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British Journal of Pharmacology logoLink to British Journal of Pharmacology
. 2000 Aug;130(8):1753–1766. doi: 10.1038/sj.bjp.0703476

Effects of BDF 9198 on action potentials and ionic currents from guinea-pig isolated ventricular myocytes

K H Yuill 1, M K Convery 1, P C Dooley 2, S A Doggrell 3, J C Hancox 1,*
PMCID: PMC1572251  PMID: 10952663

Abstract

  1. BDF 9198 (a congener of DPI 201–106 and BDF 9148) was found to be a positive inotrope on guinea-pig isolated ventricular muscle strips. The effects of BDF 9198 on action potentials and ionic currents from guinea-pig isolated ventricular myocytes were studied using the whole cell patch clamp method.

  2. In normal external solution, at 37°C, action potential duration at 50% repolarization (APD50) was 167.4±8.36 ms (n=37). BDF 9198 produced a concentration-dependent increase in APD50 (no significant increase at 1×10−10 M; and APD50 values of 273.03±35.8 ms at 1×10−9 M; n=6, P<0.01 and 694.7±86.3 ms at 1×10−7 M; P<0.001, n=7). At higher concentrations in the range tested, BDF 9198 also induced early and delayed and after-depolarizations.

  3. Qualitative measurements of INa with physiological [Na]o showed prolongation of the current by BDF 9198, and the appearance of transient oscillatory inward currents at high concentrations.

  4. Quantitative recording conditions for INa were established using low external [Na] and by making measurements at room temperature. The current–voltage relation, activation parameters and time-course of INa were similar before and after a partial blocking dose of Tetrodotoxin (TTX, 1 μM), despite a 2 fold difference in current amplitude. This suggests that voltage-clamp during flow of INa was adequately maintained under our conditions.

  5. Selective measurements of INa at room temperature showed that BDF 9198 induced a concentration-dependent, sustained component of INa (ILate) and caused a slight left-ward shift in the current–voltage relation for peak current. The drug-induced ILate showed a similar voltage dependence to peak current in the presence of BDF 9198. Both peak current and ILate were abolished by 30 μM TTX and were sensitive to external [Na].

  6. Inactivation of control INa during a 200 ms test pulse to −30 mV followed a bi-exponential time-course. In addition to inducing a sustained current component, BDF 9198 left the magnitude of the fast inactivation time-constant unchanged, but increased the magnitude of the slow inactivation time-constant. Additional experiments with a longer pulse (1 s) raised the possibility that in the presence of BDF 9198, INa inactivation may be comprised of more than two phases.

  7. No significant effects of 1×10−6 M BDF 9198 were observed on the L-type calcium current, or delayed and inward rectifying potassium currents measured at 37°C.

  8. It is concluded that the prolongation of APD50 by BDF 9198 resulted from selective modulation of INa. Reduced current inactivation induced a persistent INa, increasing the net depolarizing current during the action potential. This action of the drug indicates a potential for ‘QT prolongation' of the ECG. The observation of after-depolarizations suggests a potential for proarrhythmia at some drug concentrations.

Keywords: BDF 9198; sodium current; INa; L-type calcium current; ICa,L; delayed rectifier; IK; inward rectifier; IKl; myocyte; action potential; QT interval

Introduction

Although positive inotropes are a cornerstone in the treatment of heart failure, the inadequacy of the agents currently used in clinical practice is well recognised and has provided the impetus for the continuing development of new approaches (Dorigo et al., 1994). Prolongation of the action potential is often associated with a positive inotropic effect. One possible approach to the development of a new positive inotrope, therefore, is the synthesis of agents that prolong the action potential by inhibiting the closing of cardiac sodium channels.

Molecular biology techniques have separated five sodium channels, two of which exist in cardiac muscle, the I and h1 channels, which exhibit differential sensitivities to tetrodotoxin (TTX; e.g. Watson & Girdlestone, 1995). The opening of the TTX-resistant h1 sodium channel on cardiac muscle, which is blocked by μM TTX, produces the rapid depolarization (phase 0) of the cardiac action potential. The opening of this sodium channel in turn leads to the opening of voltage-dependent calcium channels and ensuing entry of calcium into the cell triggering calcium release from sarcoplasmic reticulum. Increasing intracellular Ca2+ concentrations leads to an increase in the force of contraction. The function of the second sodium channel in the heart, the TTX-sensitive I channel which is blocked by nM TTX, is unknown.

Low concentrations of the naturally occurring alkaloid veratridine prolong the action potential by preventing closure of the Na channel inactivation gate (Honerjager, 1982) and have marked positive inotropic effects. Higher concentrations of veratridine, however, also open Na channels (Honerjager, 1982) and can produce after-depolarizations, ectopic beats and arrhythmias (Doggrell et al., 1995; Nand et al., 1997). Veratridine also prolongs the opening of the neuronal Tetrodotoxin-sensitive I channel to promote neurotransmitter release (Honerjager, 1982). Veratridine is unsuitable as a therapeutic inotrope because of the cardiotoxicity and lack of cardioselectivity.

Cardioselective inhibitors of the TTX-resistant h1 Na channel inactivation have also been developed. The most widely studied of these are DPI 201-106 and its congener BDF 9148 (Ravens et al., 1995; Doggrell & Brown, 1997). These studies have confirmed that prolongation of the opening of the sodium channel with DPI 201-106 or BDF 9148 is associated with a prolongation of the action potential and a positive inotropic effect (Ravens et al., 1995; Doggrell & Brown, 1997). The ion channel modulatory effects of DPI 201-106 and BDF 9148 are not limited to sodium channels, however. Both DPI 210-106 and BDF 9148 inhibit the L-type calcium current (Ravens et al., 1991; 1995), and DPI 210-106 can inhibit inward and delayed rectifier potassium currents (Amos & Ravens, 1994). Animal studies suggest that DPI 201-106 may be proarrhythmic (Novosel et al., 1993). Poor bioavailability made BDF 9148 unsuitable for clinical trial and it has been superseded by BDF 9198 which has improved bioavailability (Doggrell & Brown, 1997).

The positive inotropic effect of BDF 9198 has been demonstrated on isolated human myocardium (Muller-Ehmsen et al., 1998; Schwinger et al., 1999). However there have been no reports characterizing the effects of BDF 9198 on action potentials, or sodium and other channels. In this study we report that BDF 9198 exerts a positive inotropic effect on ventricular muscle strips isolated from the guinea-pig. We have characterized the effects of BDF 9198 on ventricular action potentials (APs) from isolated guinea-pig ventricular myocytes and report AP prolongation. A major aim of our study was to investigate the effects of BDF 9198 on ionic currents from ventricular myocytes. We report modulation by BDF 9198 of the fast sodium current (INa) and also investigated the drug's effects on the L-type calcium current, and the inward and outward rectifying potassium currents.

