx}20 µM) and the features of the drug-induced hKv1.5 block have been previously described in detail [3,4]. Diltiazem, at micromolar concentrations ({approx}115 µM), decreased Kv1.5 currents recorded in mouse erythroleukemia (MEL) cells [5] and Xenopus oocytes [6], but detailed characteristics of diltiazem's effects have not been studied. Furthermore, it has also been described that verapamil and nifedipine block the transient outward current (Ito) in rat ventricular myocytes [7], whereas the effects of diltiazem on either Ito or Kv4.3 channels are unknown.

Kv1.5 channels are highly expressed in human atria and conduct the ultra-rapid delayed rectifier current (IKur) that is present in atrial but not in ventricular tissue. Thus, IKur contributes exclusively to atrial action potential repolarization [8]. Human atrial tissue also exhibits a robust 4-aminopyridine sensitive component of the transient outward current (Ito1) [9]. Human cardiac Ito1 is carried by Kv4.3 {alpha}-subunits [10], co-assembled with KChIP2s auxiliary β-subunits [11]. Therefore, both Ito1 and IKur contribute to the duration of the human atrial action potential [12]. The present study was undertaken to analyze the effects of diltiazem on hKv1.5 and Kv4.3 channels. The results demonstrated that diltiazem, at therapeutic concentrations, decreased hKv1.5 and Kv4.3 currents by binding to the open and the inactivated states of the channels.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Acknowledgments
 References
 
2.1 Transmembrane action potentials
Action potentials were recorded in left atria from male ICR (CD-1) Swiss strain mice (35–40 g) perfused with Tyrode solution (34 °C) and driven at 3 Hz using microelectrode techniques [13,14]. The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, Revised 1996).

2.2 Cell culture
Ltk– cells stably expressing hKv1.5 channels and CHO cells were grown in Hams-F12 with 10% fetal bovine serum in a 5% CO2 atmosphere as previously described [13,14]. The transient expression of Kv4.3 channels on CHO cells has been also described [14,15]. Briefly, the cells were transfected with the cDNA encoding Kv4.3 channels (3 µg) together with the cDNA encoding the CD8 antigen (0.5 µg) using lipofectamine (Gibco). Before experimental use, cells were incubated with polystyrene microbeads precoated with anti-CD8 antibody (Dynabeads M450; Dynal, Norway). Most of the cells that were beaded also had channel expression [14].

2.3 Solutions and drugs
Tyrode's solution contained (mM): NaCl 137, KCl 5.4, MgCl2 1.0, CaCl2 1.8, NaH2PO4 0.42, NaHCO3 11 and dextrose 10. Ltk– and CHO cells were superfused with an external solution containing (mM): NaCl 130, KCl 4, CaCl2 1, MgCl2 1, HEPES 10 and glucose 10 (pH=7.4 with NaOH). The internal solution contained (mM): K-aspartate 80, KCl 42, KH2PO4 10, MgATP 5, phosphocreatine 3, HEPES 5 and EGTA 5 (pH=7.2 with KOH). (2S,3S)-cis-diltiazem (Sigma) was dissolved in water to yield 0.01 M stock solutions.

2.4 Recording Techniques
Currents were recorded at 21–23 °C using the whole-cell configuration of the patch-clamp with Axopatch 200 B amplifiers and PCLAMP 9.0 software (Axon Instruments, Foster City, CA, USA). hKv1.5 and Kv4.3 currents were sampled at 2 and 4 kHz, respectively and filtered at half the sampling frequency. Micropipette resistance was kept <3.5 M{Omega} when filled with the internal solution and immersed in the external solution. Maximum hKv1.5 current amplitudes averaged 1.7±0.2 nA (n=20) and access resistance and cell capacitance 4.3±0.2 M{Omega} and 8.5±0.4 pF, respectively (n=20). CHO cells capacitance averaged 14.9±0.7 pF and access resistance 4.5±0.2 M{Omega} (n=20). Maximum Kv4.3 currents averaged 2.7±0.4 nA (n=10). Typically, {approx}80% of capacitance and series resistance could be compensated, which leads to mean uncompensated access resistances of 2.6±0.4 M{Omega} and 1.7±0.1 M{Omega} for Ltk– and CHO cells, respectively. Thus, under these conditions no significant voltage errors (<5 mV) due to series resistance were expected with the micropipettes used.

2.5 Pulse protocols and analysis
The holding potential was maintained at –80 mV and the cycle time for any protocol was 10 s to avoid accumulation of inactivation and/or block. The protocol to obtain current–voltage relationships consisted of 250 ms (Kv4.3) or 500 ms (hKv1.5) pulses that were imposed in 10 mV increments between –80 and +60 mV for hKv1.5 and between –90 and +50 mV for Kv4.3, respectively. Deactivating hKv1.5 "tail" currents were recorded on return to –40 mV. The activation curves of hKv1.5 channels were constructed by plotting tail current amplitudes as a function of the membrane potential. Voltage dependence of Kv4.3 channel activation or conductance (G) was determined from the relationship:



Formula 1

where Itp is the current amplitude at the test potential (Vm) and VR is the reversal potential (–72.4±6.4 mV, n=6).

