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Cardiovascular Research 1999 42(2):510-520; doi:10.1016/S0008-6363(99)00043-7
© 1999 by European Society of Cardiology
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Copyright © 1999, European Society of Cardiology

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

Eva Delpóna,*, Ricardo Caballeroa, Carmen Valenzuelaa, Mónica Longobardoa, Dirk Snydersb and Juan Tamargoa

aDepartment of Pharmacology, School of Medicine, Universidad Complutense, 28040-Madrid, Spain
bLaboratory for Molecular Biophysics, Physiology and Pharmacology, Universitaire Instelling Antwerpen, Universiteitsplein 1, B-2610 Wilrijk, Belgium

edelpon{at}eucmax.sim.ucm.es

* Corresponding author. Tel.: +34-91-394-1474; fax: +34-91-394-1470

Received 15 October 1998; accepted 26 January 1999


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

KEYWORDS Benzocaine; Drug–channel interactions; Local anesthetic; K+ channels; Mouse


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Benzocaine is a neutral (pKa=2.5) local anesthetic (LA) that at physiological pH predominates in its uncharged form. It produces a tonic inhibition of Na+ channels but little use-dependent block during repetitive depolarizations while, tertiary amine LAs and their quaternary derivatives elicit both tonic and use-dependent block [1]. Recently it has been demonstrated that the enantiomers of bupivacaine, a tertiary amine LA, block hKv1.5, a Shaker-like K+ channel, at concentrations lower than those needed for Na+ channel block [2]. Furthermore, ropivacaine and mepivacaine, two LAs chemically related to bupivacaine, also block Kv1.5 channels [3,4]. These channels generate the ultrarapid delayed rectifier current (IKur) in human cardiac atrial myocytes, which is involved in the control of atrial action potential duration [5]. The blockade of hKv1.5 channels produced by LAs and antiarrhythmic agents is similar to that of internal tetraethylammonium (TEA) and its derivatives, collectively called quaternary ammonium compounds [6,7]. In summary, the cationic form of these drugs blocks the ionic current only after channel opening by binding at a site in the internal mouth of the ionic pore. The binding of these drugs is determined by an electrostatic component reflecting the electrical binding distance and a hydrophobic component that determines the affinity [7]. Moreover, site-directed mutagenesis confirmed that the midsection of the transmembrane segment cells is implicated in these hydrophobic interactions [8,9].

The present work was undertaken to study the effects of benzocaine on hKv1.5 channels. We found that benzocaine reduced the current and modified the time- and voltage-dependence of hKv1.5 channel gating. Moreover, at nanomolar concentrations it affected the channel gating without obvious block. Increasing the extracellular K+ concentration also modified the time- and voltage-dependence of hKv1.5 channel gating similarly to benzocaine. Finally, our results suggest that the slow inactivation of hKv1.5 channels did not resemble the N- or C-type inactivation described in Shaker channels.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Cell culture
hKv1.5 Channels were stably expressed in mouse L-cell line using procedures previously described elsewhere [8–10]. Transfected cells were cultured in DMEM supplemented with 10% horse serum and 0.25 mg/ml G418 (Gibco). Prior to experimental use, cultures were incubated with 2 µM dexamethasone for 24 h as expression of the channel was under control of a dexamethasone-inducible promoter [10].

2.2 Recording techniques
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). To obtain 140 mM or 0 mM extracellular K+ concentrations ([K+]o) equimolar substitution of KCl for NaCl was used. Recording pipettes were filled with an "internal" solution containing (mM): K-aspartate 80, KCl 42, KH2PO4 10, MgATP 5, phosphocreatine 3, HEPES 5 and EGTA 5 (pH=7.2 with KOH). In some experiments the total internal [K+] was lowered 50% by the equimolar substitution of K-aspartate by TrisCl. Benzocaine was initially dissolved in dimethyl sulfoxide (DMSO) to yield 0.1 M stock solutions. Further dilutions were carried out in external solution to obtain the desired final concentrations. DMSO did not affect the current at concentrations up to 0.1%.

hKv1.5 Currents were measured using the whole-cell configuration of the patch clamp technique. Recordings were performed at 24–25°C using Axopatch-1D patch clamp amplifiers and PCLAMP 6.1 software (Axon Instruments). Borosilicate pipettes used had a tip resistance of 1.5–2.5 M{Omega} when filled with the internal solution and immersed in the external solution. The capacitive transients elicited by symmetrical 10 mV steps from –80 mV were recorded for subsequent calculation of capacitive surface area, access resistance and input impedance. Maximum outward current amplitudes at +60 mV averaged 1.5±0.1 nA, mean uncompensated access resistance was 3.3±0.5 M{Omega} and cell capacitance 10.2±0.9 pF (n=22). Thereafter, capacitance and series resistance compensation were optimized and {approx}80% compensation was usually obtained. Thus, no significant voltage errors (<5 mV) due to uncompensated series resistance were expected. Voltage-clamp command pulses were generated by a 12-bit digital-to-analog converter. The current records were sampled at 3–10-times the antialias filter setting.