Methods

Ventricular strip contractility

Guinea-pigs were stunned and exsanguinated. The heart was rapidly removed and placed in Krebs solution that was saturated with 5% CO2 in oxygen at 37°C, and the free walls of the right and left ventricles were excised. All experiments were performed in the presence of a modified Krebs solution [composition (mM): NaCl, 116; KCl, 5.4; CaCl2, 2.5; MgCl2, 1.2; NaH2PO4, 1.2; NaHCO3, 22.0; D-glucose, 11.2] containing guanethidine at 10−5 M to prevent the release of noradrenaline from nerve endings, and atropine at 10−6 M to block muscarinic receptors at 37°C. We have previously described the method for recording ventricular contractility (Nand & Doggrell, 1999). Strips were prepared from the ventricle free wall and mounted longitudinally between two platinum electrodes under 1 g tension, in 5 ml organ baths in Krebs solution being vigorously bubbled with 5% CO2 in oxygen and allowed to equilibrate for 75 min. Contractile responses were measured isometrically with force displacement transducers (Grass model FT03.C) and displayed on a polygraph (Grass model 79B). After stimulation at 4 Hz (5 ms duration, 10 V) for 6 min, BDF 9198 at 10−8 M was added. The cumulative addition of BDF 9198 (3×10−8, 10−7 M etc.) occurred on a 5 min cycle until conclusion of the experiment at a concentration of 1×10−6 M. The augmentation of the contractile response in the presence of each concentration of BDF 9198 was calculated as per cent change and mean values±s.e.m. were determined.

Myocyte isolation

Myocytes were isolated from both ventricles of male guinea-pig (∼400 g) hearts, using an enzymatic dispersion method described previously (Levi & Issberner, 1996). The cells were stored at room temperature in solution containing 1 mM Ca2+ until required for use. Cells remained viable for up to 8 h after isolation.

Electrophysiology

Isolated cells were placed in a Perspex chamber mounted on an inverted microscope (Nikon Diaphot 300), allowed to settle, and then superfused at 37°C with a Tyrode's solution containing (in mM): NaCl 140; KCl 4, CaCl2 2.5; MgCl2 1; Glucose 10; HEPES 5 (adjusted to 7.45 with NaOH). All experiments were carried out at 37°C, with the exception of selective INa recordings which were conducted at room temperature (20–22°C). External superfusate was changed using a rapid solution-switching device (Levi et al., 1996). Patch pipettes (Corning 7052 glass, AM Systems, Everett, U.S.A.) were pulled (Puller, Model P-87, Sutter Instrument, U.S.A.) and polished (Narishige MF-83 microforge) to resistances of between 2 and 3 MΩ in all experiments except for the selective INa recordings. For these, pipettes had resistances of between 1 and 2 MΩ. Patch pipette filling solutions and external solutions are detailed in Table 1 and recording conditions summarized below.

Table 1.

Composition of internal and external solutions used in whole-cell recordings

graphic file with name 130-0703476t1.jpg

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As INa is large and fast, special conditions were necessary in order to record it quantitatively. Thus these experiments were conducted at room temperature in order to slow the kinetics of the current. Low resistance patch-pipettes were used to facilitate intracellular dialysis of cells with the pipette solution and to minimize voltage errors due to uncompensated series resistance. Caesium-based internal and external experimental solutions containing similar levels of sodium were used to reduce the gradient for sodium entry (thereby reducing the size of the current), and to block any contamination from potassium currents. Nifedipine was used externally to block interfering calcium current. INa was measured selectively as the difference current following application of TTX (30 μM). For the solutions used for selective sodium current recording described in Table 1 the liquid junction potential (LJP) between the pipette and the external solution was −1.06±0.33 mV (n=6 pipettes; mean±s.e.m). Due to the small size of the LJP value, no corrections to the data were made.

Delayed rectifier (IK) and inward rectifier (IK1) currents were recorded using a potassium based sodium-free pipette solution, and N-methyl-D-glucamine (NMDG) externally. Replacing the external sodium with NMDG inhibited contaminating inward Na-Ca exchange tail currents that can otherwise occur on repolarization from positive test potentials. Nitrendipine or nifedipine were used externally to block interfering calcium current. BaCl2 was included in external solutions for recording IK to inhibit any interference from IK1. L-type calcium current (ICa,L) was recorded using a Cs-based pipette solution to inhibit potassium current interference, and an external NMDG solution. In all voltage clamp experiments, 1.2-bis (2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid (BAPTA, K salt) was included in the pipette filling solutions to inhibit interfering internal Ca (Cai)-activated currents. Current clamp (I Clamp) and standard voltage clamp experiments (qualitative measurements of INa) were conducted using standard internal and external solutions (Table 1).

Whole cell voltage-clamp recordings were made using an Axopatch 200 amplifier (Axon Instruments, California, U.S.A.) with a CV-201 head-stage, and whole cell current-clamp recordings were made using an Axopatch 1D amplifier with a CV-4 head-stage. Cell capacitance was measured by either analysing the charging transients elicited by a 5 mV voltage step or reading the capacitance value from the dial on the Axopatch amplifier after compensating for series resistance and cell capacitance. These methods have been shown previously to give similar values for cell capacitance (Hancox et al., 1993). Series resistance values were in the range of 2–4 MΩ; for six cells a mean value of 3.08±0.35 MΩ was obtained. Typically, 75–80% of series resistance could be compensated. Voltage errors during recordings of peak INa were estimated for six cells and ranged between 1.5 and 4 mV (mean=2.29±0.38 mV). The measurement of INa as difference current sensitive to 30 μM TTX in most experiments minimized the likelihood that current records were contaminated with residual uncompensated capacitative current.

Data analysis and statistics

Voltage and current clamp protocols were generated using the program Winwcp (version 1.7a; written and supplied by John Dempster of Strathclyde University, U.K.) via a CED (Cambridge Electronic Design, U.K.) 1401 digital interface or a Digidata 1200B interface (Axon Instruments, U.K.). Data were recorded on-line at 2 kHz, except INa data for which a recording frequency of 10 kHz was used. Data were stored on the hard disk of an IBM compatible PC, and analysed using Winwcp. Figures were constructed using FigP (Biosoft), and statistical analysis performed using EXCEL (Microsoft). Data are shown as mean±s.e.m. and were compared statistically using Students' t-test or ANOVA with a post-hoc Bonferroni correction. A P value of <0.05 was taken as significant.