To obtain the inactivation curves of hKv1.5 and Kv4.3 channels, a two-step voltage-clamp protocol was used. The first 10-s (hKv1.5) or 250-ms (Kv4.3) conditioning pulse from –80 to potentials between –90 and +50 mV was followed by a test pulse to +60 mV (hKv1.5) or to +40 mV (Kv4.3). Inactivation curves were constructed plotting the current amplitude as a function of the voltage command of the conditioning pulse.

To describe the time course of current activation and/or inactivation upon depolarization, as well as the tail currents upon repolarization, an exponential analysis was used [13–15].

To obtain the IC50 (concentration of drug that produces the half-maximum blockade) and the Hill coefficient, nH, the fractional block obtained at various drug concentrations [D] was fitted to the equation:



Formula 2

Under some circumstances, a Hill equation with two terms was needed to fit the data, the equation being:



Formula 3

where the sum of B1 and B2 represents the total amount of block reached.

Apparent rate constants for association (k) and dissociation (l) were obtained from fitting:



Formula 4

where {tau}Block is the time constant of development of block.

2.6 Statistical methods
Data obtained in the absence and the presence of diltiazem were compared in a paired manner. For comparisons at a single voltage, differences were analyzed using the Student's t-test. To analyze block at multiple voltages, two-way analysis of variance was used, followed by Newman–Keuls test. Results were expressed as mean±S.E.M. A P-value of less than 0.05 was considered as significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Acknowledgments
 References
 
3.1 Effects of diltiazem on hKv1.5 currents
Fig. 1A shows hKv1.5 current traces recorded by applying 500-ms pulses from –80 to +60 mV in the absence and the presence of 10 nM diltiazem. At this concentration, diltiazem did not significantly modify the peak current amplitude (2.7±1.4%, n=12) and the time constant of current activation (0.9±0.1 vs. 0.9±0.1 ms, n=12, P>0.05). In control conditions one component was required to describe the time course of the slow and partial inactivation of the current ({tau}c=145.9±17.7 ms). In the presence of diltiazem, the current decline was also fitted to a monoexponential function and diltiazem did not modify the time course of this process ({tau}DTZ=131.4±10.1 ms, n=12, P>0.05). However, it increased the amplitude of the decaying component from 685±122 to 911±144 pA (n=12, P<0.01). Thus, diltiazem reduced hKv1.5 currents at the end of the pulses to +60 mV by 22.8±2.1% (n=12), this effect being reversible upon washout during 5–7 min with drug-free solution (Fig. 1A). The time course of tail currents elicited upon repolarization to –40 mV after 500-ms pulses to +60 mV was fitted by a biexponential function, the fast ({tau}f) and the slow ({tau}s) time constants averaging 20.3±3.1 and 90.6±15.6 ms, respectively (Fig. 1B). Diltiazem slowed the tail current deactivation, increasing the {tau}f to 26.2±3.7 ms (P<0.05, n=12). This effect was more marked as the drug concentration was increased, in fact; 10 µM diltiazem increased the {tau}f to 41.6±5.5 ms (P<0.05, n=6), leading to a clear crossover of the tail current (Fig. 1B).


Figure 1
View larger version (24K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 Effects of diltiazem on hKv1.5 currents. (A) Current traces obtained by applying 500-ms pulses to +60 mV in the absence, presence and after washout of 10 nM diltiazem. The lower part shows the plot of the ratio between the diltiazem-sensitive current during the depolarizing pulse (IC–IDTZ) and the current in control conditions. The continuous line represents the fit to a monoexponential function to obtain the {tau}block. (B) Tail currents obtained upon repolarization to –40 mV after 500-ms pulses to +60 mV in the absence and presence of diltiazem. (C) Reduction of total hKv1.5 charge crossing at +60 mV (open symbols) and of current amplitude elicited at +50 mV (closed symbols) were plotted as a function of the diltiazem concentration. The data were fitted with a Hill equation of one (dashed line) or two (continuous line) components (Eqs. (2) and (3)Go, respectively). (D) Normalized hKv1.5 current amplitude mean±S.E.M elicited by pulses to +60 mV in 20 cells as a function of the time after seal breaking. (E) Currents elicited by pulses to +60 mV in the absence and in the presence of increasing concentrations of diltiazem. (F) 1/{tau}Block as a function of the diltiazem concentration for data obtained at concentrations in the range between 0.1 and 500 µM. The straight line is the least-squares fit to Eq. (4) and the dotted line the 95% confidence interval of the fit. In panels A, B and E, the dotted line represents the zero current level. In panels C and F, each point represents the mean±S.E.M of >5 experiments.