2.3 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 block. The protocol to obtain current–voltage (I–V) relationships and activation curves consisted of 500 ms pulses that were imposed in 10 mV increments between –80 mV and +60 mV, with additional interpolated pulses to yield 5 mV increments between –30 and +10 mV (activation range of hKv1.5). The "steady-state" I–V relationships were obtained by plotting the current level after 500 ms as a function of the membrane potential. Between –80 and –40 mV, only passive linear leak was observed and least squares fits to these data were used for passive leak correction. Deactivating "tail" currents were recorded on return to –40 mV. The activation curve was constructed by plotting the tail current amplitude values elicited on return to –40 mV after 500 ms depolarizations to various test potentials (from –80 to +60 mV) as a function of the membrane potential.

Activation curve for each individual experiment has been fitted with a Boltzmann distribution according to the following equation:

Formula (1)
where A is the amplitude term, Vh is the midpoint of activation (in mV), Vm is the test potential and k represents the slope factor for the activation curve (in mV). Thereafter, the mean value for Vh and k values for each group of experiments was calculated. Under some circumstances a Boltzmann distribution with two terms was needed to fit the experimental data, the equation being:

Formula (2)

To describe the time course of the tail currents upon repolarization exponential analysis was used as an operational approach, fitting the tail currents to an equation of the form:

Formula
where {tau}1, {tau}2 and {tau}n are the system time constants, A1, A2 and An are the amplitudes of each component of the exponential, and C is the baseline value. The Chebyshev transform and Simplex least-squares algorithm provided in CLAMPFIT (PCLAMP 6.1) and costumer made programs (Dr. D. Snyders) were used for the exponential fitting. This curve fitting procedure used a nonlinear least-squares (Gauss–Newton) algorithm; results were displayed in linear and semilogarithmic format, together with the difference plot. Goodness of fit was judged by the {chi}2 criterion and by inspection for systematic nonrandom trends in the difference plot.

Fractional block was defined as:

Formula

A first-order blocking scheme was used to describe drug-channel interaction kinetics; apparent dissociation constant, KD (concentration for 50% block or EC50), and Hill coefficient, nH, were obtained from fitting the fractional block, f, at various drug concentrations [D] to the equation:

Formula (3)

2.4 Statistical methods
Data obtained after drug exposure were compared with those obtained under control conditions in a paired manner. For comparisons at a single voltage or drug concentration differences were analyzed using the Student’s t-test. To analyze block at multiple voltages or drug concentrations, two-way analysis of variance was used. Results are expressed as mean±standard error of the mean (SEM). A P-value of less than 0.05 was considered significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Low concentrations of benzocaine increase the hKv1.5 current
Fig. 1 shows hKv1.5 currents elicited in response to 500 ms pulses to +60 mV (Panel A) and to –30 mV (Panel C) in the absence and in the presence of 10 nM benzocaine. At +60 mV hKv1.5 currents rose rapidly to a peak ({tau}=1.7±0.1 ms, n=9) and displayed slow and partial inactivation as previously described [10]. Benzocaine slightly increased the peak current, whereas it decreased the steady-state current amplitude at the end of the pulse (5.9±0.6%, n=5), indicating that the drug increased the current decline during the pulse. In contrast, at –30 mV, benzocaine increased the current amplitude by 2.8±0.8-fold over control values (n=5, P<0.001). Fig. 1D shows the activation curve in the absence and in the presence of 10 nM benzocaine. Benzocaine did not modify the k value, but shifted the Vh from –18.1±1.2 mV to –26.6±1.5 mV (n=5, P<0.05). Thus, the benzocaine-induced increase of the current at –30 mV was the consequence of a parallel shift of the activation curve of hKv1.5 channels to more negative potentials. However, at membrane potentials at which the activation curve reached saturation (>–10 mV) the tail current amplitude obtained in the presence of benzocaine was slightly greater than that obtained in the absence of drug, an effect which cannot be explained by the hyperpolarizing shift. In control conditions tail currents at –40 mV were fitted with a biexponential function. Fig. 1B shows superimposed tail currents in the absence and in the presence of 10 nM benzocaine. Benzocaine slightly increased the peak tail current and slowed the time course of tail current decline. In fact, it increased the fast ({tau}f) and slow ({tau}s) time constants of tail current decline from 28.4±2.4 ms and 85.0±8.8 ms to 39.1±3.3 ms and 136.9±13.6 ms, respectively (n=6, P<0.05). However, benzocaine did not modify the relative contribution of each component of deactivation. Thus, in the absence and in the presence of 10 nM benzocaine the fast component of deactivation averaged 53.6±4.6% and 46.1±6.6%, respectively (n=6, P>0.05). These results suggest that at very low concentrations benzocaine modifies the time- and voltage-dependent properties of hKv1.5 channels (i.e., "gating" properties), resulting in an increase in current amplitude at least at certain voltages ("agonist effect").