Chemicals and drugs

BDF 9198 (4-(3-(1-diphenylmethyl-azetidine-3-oxy)-2-hydroxy-propylamin)-1-H-indol-2-carbonitrile (donated by Beiersdorf–Lilly, Hamburg, Germany) was made as a 1 mM stock solution in ethanol, serially diluted using deionized water (Milli-Q, Millipore, U.S.A.), and then aliquoted and stored at −20°C. It was added to the experimental solutions to give final concentrations between 1×10−10 and 1×10−5 M. All isolation solutions were made from Aristar grade chemicals, supplied by British Drugs House (BDH). Type 1A collagenase was supplied by Worthington, U.S.A. and Type XIV protease was supplied by Sigma. Analar grade chemicals (BDH) were dissolved in deionized water to make external solutions, and Aristar grade chemicals (BDH) were used to make internal solutions. NMDG (Sigma) replaced NaCl in the sodium-free external solution. Nitrendipine (Research Biochemicals International) and Nifedipine (Sigma) were dissolved in dimethylsulphoxide (DMSO) to give 10 mM stock solutions. Both were shielded from light as they are light sensitive. Barium chloride (BaCl2) was dissolved in deionized water to give a stock solution of 1 M. Tetrodotoxin (TTX) obtained from Tocris was made as a 3 mM stock solution in deionized water. Aliquots of nitrendipine, nifedipine, BaCl2 and TTX were added to the appropriate test solutions to give the final concentrations shown in Table 1.

Results

Effects of BDF 9198 on contractility

Before investigating effects of BDF 9198 on electrophysiological properties of guinea-pig myocytes, we first determined whether or not the compound was positively inotropic on ventricular tissue from this species. Electrically stimulated contractions of left and right ventricular muscle strips were measured as described in Methods and concentrations of BDF 9198 between 1×10−8 and 1×10−6 M were applied. The drug produced a concentration-dependent augmentation of isometric force generation (Figure 1). The augmentation was similar between strips from left and right ventricles, and was maximal at 3×10−7 M (Figure 1; there was no significant difference between per cent augmentation at 3×10−7 and 1×10−6 M).

Figure 1.

Figure 1

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Effect of BDF 9198 on muscle strip contractility. Percentage (%) augmentation of the electrical stimulation force–responses of right and left ventricular muscle strips produced by a range of concentrations of BDF 9198. Per cent augmentation is plotted against the logarithm of the molar concentration of BDF 9198. Each value is the mean±s.e.m. from five strips from separate guinea-pigs.

Effects of BDF 9198 on action potential duration (APD).

Ventricular action potentials were recorded in ‘current clamp' mode, with internal and external solutions as described in Table 1. Brief (<10 ms) suprathreshold current pulses were applied every 3 s, and action potentials recorded in normal Tyrode's solution and after addition of BDF 9198. APD was measured at 50% repolarization (APD50). In normal Tyrode's solution the mean APD50 was 167.4±8.36 ms (n=37 cells) and external application of BDF 9198 produced a dose-dependent action potential lengthening (Figure 2). For a given drug concentration the effect on APD reached a steady-state within ∼2–3 min of application to the cell under study. 1×10−10 M caused no significant lengthening of the action potential. At 1×10−9 M there was an increase in the APD50 (to 273.03±35.84, n=6; P<0.01 compared to control). 1×10−8 M BDF 9198 increased the APD50 to 440.6±53.3 ms (n=6; P<0.001 compared to control). At 1×10−7 M, BDF 9198 caused a profound lengthening of the action potential plateau, increasing the APD50 to 694.7±86.3 ms (n=7; P<0.001). At the higher concentration of 1×10−6 M, the AP duration was increased further. However the degree of prolongation at this concentration was such that cells started to round up before steady state APD50 values could be reliably made.

Figure 2.

Figure 2

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Effect of BDF 9198 on action potentials. Action potentials were recorded in ‘current clamp' mode. (a) Action potential with normal external Tyrode's solution (Ctl) and following exposure to 1×10−8 M BDF 9198. (b) In a different cell, the effect of 1×10−7 M BDF 9198. The drug produced a dose-dependent prolongation of the action potential. (c) An action potential from another cell in the presence of 1×10−7 M BDF 9198, followed by a delayed ‘after-depolarization'. (d) From another cell, an ‘early after-depolarization recorded in the presence of 1×10−7 M BDF 9198.

BDF 9198 was also observed to induce depolarizations which occurred prior to, and following action potential repolarization, and are therefore classifiable as ‘early after-depolarizations' (EADs) and ‘delayed' after-depolarizations (DADs; Ferrier, 1977; January & Fozzard, 1988). These were observed after cells had been exposed to the drug for periods of ∼ 4 min or longer. At 1×10−7 M, four of the cells exhibited DADs with prolonged BDF 9198 exposure, one exhibited EADs, one exhibited both DADs and EADs, and one showed neither.

The action potential prolongation observed in Figure 2 could have resulted from the drug enhancing inward (depolarizing) currents during the action potential plateau phase, reducing outward (repolarizing) currents, or a combination of these actions. Therefore, we investigated the effects of BDF 9198 on major inward and outward current systems, under whole cell voltage clamp.

Effect of BDF 9198 on INa

It is widely accepted that, with normal levels of external Na, and at a physiological temperature, cardiac sodium current is difficult to voltage-clamp adequately, due to the size and speed of the current. Nonetheless, we regarded it worthwhile in initial experiments to determine qualitatively the effects of BDF 9198 on INa measured with standard internal and external solutions. Figure 3a shows a typical INa elicited by a 250 ms duration voltage step from −80 to −40 mV. Under control conditions a large (off-scale) rapidly activating and inactivating current was elicited (left panel). After application of BDF 9198 at 1×10−6 M (right panel) there was a clear increase in the duration of the current, with the current profile appearing similar to an inverted action potential (and saturating the voltage clamp). This current profile shared similarities with that reported previously for the related compound BDF 9148 (Ravens et al., 1991). The prolonged INa was followed by a subsequent oscillatory current which resembles an Iti (Lederer & Tsien, 1976; Eisner & Lederer, 1979), and is consistent with proarrhythmic potential for the drug suggested by the action potential recordings.

Figure 3.