 
The blockade at the end of 500-ms pulses to +60 mV was used as an index of block and represented as a function of the diltiazem concentration (Fig. 1C). The percentage of block remained almost constant for concentrations ranging from 0.01 to 1 nM, and thereafter, it increased as the concentration of diltiazem increased. However, the high affinity of diltiazem for hKv1.5 channels cannot be attributed to a time-dependent rundown of the current, since under our experimental conditions current amplitude remained unchanged during the time of recordings (Fig. 1D). The fit to a Hill equation obtained when assuming two components (4.8±1.5 nM and 42.3±3.6 µM) (Eq. (3), continuous line in Fig. 1C) was better than that obtained when assuming a single component [23.4±5.5 µM (nH=0.7±0.1)] (Eq. (2), dashed line). The ratio between the diltiazem-sensitive current during the depolarizing pulse and the current in control conditions [(IC–IDTZ)/IC] is shown in the lower part of Fig. 1A. The blockade increased during the application of the pulse. The onset of block was fitted by a monoexponential function (solid curve), to determine the time constant of development of block, which at 10 nM averaged 159±25 ms (n=12). Fig. 1E shows superimposed outward currents elicited by pulses to +60 mV in the absence and the presence of increasing concentrations of diltiazem, and Fig. 1F shows the plot of the 1/{tau}Block as a function of the diltiazem concentration for data obtained at concentrations between 0.1 and 500 µM. The straight line is the least-squares fit to Eq. (4). Slope and intercept with the ordinate axis for the fitted relation yielded apparent association and dissociation rate constants of (1.6±0.2)x106 M–1 s–1 and 46.8±4.8 s–1, respectively.

The upper panel of Fig. 2 shows representative hKv1.5 current traces obtained in the presence and the absence of 10 nM diltiazem, and Fig. 2A the current–voltage relationship (500 ms isochronal). Diltiazem significantly decreased the current amplitude at potentials positive to –10 mV. The IDTZ/ICON ratio was plotted as a function of the membrane potential. The blockade increased steeply in the voltage range coinciding with that of channel activation (between –20 and 0 mV) and thereafter it remained constant. The hKv1.5 charge (estimated from the integral of the current traces) as a function of the membrane potential in the absence and the presence of 10 nM diltiazem was plotted in Fig. 2B. Diltiazem significantly decreased the charge at potentials positive to –10 mV (n=12, P<0.05). The blockade increased steeply in the voltage range coinciding with that of channel activation and, thereafter, it remained constant. Furthermore, blockade of the charge at +60 mV (18.6±1.4%, n=12, P>0.05) was not statistically different from the blockade measured at the end of the pulse. When the decrease in the charge at +60 mV was used as an index of block, the IC50 values obtained for the biphasic curve (open symbols in Fig. 1C) averaged 28.7±2.9 nM and 63.7±9.8 µM, respectively, whereas the IC50 obtained for the monophasic curve averaged 20.2±6.8 µM (nH=0.8±0.2). Fig. 2C shows the activation curves in the absence and the presence of 10 nM diltiazem. Under control conditions, the activation curve yielded Vh and k values of –16.5±2.1 and 4.3±0.5 mV, respectively (n=12). Diltiazem decreased the tail current amplitude at potentials positive to 0 mV (16.9±2.4% at +60 mV, P<0.05) and shifted the Vh to more negative potentials (–20.4±2.6 mV, P<0.001) without modifying the k value (3.9±0.5 mV, P>0.05). Fig. 2D shows the effects of diltiazem on the voltage dependence of hKv1.5 channels slow inactivation. Diltiazem shifted the half-inactivation voltage from –22.4±0.7 to –28.2±1.9 mV (n=9, P<0.001) without modifying the slope of the curve (k=3.3±0.3 mV). Fractional block demonstrated that blockade increased in the voltage range of channel inactivation, reaching a 30.3±10.1% at –30 mV.


Figure 2
View larger version (44K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 The upper inset shows current traces obtained by applying 500-ms pulses to potentials ranging from –80 to +60 mV followed by the tail currents obtained upon repolarization to –40 mV in the absence (left panel) or the presence (right panel) of 10 nM diltiazem. (A) Current–voltage relationships (500 ms isochronal) of hKv1.5 channels in the absence and in the presence of diltiazem. (B) Relationship between the current–time integral and the membrane potential in the absence and in the presence of diltiazem. (C) Activation curves in the absence and the presence of diltiazem. The continuous lines represent the fit of the data to a Boltzmann equation and the dashed line the activation curve in the presence of diltiazem normalized to the control amplitude. (D) Inactivation curves in the absence and in the presence of 10 nM diltiazem. Continuous lines represent the fit of the data to a Boltzmann equation. Squares in panels A, B, C and D represent the fractional block (IDTZ/ICON) at each potential. *P<0.05 vs. control data. Each data point represents the mean±S.E.M of >5 experiments.