Figure 1
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  		Fig. 1 Effects of 10 nM benzocaine on hKv1.5 currents. (A) Superimposed current tracings for 500 ms depolarizing pulses from a holding potential of –80 mV to +60 mV in control conditions and in the presence of benzocaine. (B) Tail currents elicited on return to –40 mV after 500 ms depolarizing pulses to +60 mV in control conditions and in the presence of benzocaine. Continuous line represents the biexponential fit of the tail current decay. (C) Superimposed tracings for steps to –30 mV from a holding potential of –80 mV. (D) Activation curve of hKv1.5 channels in control conditions (bullet) and in the presence of benzocaine ({circ}). The lines illustrate the Boltzmann fit for each condition. Each data point represents the mean and vertical lines the SEM of six experiments. The dotted line in panels A–C represents the zero current level.

 
3.2 Blocking effects of benzocaine on hKv1.5 channels
Fig. 2 shows currents elicited by 500 ms pulses applied in 20 mV increments from –80 to +60 mV in the absence (panel A) and in the presence of benzocaine 700 µM (panel B). Benzocaine reduced more the steady-state than the peak current which resulted in a slight increase in the amplitude of the inactivating component. In fact, currents elicited by 500 ms pulses to +60 mV inactivated by 20.3±0.6% and by 33.1±1.1% (n=8, P<0.01) in the absence and in the presence of drug, respectively. Blocking effects of benzocaine reached steady-state in 7 min and were largely reversible upon washout. Benzocaine reduced the steady-state currents in a dose-dependent manner (Fig. 3). Using the reduction of current at +60 mV as an index of block, the apparent affinity (KD) for benzocaine blockade of hKv1.5 (calculated assuming nH=1.0) averaged 901±81 µM (n=20). For comparison the KD for benzocaine blockade of batrachotoxin-modified Na+ channels is 200 µM [11]


Figure 2
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  		Fig. 2 Effects of 700 µM benzocaine on hKv1.5 currents ([K+]o=4 mM). Superimposed current traces are shown for 500 ms depolarizing pulses from –80 mV to voltages between +60 to –80 mV in 20 mV steps (diagram on top). Traces were obtained in the absence (A) and in the presence of benzocaine (B). (C) Superimposed tail currents elicited upon return to –40 mV after 500 ms depolarizing pulses to +60 mV from a holding potential of –80 mV in control conditions and in the presence of benzocaine. The continuous line represents the biexponential fit of the tail current decay. (D) Current–voltage relationship (500 ms isochronal) in control conditions (bullet) and in the presence of benzocaine ({circ}). In panels A–D the dotted line represents the zero current level. (E) Fractional block (f=1–IBZ/ICON) from the data shown in D. The dotted line represents the activation curve for this group of experiments. (F) Activation curves of hKv1.5 channels in the absence and in the presence of benzocaine. The dotted line represents the fit to the control data with a single Boltzmann component (Eq. (1) in Methods). The dashed line shows that this approach was not optimal for the data in the presence of benzocaine, whereas a better fit was obtained with a sum of two Boltzmann components (solid line, Eq. (2) in Methods). Each data point in panels D, E and F represents the mean and vertical lines the SEM of nine experiments.

 

Figure 3
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  		Fig. 3 Percentage of benzocaine-induced block measured at the end of 500 ms pulse to +60 mV as a function of the drug concentration. Each bar represents the mean and vertical lines the SEM of 6–9 experiments.