Figure 3

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Effect of BDF 9198 on INa with normal [Na]o and establishment of selective INa recording conditions with reduced [Na]o. (a) Recordings of INa (upper traces) using standard Tyrode's solution externally, and a potassium based pipette filling solution. Lower traces show voltage protocol (250 ms step from −80 to −40 mV). BDF 9198 produced an increase in the duration of INa. Note that in the record showing effects of 10−6 M BDF 9198, an oscillatory inward current followed the large (poorly clamped) INa. (b) Establishment of selective INa recording conditions (see Methods). Left hand panel (Ctl) shows currents (upper traces) activated on step depolarization from −140 mV to −50, −40 and −30 mV (lower traces). In the same cell, TTX (30 μM) was applied and the TTX-subtraction (TTX-S) currents are shown in the right panel. (c) The I–V relation shows close similarity between Ctl and TTX-S currents.

Establishing selective recording conditions for INa

In order to investigate the effects of BDF 9198 on INa in a selective and quantitative manner, it was necessary to establish conditions that would permit accurate whole cell measurements of INa. For these experiments solutions were chosen (see Table 1) that eliminate interfering currents and reduce the amplitude of the current (by reducing [Na]o to 10 mM). Measurements were conducted at room temperature (20–22°C) in order to slow INa kinetics. Low resistance pipettes were used to minimise potential voltage errors which might arise during flow of INa (see Methods for estimated values).

Previous studies (e.g. Makielski et al., 1987; Miyamoto et al., 1991; Feng et al., 1996) have reported a negative shift in the voltage-dependent inactivation kinetics of INa under conditions of internal fibre/cell dialysis. This necessitates applying depolarizing test-pulses from a highly negative membrane potential in order to avoid partial inactivation of INa. In our INa experiments, therefore, whilst cell membrane potential was held at −80 mV, a 2 s pre-pulse to −140 mV preceded each depolarizing test pulse.

Figure 3b (left panel) shows sample currents elicited by step depolarizations from −140 mV to −50, −40 and −30 mV. Application of 30 μM TTX completely eliminated the observed inward currents and subtraction of control recordings from those in the presence of TTX provided ‘TTX-subtraction' current measurements (TTX-S; Figure 3b right panel). TTX-S and control currents were very similar, suggesting that we were successful in measuring INa. This is further highlighted by close similarity in the current–voltage (I–V) relations for the control and TTX-S currents shown in Figure 3c (example from one cell). Peak INa values between 3 and 5 nA were observed under these recording conditions and I–V relations for individual cells crossed the voltage axis between 0 and +12 mV.

Previously, experiments in which the I–V relations for INa were compared between ‘control' and partially inhibited current have been used to assess the quality of the voltage clamp during flow of INa (e.g. Brown et al., 1981; Follmer et al., 1987). We used an approach similar to that described by Follmer et al: the effects were determined of a partial blocking dose (1 μM) of TTX on the I–V relation for, and activation and inactivation time-courses of INa. Figure 4a shows recordings of INa elicited by a step-depolarization from a prepulse potential of −140 to −30 mV, in the presence and absence of TTX. 1 μM TTX (middle panel) reduced the peak INa to less than 50% of the control current (left panel). A higher dose of 30 μM TTX, abolished the INa completely (right panel). The currents obtained in control conditions and after the partial blocking dose of 1 μM TTX were subtracted from those in the presence of 30 μM TTX, to give TTX-S INa from which I–V relations were constructed. The mean I–V relations for TTX-S control current and current sensitive to 1 μM TTX are shown in Figure 4b. Each data-set was fitted by a modified Boltzmann equation of the form:

graphic file with name 130-0703476e1.jpg

where INa represents current density at test potential Vm, Gmax is maximal INa conductance, Vrev is the reversal potential, V0.5 is the membrane potential exhibiting half maximal current activation, and k is the slope factor which describes the steepness of activation for the current. The Boltzmann fit to the data gave a value for V0.5 of −39.4±0.49 mV and k=4.92±0.42 mV in control solution. This was not significantly different after the application of 1 μM TTX, where V0.5=−40.7±0.45 mV and k=4.4±0.30 mV in six cells (P>0.05). Thus, it was evident that although 1 μM TTX reduced the magnitude of current at all test potentials, there was no significant effect on the activation parameters derived from the curve-fit to the I–V data. Furthermore, our observed V0.5 values compare favourably to that reported previously for human atrial INa (−38.6 mV; Feng et al., 1996) and the mean voltage for half maximal Na conductance reported for cat atrial INa (−41.8 mV) by Follmer et al. (1987).

Figure 4.

Figure 4

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Effects of a partial blocking dose of Tetrodotoxin on INa. (a) Selective INa recordings (upper traces) elicited by a step depolarization from −140 mV to a test potential of −30 mV (lower traces). In control conditions (Ctl; left panel) a rapidly activating and inactivating INa was observed. 1 μM TTX produced a partial block of INa (middle panel); a higher concentration of 30 μM TTX (right panel) completely abolished INa. (b) shows the mean current-voltage (I–V) relationship for peak INa under control conditions and after the application 1 μM TTX. Curve fits to the data were made using equation 1 (Results).

To quantify the time-course of activation, we measured the time to peak (TTP) INa, over a range of test potentials. TTP was measured from the start of the voltage step to the peak of the current. Under control conditions, selected values obtained were (in ms): −40 mV: 1.43±0.18; −30 mV: 1±0.13; −20 mV: 0.70±0.11; −10 mV: 0.46±0.12. These were similar to other reported values (for a review see Fozzard & Hanck, 1996). The values for TTP after application of 1 μM TTX were not significantly different from those under control conditions (−40 mV: 1.69±0.23; −30 mV: 1.14±0.15; −20 mV: 0.73±0.05; −10 mV: 0.40±0.06; P>0.4 at each potential).

Under our recording conditions we found that control INa inactivated very rapidly, and the time-course of inactivation was well described by a bi-exponential process. We were able to quantify the time-course of inactivation by fitting the INa records with the following equation:

graphic file with name 130-0703476e2.jpg

where Af is the current described by τf and As the current described by τs, and where C represents any residual current component remaining at the end of the pulse, fitted by neither time-constant (under our conditions, C=0 for INa in the absence of BDF 9198). Under control conditions the values for τf were ∼4 fold lower than the values for τs. Both τf and τs values showed voltage-dependence, decreasing progressively at more positive test potentials (see Table 2). The relative proportion of current inactivation fitted by each τ value did not significantly alter over the voltage range studied. The time-constant values obtained from currents fitted after the application of the partial blocking dose of TTX (see Table 2), were not significantly different from control values (P>0.15 for comparisons at each voltage). Furthermore, our inactivation time-constant values were comparable to other studies in which INa was recorded using the whole cell patch clamp technique (e.g. Sakakibara et al., 1992; Conforti et al., 1993), and were comparable with (although smaller than) published data obtained at a lower temperature (10–13°C) using large bore suction pipettes (Hanck & Sheets, 1992).