 
3.2 Effects of diltiazem on Kv4.3 currents
Fig. 3A shows Kv4.3 current traces elicited when applying 250-ms pulses to +50 mV in the absence and the presence of 10 nM diltiazem. The currents rose rapidly to a peak ({tau}act=0.9±±0.1 ms, n=10), and then inactivated following a biexponential process ({tau}f=19.2±2.3 ms and {tau}s=54.5±9.9 ms at +50 mV, n=5). Diltiazem 10 nM decreased the peak current by 23.3±3.5% (n=5, P<0.05) but, at this concentration, did not modify the time course of current decay ({tau}f=18.3±2.0 ms and {tau}s=58.7±7.1 ms, n=5, P>0.05). The effects of diltiazem on Kv4.3 channels were reversible upon superfusion with drug-free external solution for 7–10 min (Fig. 3A). Fig. 3B shows Kv4.3 traces obtained in the presence of increasing diltiazem concentrations. As the concentration increased, the peak current amplitude decreased, and at concentrations ≤0.1 µM, diltiazem accelerated the time course of current decay. In fact, at 0.1 µM, diltiazem {tau}f decreased from 20.4±2.7 to 14.9±1.6 ms (n=5, P<0.05). Fig. 3C shows the concentration–response curve obtained by plotting the peak reduction at +50 mV as a function of the diltiazem concentration. The solid line represented the fit to a single Hill equation (IC50=97.7±15.1 µM, nH=0.7±0.1). However, the actions of diltiazem at concentrations ≤0.1 µM are suggestive of an open-channel block, and thus, the reduction of peak current would not represent the steady-state block. Therefore, diltiazem-induced block was also measured as the reduction of the total charge crossing the membrane estimated from the integral of the current traces elicited at +50 mV. Using this index of block, the data clearly deviated from the continuous line which represented the fit to a Hill equation of a single component (IC50=22.9±9.9 µM, nH=0.4±0.1) because the concentration–response exhibited two components described by two IC50 values that averaged 62.6±11.1 nM and 109.9±12.8 µM, respectively. The high affinity of diltiazem for Kv4.3 channels cannot be attributed to a time-dependent rundown of the current, since current amplitude remained unchanged during the time of recordings (Fig. 3D).


Figure 3
View larger version (20K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 Effects of diltiazem on Kv4.3 currents. (A) Current traces obtained by applying 250-ms pulses to +50 mV in the absence, the presence, and after washout of 10 nM diltiazem. (B) Current traces obtained in the absence and the presence of increasing diltiazem concentrations. In panels A and B, the dotted line represents the zero current level. (C) Reduction of total Kv4.3 charge crossing at +50 mV (open symbols) and reduction of peak current amplitude elicited at +50 mV (closed symbols) were plotted as a function of the diltiazem concentration. The data were fitted with a Hill equation of one (continuous line) or two (dashed line) components (Eqs. (2) and (3)Go, respectively). (D) Normalized Kv4.3 current amplitude mean±S.E.M in 18 cells elicited by pulses to +50 mV as a function of the time after seal breaking. (E) Ratio between the diltiazem-sensitive current during the depolarizing pulse (IC–IDTZ) and the current in control conditions. The continuous line represents the fit to a monoexponential function to obtain the {tau}block. (F) 1/{tau}Block as a function of the diltiazem concentration for data obtained at concentrations between 0.1 and 500 µM. The straight line is the least-squares fit to Eq. (4) and the dotted line the 95% confidence intervals of the fit. In panels C and F, each point represents the mean±S.E.M of >5 experiments.

 
To assess the onset block kinetics, the ratio between diltiazem-sensitive current during the pulse to +50 mV and the current in control conditions [(IC–IDTZ)/IC] was calculated for all the concentrations tested. As can be observed in Fig. 3E, blockade increased during the application of the pulse. The onset of block was fitted by a monoexponential function (solid curve) to determine the {tau}Block, which at 0.1 µM averaged 10.1±3.2 ms (n=7). Fig. 3F shows the plot of the 1/{tau}Block as a function of the diltiazem concentration for data obtained at concentrations in the range 0.1–500 µM. The straight line is the least-squares fit to Eq. (4) and the apparent association and dissociation rate constants resulted (1.7±0.2)x106 M–1 s–1 and 258.6±38.1 s–1, respectively.

The upper part of Fig. 4 shows Kv4.3 current traces obtained in the presence and the absence of 10 nM diltiazem with the protocol described in Methods. In Fig. 4A, the peak current–voltage relationship, demonstrated that diltiazem significantly decreased the Kv4.3 current at potentials >0 mV. Furthermore, as derived from the fractional block represented as a function of the membrane potential, the blockade steeply increased in the voltage range of channel activation, reaching a 24.9±1.4% at 10 mV and, thereafter, it remained constant. The Kv4.3 charge as a function of the membrane potential in the absence and the presence of 0.1 µM diltiazem was plotted in Fig. 4B. At concentrations ≤0.1 µM, diltiazem significantly decreased the charge at potentials positive to –10 mV (n=6, P<0.05). The fractional charge block remained unchanged at potentials between –20 and +50 mV (35.0±6.3% at –20 mV vs. 33.8±2.4 at +50 mV, n=6, P>0.05). Fig. 4C shows the conductance–voltage curves of Kv4.3 channels in the absence and the presence of 10 nM diltiazem. Conductance was determined from the peak current amplitude elicited with 250-ms pulses from –80 mV to potentials ranging –60 and +50 mV, corrected for changes in the driving force with the mean reversal potential (Eq. (1)). Diltiazem did not modify either the Vh (15.3±1.0 vs. 15.3±0.6 mV) or the k (12.0±1.8 vs. 10.9±0.5 mV) (n=6, P>0.05) values of the conductance curve.