 
3.3 Voltage-dependence of benzocaine induced block
Fig. 2D shows the I–V relationship in the absence and in the presence of 700 µM benzocaine (n=9). Benzocaine blocked hKv1.5 current at membrane potentials positive to –20 mV. To quantify the voltage-dependence of block, the fractional block was plotted as a function of the membrane potential together with the activation curve of hKv1.5 channels obtained in this group of experiments under control conditions (Fig. 2E). The blockade increased steeply coinciding with the channel opening between –20 and 0 mV. At potentials positive to 0 mV, the blockade progressively decreased with a shallow voltage-dependence, so that the blockade induced by 700 µM benzocaine decreased from 50.7±7.4% at 0 mV to 40.1±2.4% at +60 mV (n=9, P<0.01).

Fig. 2F shows the voltage-dependence of channel opening derived from the amplitude of deactivating tail currents recorded on return to –40 mV. The control data could be described with a single Boltzmann equation (dotted line), the values for Vh and k averaging –16.3±1.1 mV and 4.9±0.1 mV (n=9), respectively. Benzocaine shifted the Vh in the negative direction (Vh=–21.6±1.9 mV, P<0.05) and reduced the k value to 7.8±0.6 mV (P<0.01) (dashed line in Fig. 2F). However, data in the presence of drug deviated from the dashed line at potentials positive to –10 mV, indicating that a single Boltzmann component was inadequate to fully describe the voltage dependence. The solid line illustrates a fit with a sum of two Boltzmann components (Eq. (2) in Methods). Following this procedure, the Vh and k values of the steeper component averaged –23.9±0.5 mV and 5.2±0.1 mV, respectively, while those of the shallow component were 52.7±2.7 mV and 26.0±2.5 mV, respectively. The amplitude of the shallow component represented 29.4±2.5% of the activation process.

3.4 Time-course of deactivating tail currents in the presence of benzocaine
Fig. 2C shows the tail currents elicited on return to –40 mV after 500 ms pulses to +60 mV. Control tail currents were fitted by a biexponential function (continuous line), {tau}f and {tau}s values averaging 33.8±3.7 ms and 89.5±8.6 ms (n=9), respectively. The contribution of the fast component of deactivation averaged 48.8±6.0% (n=9). Benzocaine (700 µM) accelerated the initial phase of deactivation ({tau}f=21.1±2.8 ms, n=9, P<0.01) and prolonged the slow phase ({tau}s=117.1±9.8 ms, n=9, P<0.01). However, it did not modify the relative contribution of each component of deactivation. At 500 µM, benzocaine decreased {tau}f values from 38.9±2.8 ms to 24.9±3.2 ms (n=8, P<0.05). This acceleration of the tail current decrease was not observed with depolarizing pulses to potentials near the Vh value. In fact, 500 µM benzocaine did not accelerate the fast component ({tau}f=32.9±2.8 ms vs. 28.4±1.8 ms, n=8, P>0.05), whereas it significantly increased the {tau}s values (188.1±25.6 ms vs. 122.3±11.7, n=8, P<0.05) of the tail currents elicited on return to –40 mV after a depolarizing pulse to –10 mV.

We also examined the effects of the drug on the tail currents after the application of 5 ms and 250 ms pulses to +60 mV. Under control conditions, increasing the duration of the pulse resulted in a slowing of the fast phase of tail current decay. In fact, {tau}f after 5 ms or 250 ms pulses averaged 13.9±0.7 ms and 23.9±4.1 ms, respectively (n=6). Benzocaine (500 µM) significantly decreased the {tau}f when 250 ms depolarizing pulses were applied ({tau}f=12.6±1.9, P<0.05), whereas it did not modify the {tau}s values. In contrast, for 5 ms depolarizing pulses benzocaine did not modify the {tau}f, but significantly increased the {tau}s values (92.3±8.3 ms vs. 52.3±2.7, P<0.05).