Table 2.

graphic file with name 130-0703476t2.jpg

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The similarity between control and partially blocked INa, of the I–V relations and parameters for both activation and inactivation (despite the large difference in current amplitude) suggested that the quality of the voltage clamp was not compromised under our recording conditions.

One feature of our INa recording conditions merits further mention. Despite similar [Na] in bulk pipette and external solutions, the mean I–V relations for TTX-S INa (Figure 4b) intersected the voltage axis positive to 0 mV, the value expected for equimolar [Na]. Therefore, we performed some additional experiments with different internal and external solutions used in prior successful measurements of INa by Sakakibara et al. (1992; CsF based internal solution; TMA-C1 based external solution; 5 mM bulk [Na] in both internal and external solutions). We observed a voltage dependence for INa with these solutions very similar to that described above (V0.5=−36.7±1.2 mV; k=5.5±0.7, n=5; P>0.05). Furthermore, the mean I–V relationship with these solutions also intersected the voltage axis positive to 0 mV under our conditions (data not shown) but close to 0 mV in the experiments by Sakakibara et al. It is possible, therefore, that whilst we were successful in controlling bulk [Na] concentrations sufficiently to permit adequate voltage clamp of INa, the [Na] close to the cell membrane may not have precisely reflected concentrations in the bulk solution. Taken collectively, however, the data described above very strongly indicate that our experimental conditions permitted sufficiently accurate recordings of INa voltage-dependence and time-course, to allow quantitative investigation of the effects of BDF 9198 to be undertaken.

Effects of BDF 9198 on INa measured selectively

Figure 5a shows the effect of 1×10−6 M BDF 9198 on INa elicited by depolarizing test pulses from −140 to −30 mV. Under control conditions (left panel) INa activated rapidly (reaching a peak of ∼4.3 nA) and then inactivated completely within ∼20 ms. In the presence of 1×10−6 M BDF 9198, INa was significantly altered, with a profound effect on current inactivation. At the end of the 200 ms long voltage pulse there was still a substantial inward current remaining (middle panel). Application of 30 μM TTX completely abolished the sustained inward current produced by BDF 9198. This also allowed us to use the ‘TTX-S' current as a measure of INa both before and after exposure to BDF 9198. The effect of BDF 9198 on INa was long-lasting, and was not readily reversible on returning to control solution. This contrasted with the effect of TTX which was readily reversible (within 3 pulses following washout, data not shown).

Figure 5.

Figure 5

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Selective recordings of the effect of BDF 9198 on INa. (a) shows selective INa recordings (upper traces) at a test potential of −30 mV (lower traces). In control conditions (left panel) a rapidly activating and inactivating INa was observed. 1×10−6 M BDF 9198 produced a profound prolongation of INa (middle panel). The current in the presence of BDF 9198 was abolished by 30 μM TTX (right panel). (b) Recordings of INa (upper traces) from a different cell elicited by a step depolarization from −140 mV to a test potential of −30 mV (lower traces). The current in control conditions (left panel) was prolonged upon application of 1×10−6 M BDF 9198 (middle panel). External application of a Na-free solution abolished the current (right panel), confirming its identity as an INa.

Figure 5b shows the results obtained in additional experiments performed to confirm that Na was the charge carrier for the sustained current component induced by BDF 9198. Each of five cells were dialysed with Na-free pipette solution and pulses applied from −140 to −30 mV. 1×10−6 M BDF 9198 produced a sustained inward current at the end of the pulse (middle panel), compared to the rapidly inactivating current in control (left panel). Subsequent replacement of the external Na with NMDG eliminated both the rapid and sustained current components (right panel). The sensitivity of the sustained inward current to external Na indicates that this current component was carried by flow of Na during an INa.

Figure 6 shows families of INa (as TTX-S current; shown on an expanded time-base) elicited by 200 ms duration step depolarizations from −140 mV to a range of test potentials. Under control conditions (Figure 6a) peak INa in this cell increased in amplitude with test pulse magnitude, up to −35 mV and then decreased with steps to more positive potentials, reversing near 0 mV. In the presence of 1×10−6 M BDF 9198 (Figure 6b), significant INa was observed at potentials at which only small current had been observed in control solution and the maximal peak INa was observed at −45 mV. These observations were suggestive of a negative shift in the voltage dependence of the current. In addition, at all test potentials, BDF 9198 reduced current inactivation during the pulse, giving rise to a sustained component of INa, which was absent under control conditions.

Figure 6.

Figure 6

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Voltage and concentration-dependence of effect of BDF 9198 on INa. Families of TTX-S currents elicited by step-depolarizations from −140 mV to a range of more positive test potentials under control conditions (a) and (b) in the presence of 1×10−6 M BDF 9198-containing solution. (c) shows the mean current–voltage (I–V) relationship for peak INa and (d) for end-pulse current (ILate) under control conditions and after the application BDF 9198 1×10−6 M. Curve fits to the data were made using equation 1 (Results). (e) shows a concentration-response relation for the effect of BDF 9198. The magnitude of response at each concentration was expressed as the current density of drug-induced ILate.

The mean effects of BDF 9198 are also shown in Figure 6, which shows I–V plots (n=6) for peak current (IPeak; Figure 6c) and end-pulse current (ILate; Figure 6d). Under both control conditions and with BDF 9198, the I–V relation for IPeak was ‘bell-shaped' and crossed the voltage axis between 0 and +12 mV (with Erev more negative in the presence of BDF 9198, possibly due to raised Na in the subsarcolemmal space, cf Convery & Hancox, 1999). The mean (±s.e.m.) data for both plots were fitted with equation 1. Under our conditions, activation of INa increased steeply over the voltage range between −50 and −30 mV. For IPeak under control conditions (open circles), the Boltzmann fit to the data gave a value for V0.5 of −39.0±0.4 mV, and k=5.1±0.4 mV (similar to the values in Figure 4b). Application of 10−6 M BDF 9198 resulted in a slight left-ward shift in the I–V relation for IPeak (closed circles): V0.5 was −45.8±0.5 and k=5.2±0.5 mV (P<0.001). The I–V relation for ILate (Figure 6d) shows that under control conditions there was no discernible late component of INa (open circles). In the presence of 1×10−6 M BDF 9198 significant ILate was observable, and this showed a bell-shaped I–V relation similar in profile to, but smaller in magnitude than that for IPeak. The Boltzmann fit to ILate observed in the presence of BDF 9198 gave a V0.5 of −42.5±0.7 mV and a k of 5.4±0.6 mV, parameters which were similar to those for the fit to IPeak in the presence of the drug.