Figure 4
View larger version (39K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4 The upper inset shows Kv4.3 current traces in the absence and the presence of diltiazem obtained when applying pulses to potentials ranging from –90 to +50 mV followed by a 250-ms pulse to +40 mV. The dashed lines represent the zero current level. (A) Peak current–voltage Kv4.3 relationship in the absence and the presence of 10 nM diltiazem. (B) Relationship between the current–time integral and the membrane potential in the absence and the presence of 0.1 µM diltiazem. In panels A and B, *P<0.05 vs. control data. (C) Conductance–voltage relationships in the absence and in the presence of diltiazem. Conductance was obtained using Eq. (1) and the data were fitted to a Boltzmann function (continuous line). The dashed line the activation curve in the presence of diltiazem normalized to the control amplitude (D) Inactivation curves in the absence and in the presence of 10 nM diltiazem. Continuous lines represent the fit of the data to a Boltzmann equation and the dashed line the inactivation curve in the presence of diltiazem normalized to the control amplitude. *P<0.05 vs. data obtained at –90 mV. In panels A, B and D, squares represent the fractional block from data shown on each panel and each point represents the mean±S.E.M of six experiments.

 
Fig. 4D shows the inactivation curves in the absence and the presence of 10 nM diltiazem. Under control conditions, the Vh and the k values averaged –33.1±2.3 and 5.6±0.2 mV, respectively. Diltiazem decreased Kv4.3 current amplitude and shifted the Vh of the curve to –38.2±3.5 mV (n=6, P<0.05) without modifying the k value (5.6±0.2 mV). Fractional block was plotted as a function of the voltage of the preceding pulse, demonstrating that the blockade remained unchanged at potentials between –90 mV (22.7±4.9%) and –60 mV (25.5±6.0%, n=6, P>0.05). At more positive potentials, it augmented as the amount of inactivated channels increased, reaching a maximum at –30 mV (57.2±11.8%, P<0.05 vs. blockade at –90 mV).

3.3 Frequency-dependent effects of diltiazem on hKv1.5 and Kv4.3 channels
To evaluate whether the blockade of hKv1.5 and Kv4.3 channels increased under conditions of repetitive stimulation trains of 16 pulses to +60 mV were applied in the absence and the presence of 10 nM diltiazem (Fig. 5A and B). During the train 200-ms pulses were applied at 1 or 2 Hz, whereas 100-ms pulses were applied at 5 Hz. Trains were separated from each other by 2-min intervals. Peak current amplitude decreased under control conditions because of the accumulation of the slow (hKv1.5) and fast (Kv4.3) inactivation. Fig. 5A and B shows the ratio of the peak hKv1.5 and Kv4.3 current amplitudes in the presence of diltiazem (IDTZ/ICON) as a function of the number of pulse during trains. A certain amount of block was apparent from the first depolarization applied, i.e., "tonic block" (Table 1). Thereafter, during the train, the blockade increased until a steady state was achieved (Table 1, Fig. 5A and B). The onset kinetics of the frequency-dependent block was analyzed by fitting the relative current against the number of consecutive pulses of the trains (continuous lines) to a monoexponential function, the rate constants of the onset kinetics (K) being summarized in Table 1. These results indicated that under conditions of repetitive stimulation the diltiazem-induced block of hKv1.5 and Kv4.3 channels significantly increased.


Figure 5
View larger version (25K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5 Ratio of the amplitude of the peak hKv1.5 (panel A) and Kv4.3 (panel B) current in the presence and the absence of diltiazem when trains at 1, 2 and 5 Hz were applied. Continuous lines represent the best monoexponential fits of the data. *P<0.05 vs. tonic block. Each data point represents the mean±S.E.M of >5 experiments.

 

View this table:
[in this window]
[in a new window]

  		Table 1 Frequency-dependent effects of diltiazem on hKv1.5 and Kv4.3 channels

 
3.4 Effects on transmembrane action potentials
The effects of 10 nM diltiazem on the action potentials recorded in isolated mouse atria were also examined. Fig. 6A shows action potentials recorded in the absence and the presence of diltiazem. Diltiazem did not modify the resting membrane potential (–80.7±1.6 mV) or the action potential amplitude (104±0.8 mV, n=6). In contrast, diltiazem slightly, but significantly, lengthened the action potential duration measured at 50% and 90% of repolarization (Fig. 6B).