3.5 Effects of benzocaine on hKv1.5 channels in high external K+
To assess a potential interaction between benzocaine-induced block and C-type inactivation, we analyzed the effects of benzocaine in 140 mM [K+]o (Fig. 4). Perfusion with 140 mM [K+]o after the normal external solution decreased the maximum outward current amplitude elicited by depolarizations to +60 mV by 26.1±1.8% (n=9) (see inset) and markedly increased the peak tail current amplitude which became inward. Moreover, at 140 mM [K+]o increased the amplitude of the inactivating component (see inset). In fact, currents elicited by 500 ms pulses to +60 mV inactivated by 29.3±1.5% at 4 mM [K+]o and by 37.7±2.3% at 140 mM [K+]o (n=16, P<0.01). In contrast, the time constant of inactivation during 500 ms depolarization to +60 mV was not modified ({tau}4mM=171.4±11.8 ms vs. {tau}140mM=186.0±9.9 ms, n=18, P>0.05). Under these conditions (Fig. 4A), benzocaine blocked hKv1.5 currents at the end of 500 ms depolarizing pulses to +60 mV by 30.9±3.6% (n=6) as compared with a 30.8±2.7% (n=12, P>0.05) inhibition at 4 mM [K+]o. Moreover, the drug also decreased the peak tail current amplitude elicited on return to –40 mV by 28.8±6.4% (n=6), an inhibition similar to that observed at 4 mM [K+]o (31.7±4.2%, n=12, P>0.05). These results indicated that in the presence of benzocaine the currents were scaled down by the same factor at 4 mM [K+]o as at 140 mM [K+]o.


Figure 4
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  		Fig. 4 Effects of benzocaine on hKv1.5 currents ([K+]o=140 mM). (A) Superimposed current traces are shown for 500 ms pulses from –80 mV to voltages between –80 to +60 mV in steps of 20 mV. Tail currents were obtained on return to –40 mV. Traces were obtained in the absence and in the presence of 500 µM benzocaine. The inset shows current traces elicited by pulses to +60 mV when the external solution contained 4 mM [K+]o and 140 mM [K+]o. Current amplitude of the trace elicited at 140 mM [K+]o was scaled to control (labeled ST) to better appreciate the increase in current inactivation. (B) Activation curves of hKv1.5 channels obtained at 4 mM [K+]o (dashed line), at 140 mM [K+]o (continuous line) or after perfusion with 140 mM [K+]o plus 500 µM benzocaine (dotted line). The activation curve in the presence of normal external solution was fitted with a single Boltzmann component (see Fig. 2). However, activation curves in high K+ solution in the absence and the presence of benzocaine were better defined by a Boltzmann equation with two components. Each data point represents the mean and vertical lines the SEM of nine experiments. (C) Activation curves of hKv1.5 channels obtained at 140 mM [K+]o (continuous line) or after perfusion with 140 mM [K+]o plus 10 nM benzocaine (dotted line). Each data point represents the mean and vertical lines the SEM of six experiments.

 
Fig. 4B shows the activation curve obtained in this group of experiments at 4 and 140 mM [K+]o, and in the presence of high [K+]o plus 500 µM benzocaine. The Vh and the k values at 4 mM [K+]o averaged –16.5±0.7 mV and 4.7±0.2, respectively (n=9). Fitting of the activation curves at 140 mM [K+]o required a sum of two Boltzmann functions, the k values for the steeper and the shallow components averaging 4.4±0.1 mV and 27.8±2.5 mV, respectively (n=9). The shallow component represented the 20.2±2.8% of the activation process and reached the Vh value at very positive potentials (Vh=98.0±27.9 mV, n=9), indicating that the saturation level for this component of the Boltzmann distribution was not achieved. Furthermore, benzocaine in cells superfused at 140 mM [K+]o shifted the Vh of the steeper component from –20.8±0.4 mV to –24.8±0.5 mV (n=9, P<0.05), but it did not change the Vh of the shallow component (Vh=94.2±3.5 mV, P<0.05).

We also analyzed the kinetics of the tail currents after pulses of different durations (5, 250 and 500 ms) to +60 mV. Deactivation of the tail currents was always faster at 140 than at 4 mM [K+]o. In fact, {tau}f for 5, 250 and 500 ms averaged 11.6±0.8 ms, 13.1±1.4 ms and 18.9±1.6 ms, respectively (n=6). Benzocaine (500 µM) did not modify the {tau}f for any duration of the depolarizing pulse.

Because high [K+]o increased the hKv1.5 inactivation we also studied the effects of 500 µM benzocaine when 5 s pulses to +60 mV were applied. Under control conditions, the amount of inactivation averaged 69.2±2.1% (n=5). Benzocaine reduced the current amplitude measured at the end of the pulse by 37.5±8.1%, i.e., by the same amount as after 500 ms depolarizations (n=5, P>0.05), suggesting that benzocaine does not interact with the slow inactivated state of the channel.