For a range of drug concentrations, the effect of BDF 9198 on INa (elicited by a test pulse to −30 mV) was expressed as the current density of the late current (ILate), shown in Figure 6e (n=at least four cells for each concentration). There was a relatively small effect of BDF 9198 at 5×10−9 M, with a progressive increase in the magnitude of ILate up to 1×10−5 M (the highest concentration applied).

Effects of BDF 9198 on the time-course of inactivation

The presence of an ILate in the presence of BDF 9198 is consistent with modulation by the drug of the inactivation process, such that at the end of the 200 ms voltage command to −30 mV, substantial current still remained (Figures 5 and 6b,d). The decline of INa over a 200 ms test-pulse to −30 mV in the presence of BDF 9198 could be well described by a process comprised of two exponentials together with a residual, end-pulse component (C; see equation 2). For the cell shown in Figure 7a, the fast and slow time constants of inactivation in the presence of 1×10−6 M BDF 9198 were 1.50 and 55.0 ms respectively (compared to control values for the same cell of 1.48 and 12.87 ms). In a sample of six cells inactivation of INa was quantified as follows: 96±0.02% of the current was described by a τf of 1.4±0.3 ms; 4±0.01% of the current was fitted by a τs of 13.6±1.0 ms (there was no significant difference to the values obtained in Table 2, P>0.15). There was no discernible end-pulse current component (C). In the presence of BDF 9198, the relative proportions of the current fitted by τf (56±0.1%) and τs (16±0.2%) altered, without any significant alteration in the magnitude of τf (1.5±0.3 ms; P>0.1). By contrast τs was increased to 49±9.4 ms. In the presence of BDF 9198, 28±0.4% of the peak current remained at the end of the test-pulse (C). To summarize: BDF altered the relative proportions of fast to slowly inactivating INa, left the magnitude of τf unchanged, increased the magnitude of τs, and led to the presence of a significant component of INa at the end of the applied 200 ms test-pulse.

Figure 7.

Figure 7

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Effects of BDF 9198 on INa inactivation time-course. (a) shows a curve fit to the inactivation of an INa (TTX-S current) recorded under control conditions (left panel), and in the presence of 1×10−6 M BDF 9198 (Equation 2, see Results). Currents were elicited by a step depolarization from −140 to −30 mV. (b) shows inward and outward INa (TTX-S current) elicited in control conditions (left panel) by a step-depolarization from −140 to −30 mV and +30 mV (Erev for INa=0 mV in this cell). The effect of application of 1×10−6 M BDF 9198 on inward and outward INa is shown in the right panel.

It should be noted however, that experiments with a longer duration (1 s) test-pulse indicated that the sustained current component observed 200 ms after depolarization in the presence of BDF cannot be considered to represent current amplitude in the steady-state. In 5 cells, to which 1 s pulses to −30 mV were applied 25.4±2.1% of peak inward current remained 200 ms into the pulse (P>0.8 compared to 200 ms pulse data above). At 1 s however, only 11.9±1.4% of peak current remained. Additionally, it was difficult to fit adequately the current decline over 1 s with a bi-exponential process. Thus, whilst inactivation of INa in the presence of BDF 9198 may adequately be ascribed a bi-exponential decline over the first 200 ms, over a longer duration current inactivation may in fact be better described by process comprised of >2 exponential phases. Under our conditions it was difficult to apply long pulses in the presence of BDF 9198 and the time-course of current decline was not monitored for >1 s.

If the dominant effect of BDF 9198 is to interact with the Na channel to modulate the process of current inactivation, a sustained current component might be predicted in the presence of drug, whether INa was inwardly or outwardly directed. Figure 7b shows the result of experiments performed to test this proposition. For five cells we measured peak INa and Erev and then applied a pulse to a potential an equal but opposite distance from the Erev, to elicit an outward INa. Under control conditions (left panel) both inward and outward INa inactivated completely during the applied depolarization (with inactivation of the outward INa being slightly faster than that of the inward current). As expected, application of BDF 9198 led to sustained inward INa (right panel); moreover, outward INa was also modified such that a sustained current component remained at the end of the test-pulse. The results of the experiment shown in Figure 7b (displayed as TTX-S current measurements) showed that there was no significant quantitative difference between the amount of end-pulse current for inward and outward INa in the presence of BDF 9198 (22.8±2.9% and 23.6±1.9% of peak current amplitude respectively, P>0.66; n=five cells).

Effects of BDF 9198 on ICaL

The effects described for BDF 9198 on INa might be anticipated to increase the contribution of INa to membrane potential depolarization during the action potential plateau. Another important inward current that contributes to the plateau phase is ICaL (e.g Linz & Meyer, 1999). Therefore, if BDF 9198 altered ICaL then this could also contribute to the observed effects of the drug on APD50. In order to test the effects of BDF 9198 on ICaL we used selective recording conditions (see Table 1). ICaL was elicited by applying a 500 ms voltage step from a holding potential of −40 to +10 mV. Figure 8 shows an ICaL recorded under control conditions (left panel) and after the application of 1×10−6 M BDF 9198 (right panel). In eight cells there appeared to be no significant change (P>0.05) in ICaL; this precluded modulation of ICaL from contributing to action potential prolongation by the drug.

Figure 8.

Figure 8

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Effect of BDF 9198 on ICaL. ICaL elicited under selective recording conditions (see Methods) by applying a square voltage clamp pulse (b), from −40 to +10 mV. (a; left panel) shows current elicited in control solution (Ctl), (a; right panel) shows that 1×10−6 M BDF 9198 did not affect peak ICaL amplitude.

Effects of BDF 9198 on IK

We then proceeded to investigate the effects of BDF 9198 on potassium currents known to contribute to repolarization of the action potential (Carmeliet, 1993; Sanguinetti & Keating, 1997). If BDF 9198 inhibited repolarizing potassium currents, this would also contribute to action potential prolongation. We first tested the effects of BDF 9198 on the delayed rectifier current, IK, which participates in action potential repolarization over the plateau voltage range (Sanguinetti & Keating, 1997). In guinea-pig ventricular myocytes IK is a composite current, comprised of rapid (IKr) and slow (IKs) delayed rectifier sub-types (Sanguinetti & Jurkiewicz, 1990; Heath & Terrar, 1996). In order to record composite IK, we applied 2000 ms duration, progressively incrementing step-depolarizations at frequency of 0.1 Hz, from a holding potential of −40 mV. Interfering currents were inhibited using the internal and external solutions described in Table 1. Figure 9a shows representative currents elicited by depolarizing pulses to four test potentials (panel b). On repolarization to −40 mV, deactivating outward tail currents were observed. Neither the currents during the test pulses, nor the outward current tails were affected by application of 1×10−6 M BDF 9198. Figure 9c shows mean I–V relations for the IK tails observed on repolarization from the different test potentials (n=5). There was no significant effect of BDF 9198 on IK tail density at any test potential (P>0.05), indicating that the effect of BDF 9198 on action potential duration could not be attributed to IK inhibition.