Figure 6
View larger version (18K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 6 Effects of diltiazem on action potentials recorded in mouse atria driven at 3 Hz. (A) Superimposed action potentials recorded in the absence and the presence of 10 nM diltiazem. (B) Action potential duration measured at 20%, 50% and 90% of repolarization in the absence and the presence of diltiazem. Each data point represents the mean±?SEM of six experiments. *P<0.05, ***P<0.01 vs. control.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Acknowledgments
 References
 
We have analyzed the effects of diltiazem on two K+ currents involved in human atrial repolarization. Therapeutic plasma concentrations obtained after administration of slow and fast release formulations of diltiazem (120 mg) were 0.11–0.33 µM [16]. Considering that diltiazem is highly bound to plasma proteins ({approx}80%), the free plasma concentration would be 22–66 nM. Thus, this study demonstrated for the first time that, at therapeutic concentrations, diltiazem blocked hKv1.5 and Kv4.3 channels.

4.1 Concentration-dependent effects of diltiazem on hKv1.5 and Kv4.3 channels
The effects of two micromolar concentrations of diltiazem on Kv1.5 channels expressed in MEL cells were previously studied, but the concentration-, time- and voltage-dependent characteristics of the blockade were not described [5]. More recently, it has been described that 100 µM diltiazem decreased Kv1.5 currents recorded in Xenopus oocytes by 15% [6]. However, in oocytes much higher concentrations of lipophilic drugs are needed to block expressed channels [6]. On the other hand, the effects of diltiazem on either Ito or Kv4.3 are unknown. Our results demonstrated that diltiazem exhibited a high affinity for hKv1.5 and Kv4.3 channels and that the blockade of these cardiac K+ channels would appear at the same concentrations needed to block L-type Ca2+ channels [16]. Surprisingly, the concentration–response curve of diltiazem in both channels exhibited two components. This could be explained by the existence of two different populations of channels with different affinities for diltiazem on the cell expression system used. hKv1.5 channels were studied in stably transfected Ltk– cells and Kv4.3 on transiently transfected CHO cells, and no evidence was obtained for non-homogeneous channel populations when examining the effects of other drugs on these channels under the same experimental conditions [14,15,17]. It is also possible that two separated binding sites with different affinities would exist both on hKv1.5 and Kv4.3 channels. The existence of two separated binding sites for drugs on hKv1.5 channels has recently been proposed [17]. Furthermore, some results demonstrated the presence of an intracellular [18] and an extracellular binding site for local anesthetics in these channels [19]. Another possibility is that more than one diltiazem molecule would access the pore to block the K+ efflux, but probably when the concentration of bulky molecules of diltiazem near the binding site increases, the steric hindrance interactions between them would decrease the efficacy of block [15]. Therefore, although previous explanations can account for this result, further analysis at the single channel level is necessary to clarify this issue. Finally, even when diltiazem contains two adjacent chiral centres, thus existing as four stereoisomers, in this study we evaluate the effects of (2S,3S)-cis-diltiazem, which is the enantiomer used in therapeutics [16]. Therefore, the biphasic curve cannot be attributed to the separated interaction, with different affinities, of the diltiazem enantiomers with either the hKv1.5 or the Kv4.3 channel proteins. Finally, it should be stressed that the concentrations of verapamil and nifedipine required for inhibition of hKv1.5 (IC50=20 and 6.3 µM, respectively) [3,4] and Kv4.3 channels (IC50=430 and 14 µM, respectively) [20,21] are 1–2 orders higher than those of diltiazem.

4.2 State-dependence of the hKv1.5 and Kv4.3 diltiazem-induced block
Diltiazem induced a voltage-dependent block of hKv1.5 and Kv4.3 channels that increased at the voltage range of channel activation. At potentials at which channel activation reached saturation, diltiazem-induced block remained constant. This result differentiates diltiazem from verapamil and nifedipine, whose hKv1.5-blockade increased and decreased, respectively, at the same voltage range [3,4]. Diltiazem induced block of both hKv1.5 and Kv4.3 channels developed during the depolarization and no block was apparent before the channels opened. The voltage dependence and the development kinetics of block strongly suggest that diltiazem blocks the open state of both channels. Furthermore, diltiazem slowed the tail current decline and induced a frequency-dependent block of hKv1.5 and Kv4.3 channels, and accelerated the Kv4.3 current inactivation, results that strongly support the proposed open-state interaction for both channels.

Using the time constants of development of hKv1.5-block obtained in the range 0.1–500 µM, the k and l constants for diltiazem were obtained. Assuming a first-order reaction drug/channel interaction, the ratio l/k would give the apparent IC50 of 29.2 µM. This estimate was independent but similar to the IC50 calculated from the concentration–response curve. In the case of Kv4.3 channels, the ratio l/k yielded an apparent IC50 (152 µM), which is also similar to that obtained from the concentration–response curve. The similarity of the IC50 values obtained by the two independent methods supports the open-channel block model used to calculate the rate constants in each channel. Furthermore, the results indicated that the apparent rate constant of association of diltiazem was very similar for both channels, whereas the dissociation process was slower in Kv4.3 compared with hKv1.5 channels, which explains the somewhat lower affinity of diltiazem for the latter.