Figure 9.

Figure 9

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Effect of BDF 9198 on IK. (a) Records of IK (for recording conditions, see Methods) elicited by 2000 ms step-depolarizations from −40 mV to a range of test potentials (−20, 0, +20, +40 mV; b). There was no significant difference between Control (Ctl) IK (a; left panel) and that in the presence of 1×10−6 M BDF 9198 (a; right panel). (c) shows the mean I-V relations for IK tails before and after BDF 9198.

Effects of BDF 9198 on IKI

Inward rectifier potassium current (IKI) participates in late repolarization of the ventricular action potential (Shimoni et al., 1992; Carmeliet, 1993). We used the internal and external solutions described in Table 1 to record IKI selectively. In order to elicit IKI we applied a ramp protocol (Figure 10b) comprised of a step depolarization to 0 mV from a holding potential of −40 mV, followed by a descending ramp over 2000 ms to −120 mV. The protocol was applied every 3 s. In control conditions a clear inwardly rectifying current, typical of IKI was observed (Figure 10a, left panel). The application of 1×10−6 M BDF 9198 had no significant effect on the current (right panel). This was confirmed by constructing mean I–V relations for IKI (Figure 10c) in which the control data (circles) and BDF 9198 data (squares) are superimposed (n=6; P>0.05 at all potentials). These findings indicate that BDF 9198 did not inhibit IKI, and taken together with the data in Figure 9, suggest that any action potential prolongation by BDF 9189 up to 1×10−6 M did not result from outward potassium current inhibition.

Figure 10.

Figure 10

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Effect of BDF 9198 on IK1. (a) shows IK1 (for recording conditions, see Methods) under control conditions (left panel) elicited by a 2000 ms ramp protocol from 0 to −120 mV, applied from a holding potential of −40 mV (panel b). (Right panel) shows current elicited by the same protocol after applying 1×10−6 M BDF 9198. There was little observable effect of BDF 9198 1×10−6 M (c) shows the mean I-V relation for IK1 before and after BDF 9198

Discussion

Effects of BDF 9198 on action potentials and underlying ionic currents

BDF 9198 has recently been reported to exert a positive inotropic effect on human myocardium (Muller-Ehmsen et al., 1998; Schwinger et al., 1999), an effect similar to that exerted by the related compounds BDF 9148 and DPI 201-106 (e.g. Scholtysik et al., 1985; Brasch & Iven, 1991; Hoey et al., 1993). The present study demonstrates that BDF 9198 is also a positive inotrope on guinea-pig myocardium, showing a similar concentration-response profile to that observed with human tissue (Muller-Ehmsen et al., 1998; Schwinger et al., 1999). To date, however, there has been no detailed study which describes the electrophysiological effects of BDF 9198. The principle findings of the present study were that, under selective recording conditions, BDF 9198 slows inactivation of INa recorded under voltage clamp, and like the related compounds BDF 9148 and DPI 201-106 (Brasch & Iven, 1991; Ravens et al., 1991; Hoey et al., 1994; Raap et al., 1997) the drug causes action potential prolongation. It is notable that the extent of action potential prolongation produced by BDF 9198 in the present study was markedly greater than that observed for BDF 9148 or DPI 201-106 in the guinea-pig heart. Ravens et al. (1991) observed a 1.2–1.3 fold increase in APD90 with 1×10−6 M BDF 9148, whilst we observed a greater than 3 fold increase in APD50 at a lower concentration (1×10−7 M). Brasch and Iven (1991) also observed comparatively modest increases in APD with BDF 9148 compared to that reported for its sister compound in the present study. Neither of these previous studies reported after-depolarizations in the presence of BDF 9148, suggesting that the degree of internal calcium loading during exposure to DPI 201-106 or BDF 9148 was smaller than that which occurred under the conditions of the present study.

The observed differences in effects on APD of BDF 9198, on the one hand, and BDF 9148 and DPI 201-106, on the other, can be explained by considering the comparative effects of the compounds on ionic currents underlying the action potential. In addition to prolonging INa, both DPI 201-106 and BDF 9148 exhibit inhibitory effects on ICa,L with 30–50% inhibition at 1×10−6 M (Ravens et al., 1991). The reduction in depolarizing current resulting from this action is likely to offset, to an extent, the Na-channel effects of the compound. Under our conditions, we observed no significant alteration in peak ICa,L at 1×10−6 M BDF 9198, thus the increased depolarizing drive of INa would not have been countered by reduced ICa,L. Ravens et al. (1991) considered the relative effects of INa prolongation and ICa,L reduction in their study, noting that whilst BDF 9148 exerted more potent effects on INa than ICa,L, DPI 201-106 affected the currents equally, whilst producing a greater action potential prolongation. This was suggestive of additional drug effects on other ion channel types. Subsequently, differential effects of these compounds on IKs, and inward rectifier, IKI were demonstrated (Amos & Ravens, 1994). DPI 201-106 produced moderate reductions in both IKI and IKs, whilst BDF 9148 did not alter IKI and only affected IKs at higher concentrations. In the present study, both IKI and composite IK were unaffected by 1×10−6 M BDF 9198–a concentration 10 fold higher than that required to prolong the action potential to over 600 ms. These observations are suggestive of selectivity of BDF 9198 for INa without concomitant changes in IK, IKI or ICa,L. These data suggest that the actions of BDF9198 solely on INa, underlie the action potential prolongation observed under current clamp conditions.

Effects on INa

As described in Results, the properties of INa we observed under selective conditions correlate well with other published data. The V0.5 estimated from the fit to our control I–V data compares favourably with that reported for both feline ventricular myocytes (−33.2 mV; Shacklow et al., 1995) and human atrial cells (−38.9 mV, Sakakibara et al., 1992; −38.6 mV, Feng et al., 1996). Both Sakakibara et al. (1992) and Shacklow et al. (1995) found that inactivation of INa was best described by a bi-exponential decline. Our control values for τf and τs at −30 mV are close to those reported by Sakakibara et al, who also observed that τf contributed to over 90% of current inactivation at this potential.