Finally, diltiazem modified the voltage dependence of hKv1.5 and Kv4.3 channel inactivation and, in Kv4.3 channels the blockade significantly augmented with channel inactivation. These results suggested that diltiazem also binds to the inactivated state of hKv1.5 and Kv4.3 channels. Affinity for both the open and the inactivated state has been previously described for flecainide and quinidine on Kv4.2 [22] and for irbesartan on Kv4.3 channels [15].

4.3 Clinical implications
In anesthetized dogs, despite similar effects on blood pressure, PR interval and intra-atrial conduction, diltiazem did not share verapamil's ability to decrease the atrial effective refractory period or the atrial fibrillation cycle length and did not promote atrial fibrillation [23]. These differences could be explained because verapamil and diltiazem exhibited a different blocking profile of Ca2+ and/or K+ channels [24]. Here we demonstrated that, at room temperature (a possible limitation of the study), diltiazem blocks Kv4.3 and hKv1.5 channels, the latter being present only in the atria [8]. In contrast, previous reports demonstrated that verapamil is a weak blocker of hKv1.5 and Kv4.3 channels (at the low micromolar range), but a potent blocker of HERG channels [2,3]. The effects of nanomolar concentrations of diltiazem on hKv1.5 and on Kv4.3 are moderate but are likely to be enhanced during tachyarrhythmias due to the frequency dependence of its action. Speculatively, blockade of Kv1.5 and Kv4.3 currents in the human atria could attenuate the shortening of the action potential duration caused by the inhibition of L-type Ca2+ channels by diltiazem. In fact, we demonstrated that 10 nM diltiazem (a concentration somewhat lower than the free plasma concentrations reported in humans) lengthened the atrial action potentials in mice. These effects can be explained by the blockade of Ito and IKslow that contributed to the repolarization in mouse heart and are generated by Kv4.2 and Kv1.5 channels, respectively [25]. Thus, these K+ channel-blocking properties exhibited by diltiazem could protect against an atrial fibrillation-promoting effect and raise the question of whether diltiazem might be a better choice than verapamil for rate control among patients with atrial fibrillation in whom a subsequent cardioversion is considered [24].


   Acknowledgments
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
References
 
We thank Guadalupe Pablo for her excellent technical assistance. This work was supported by SAF2002-02304, and Pfizer Foundation Grants.