Ravens et al. (1991) observed induction of a sustained INa by BDF 9148 with physiological [Na]o and showed this to be blocked by TTX. This observation is qualitatively similar to our own, regarding BDF 9198. Due to the differing [Na]o, in the two studies, it is difficult to make direct comparisons between their concentration–response data for BDF 9148 and our own for BDF 9198. At 1×10−6 M BDF 9148 Ravens et al. (1991) reported a sustained INa of just under 20pA/pF in magnitude (their Figure 4). With a 10 fold reduction in [Na]o we observed an ILate of ∼10–12pA/pF. This, together with the profound effects on APD50 does suggest that BDF 9198 may be more potent in modulating INa than its sister compound. A more recent study by Ravens and co-workers, with a lowered [Na]o (Hoey et al., 1994) reported no significant effects of DPI 201-106 or BDF 9148 on the I–V relation for IPeak. Other data, from expressed cardiac Na channel α-subunits (Krafte et al., 1994), also suggested no shift in the I–V relation, but noted a small (8%) reduction in peak current with BDF 9148. The slight left-ward shift of the I–V relation for IPeak we observed with BDF 9198 differs from these observations, and accounts for the larger peak currents we observed at negative potentials (e.g at −50 mV, see Figure 6b) and reduced current amplitudes at potentials on the ascending limb of the I–V curve (Figure 6b,c). Overall, we did not observe statistically significant alterations in the absolute IPeak density with exposure to BDF 9198. The lack of effect of a partial blocking dose of TTX on the I–V profile for INa, despite a marked alteration in current amplitude would argue against any left-ward shift of the I–V produced by BDF 9198 resulting from an experimental artifact. However, even if present, the relatively small observed shift in voltage dependence of IPeak is unlikely to have made a major contribution to the overall effect of BDF 9198 on the ventricular action potential.

The reported modification of Na currents through expressed Na channel α subunits by congeners of BDF 9198 suggests that the α subunit is the site of interaction between this family of compounds and the Na channel (Krafte et al, 1991; 1994). Mutagenesis experiments suggest that the III–IV linker region of the α subunit is critical for inactivation of INa, whilst TTX binds in the vestibule of the channel pore (see Fozzard & Hanck, 1996 for a review). It is possible, therefore, that BDF 9198 exerts its effects on INa inactivation by modulating the ability of the III–IV linker to act as the blocking particle normally responsible for inactivation. Such an action would produce an altered inactivation profile of the current, but would not be expected to affect the ability of TTX to inhibit current flow through BDF 9198-modulated channels. It is noteworthy that DPI 201-106 modifies inactivation time–course of current carried by cloned cardiac but not neuronal Na channel alpha-subunits expressed in Xenopus oocytes (Krafte et al., 1991). Thus, the portion of the channel to which such agents bind may differ in its molecular composition between cardiac and neuronal tissue. Expression studies, using modified α subunits are necessary to test such a hypothesis.

Implications of the study

Our data indicate that BDF 9198 is a potent modulator of cardiac INa at concentrations which do not affect other important ionic currents. As BDF 9198 is positively inotropic on the human myocardium (Muller-Ehmsen et al., 1998), questions therefore arise regarding the possible therapeutic value of the drug. It is difficult to predict with precision from experiments on isolated cells what effects the compound may have in vivo, but our results do raise issues worthy of consideration. DPI 201-106 has been reported to produce some prolongation of the QT-interval of the electrocardiogram in healthy subjects (Ruegg & Nuesch, 1995). The more extensive in vitro APD prolongation caused by BDF 9198 may translate into greater QT prolongation, with an associated risk of pro-arrhythmia mediated by after-depolarizations and Torsade de pointes (Roden, 1990; Levi et al., 1997). Such a pharmacological proarrhythmic risk may be analagous to forms of genetic Long-QT syndrome (LQTS) involving Na channel mutations which give rise to persistent INa (Bennett et al., 1995; Dumaine et al., 1996). In this regard, the selectivity of BDF 9198 for INa may make it of investigational value for experimental models of INa-linked QT prolongation. The reported effects of DPI 201-106 and BDF 9148 on additional membrane currents make them less suitable for such an application. The actual likelihood of BDF 9198-linked proarrhythmia in vivo would depend on whether satisfactory inotropic effects of BDF 9198 could be obtained at concentrations producing only moderate changes to the ventricular APD and, thereby, QT interval.

Acknowledgments

This study was funded by a Wellcome Trust Career Development award to J.C. Hancox. S.A. Doggrell's research is supported by the Auckland Medical Research Foundation. We gratefully acknowledge the skilled support of Lesley Arberry in performing myocyte isolations and an anonymous referee for valuable experimental suggestions.

Abbreviations

Af

inactivating current described by the fast time constant

ANOVA

analysis of variance

APD50

action potential duration at 50% repolarization

APs

action potentials

As

inactivating current described by the slow time constant

α subunit

(sodium channel) alpha-subunit

BAPTA

1.2-bis (2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid

BDF 9148

(4-(3-(1-diphenylmethyl-azetidine-3-oxy)-2-hydroxypropoxy)-1-H-indol-2-carbonitrile

BDF 9198

(4-(3-(1-diphenylmethyl-azetidine-3-oxy)-2-hydroxy-propylamin)-1-H-indol-2-carbonitrile

C

residual current at end of voltage pulse

Cai

internal calcium transient

Ctl

control

DADs

delayed after-depolarizations

DMSO

dimethylsulphoxide

DPI-201-106

4-[3-(4-diphenylmethyl-1-piperazinyl)-2-hydroxypropoxy]-1H-indole-2-carbonitrile

EADs

early after-depolarizations

Gmax

maximal INa conductance

h1

human cardiac sodium channel

I

tetrodotoxin sensitive cardiac sodium channel

ICa,L

L-type calcium current

IClamp

current clamp

IK

delayed rectifier potassium current

IKl

inward rectifier potassium current

ILate

sustained current remaining at end of voltage pulse

INa

sodium current

IPeak

peak current at start of voltage pulse

Iti

transient inward current

I-V

current–voltage relation

k

slope factor

LJP

liquid junction potential

LQTS

long QT syndrome

[Na]I

Internal sodium concentration

[Na]o

external sodium concentration

NMDG

N-methyl-D-glucamine

s.e.m.

standard error of the mean

τf

fast time constant

τs

slow time constant

TTP

time to peak

TTX

tetrodotoxin

TTX-S

tetrodotoxin-subtraction

V0.5

membrane potential exhibiting half maximal current activation

Vm

membrane test potential

Vrev

reversal potential

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