   Notes
 
1 These authors contributed equally to this work. Back

Time for primary review 21 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 

  1. Abernethy D.R., Schwartz J.B. Calcium-antagonist drugs. N. Engl. J. Med. (1999) 341:1447–1457.[Free Full Text]
  2. Zhang S., Zhou Z., Gong Q., Makielski J.C., January C.T. Mechanism of block and identification of the verapamil binding domain to HERG potassium channels. Circ. Res. (1999) 84:989–998.[Abstract/Free Full Text]
  3. Rampe D., Wible B., Fedida D., Dage R.C., Brown A.M. Verapamil blocks a rapidly activating delayed rectifier K+ channel cloned from human heart. Mol. Pharmacol. (1993) 44:642–648.[Abstract]
  4. Zhang X., Anderson J.W., Fedida D. Characterization of nifedipine block of the human heart delayed rectifier, hKv1.5. J. Pharmacol. Exp. Ther. (1997) 281:1247–1256.[Abstract/Free Full Text]
  5. Grissmer S., Nguyen A.N., Aiyar J., Hanson D.C., Mather R.J., Gutman G.A., et al. Pharmacological characterization of five cloned voltage-gated K+ channels, types Kv1.1, 1.2, 1.3, 1.5, and 3.1, stably expressed in mammalian cell lines. Mol. Pharmacol. (1994) 45:1227–1234.[Abstract]
  6. Rolf S., Haverkamp W., Borggrefe M., Musshoff U., Eckardt L., Mergenthaler J., et al. Effects of antiarrhythmic drugs on cloned cardiac voltage-gated potassium channels expressed in Xenopus oocytes. Naunyn-Schmiedeberg’s Arch. Pharmacol. (2000) 362:22–31.[CrossRef][Web of Science][Medline]
  7. Jahnel U., Klemm P., Nawrath H. Different mechanisms of the inhibition of the transient outward current in rat ventricular myocytes. Naunyn-Schmiedeberg’s Arch. Pharmacol. (1994) 349:87–94.[Web of Science][Medline]
  8. Wang Z., Fermini B., Nattel S. Sustained depolarization-induced outward current in human atrial myocytes. Evidence for a novel delayed rectifier K+ current similar to Kv1.5 cloned channel currents. Circ. Res. (1993) 73:1061–1076.[Abstract/Free Full Text]
  9. Escande D., Coulombe A., Faivre J.F., Coraboeuf E. Characteristics of the time-dependent slow inward current in adult human atrial single myocytes. J. Mol. Cell. Cardiol. (1986) 18:547–551.[CrossRef][Web of Science][Medline]
  10. Dixon J.E., Shi W., Wang H.S., McDonald C., Yu H., Wymore R.S., et al. Role of the Kv4.3 K+ channel in ventricular muscle. A molecular correlate for the transient outward current. Circ. Res. (1996) 79:659–668.[Abstract/Free Full Text]
  11. Rosati B., Pan Z., Lypen S., Wang H.S., Cohen I., Dixon J.E., et al. Regulation of KChIP2 potassium channel beta subunit gene expression underlies the gradient of transient outward current in canine and human ventricle. J. Physiol. (2001) 533:119–125.[Abstract/Free Full Text]
  12. Courtemanche M., Ramirez R.J., Nattel S. Ionic mechanisms underlying human atrial action potential properties: insights from a mathematical model. Am. J. Physiol. (1998) 275:H301–H321.[Web of Science][Medline]
  13. Caballero R., Delpón E., Valenzuela C., Longobardo M., Tamargo J. Losartan and its metabolite E3174 modify cardiac delayed rectifier K+ currents. Circulation (2000) 101:1199–1205.[Abstract/Free Full Text]
  14. Caballero R., Delpon E., Valenzuela C., Longobardo M., Gonzalez T., Tamargo J. Direct effects of candesartan and eprosartan on human cloned potassium channels involved in cardiac repolarization. Mol. Pharmacol. (2001) 59:825–836.[Abstract/Free Full Text]
  15. Moreno I., Caballero R., Gonzalez T., Arias C., Valenzuela C., Iriepa I., et al. Effects of irbesartan on cloned potassium channels involved in human cardiac repolarization. J. Pharmacol. Exp. Ther. (2003) 304:862–873.[Abstract/Free Full Text]
  16. Buckley M.M., Grant S.M., Goa K.L., McTavish D., Sorkin E.M. Diltiazem. A reappraisal of its pharmacological properties and therapeutic use. Drugs (1990) 39:757–806.[Web of Science][Medline]
  17. Caballero R., Moreno I., González T., Valenzuela C., Tamargo J., Delpón E. Putative binding sites for benzocaine on a human cardiac cloned channel (Kv1.5). Cardiovasc. Res. (2002) 56:104–117.[Abstract/Free Full Text]
  18. Decher N., Pirard B., Bundis F., Peukert S., Baringhaus K.H., Busch A.E., et al. Molecular basis for Kv1.5 channel block: conservation of drug binding sites among voltage-gated K+ channels. J. Biol. Chem. (2004) 279:394–400.[Abstract/Free Full Text]
  19. Longobardo M., González T., Navarro-Polanco R., Caballero R., Delpón E., Tamargo J., et al. Effects of a quaternary bupivacaine derivative on delayed rectifier K+ currents. Br. J. Pharmacol. (2000) 130:391–401.[CrossRef][Web of Science][Medline]
  20. Calmels T.P., Faivre J.F., Cheval B., Javre J.L., Rouanet S., Bril A. hKv4.3 channel characterization and regulation by calcium channel antagonists. Biochem. Biophys. Res. Commun. (2001) 281:452–460.[CrossRef][Web of Science][Medline]
  21. Gotoh Y., Imaizumi Y., Watanabe M., Shibata E.F., Clark R.B., Giles W.R. Inhibition of transient outward K+ current by DHP Ca2+ antagonists and agonists in rabbit cardiac myocytes. Am. J. Physiol. (1991) 260:H1737–H1742.[Web of Science][Medline]
  22. Caballero R., Pourrier M., Schram G., Delpón E., Tamargo J., Nattel S. Effects of flecainide and quinidine on Kv4.2 currents: voltage dependence and role of S6 valines. Br. J. Pharmacol. (2003) 138:1475–1484.[CrossRef][Web of Science][Medline]
  23. Benardeau A., Fareh S., Nattel S. Effects of verapamil on atrial fibrillation and its electrophysiological determinants in dogs. Cardiovasc. Res. (2001) 50:85–96.[Abstract/Free Full Text]
  24. Fareh S., Benardeau A., Nattel S. Differential efficacy of L- and T-type calcium channel blockers in preventing tachycardia-induced atrial remodeling in dogs. Cardiovasc. Res. (2001) 49:762–770.[Abstract/Free Full Text]
  25. Nerbonne J.M., Nichols C.G., Schwarz T.L., Escande D. Genetic manipulation of cardiac K+ channel function in mice: what have we learned, and where do we go from here? Circ. Res. (2001) 89:944–956.[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?



This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Caballero, R.
Right arrow Articles by Delpón, E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Caballero, R.
Right arrow Articles by Delpón, E.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?