In another group of experiments we analyzed the effects of 10 nM benzocaine at 140 mM [K+]o. Under these conditions, benzocaine decreased the current amplitude at the end of the 500 ms depolarizations to +60 mV by 16.7±2.6% (n=6), and it slowed the decline of tail currents elicited on return to –40 mV increasing both the {tau}f (from 16.4±2.7 ms to 34.3±7.9 ms, n=5, P<0.05) and the {tau}s (from 123.9±14.9 ms to 255.4±46.6, P<0.05) values. Fig. 4C shows the activation curve obtained in six cells at 140 mM [K+]o in the absence and in the presence of 10 nM benzocaine. It can be observed that benzocaine shifted into the negative direction the Vh of the steeper component of the activation curve (–21.8±1.1 mV vs. –28.9±1.4 mV, n=6, P<0.001), whereas it did not modify the Vh of the shallow component. Moreover, it is evident that the increase in tail current amplitude observed at 4 mM [K+]o at potentials positive to –10 mV was not observed at 140 mM [K+]o.

3.6 Effects of benzocaine in the absence of external K+
To study whether there was competition between the gating effects of the [K+]o and benzocaine, cells were superfused at 4 mM [K+]o and then at 0 mM [K+]o in the absence and in the presence 500 µM of benzocaine. At 0 mM [K+]o, currents at the end of the 500 ms pulse to +60 mV decreased slightly (16.4±4.9%, n=5) (Fig. 5A), which confirmed the role of [K+]o in modulating the gating of hKv1.5 channels (the current did not increase but decreased). Benzocaine decreased the current at the end of the pulse by a similar amount (34.0±3.3%) as that obtained at 4 or at 140 mM [K+]o. At 0 mM [K+]o the amplitude of the deactivating tail currents increased (Fig. 5A) and, more interesting, the subsequent decay was markedly slower than at 4 mM [K+]o. Fig. 5B shows the normalized tail currents to compare the kinetic differences and Table 1 summarizes the {tau}f and {tau}s values of the decline of tail currents elicited on return to –40 mV after application of 5, 250 and 500 ms pulses to +60 mV. At 0 mM [K+]o the time course of the decline of tail currents was slower than at 4 mM [K+]o and thus, both the {tau}f and {tau}s values were significantly prolonged. Addition of benzocaine accelerated the initial component, thus, the corresponding {tau}f values were similar to those obtained in the presence of benzocaine at 4 mM [K+]o.


Figure 5
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  		Fig. 5 Effects of benzocaine in the absence of external K+. (A) Superimposed current traces elicited by 500 ms pulses to +60 mV from –80 mV followed by repolarization to –40 mV. Currents were obtained at 4 mM, at 0 mM [K+]o or after superfusion with 0 mM [K+]o plus 500 µM benzocaine. (B) Tail currents recorded on return to –40 mV after 500 ms depolarizing pulses to +60 mV obtained at 4 mM [K+]o, at 0 mM [K+]o alone or in combination with 500 µM benzocaine. Tail amplitudes were normalized in order to better show the kinetic modifications. (C) Activation curves of hKv1.5 channels obtained at 4 mM [K+]o in this group of experiments (dashed line), at 0 mM [K+]o (bullet) or after superfusion with a solution containing 0 mM [K+]o plus 500 µM benzocaine ({circ}). The activation curve at 4 mM [K+]o was fitted with a single Boltzmann component (see Fig. 2), while the activation curves at 0 mM [K+]o both in the absence or presence of benzocaine were better defined by a Boltzmann equation with two components (continuous line). Each data point represents the mean and vertical lines indicate the SEM of five experiments. (D) Activation curves of hKv1.5 channels obtained at 0 mM [K+]o in the absence (bullet) or in the presence of 10 nM benzocaine ({circ}). Each data point represents the mean and vertical lines the SEM of seven experiments.

 

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  		Table 1 Time-course of deactivating tail currents in the presence of benzocaine in cells at [K+]o 0 mMa

 
In five cells, the Vh and k values at 4 mM [K+]o were –20.7±0.8 mV and 4.5±0.1 mV, respectively (Fig. 5C). However, the voltage dependence of the activation process at 0 mM [K+]o in the absence and in the presence of 500 µM benzocaine was better defined by a fit with a sum of two Boltzmann components (solid lines), even when the amplitude of the shallow component represented only 9.2±0.6% of the total activation process. Benzocaine did not modify the shallow component (Vh=142.7±3.1 mV; k=40.3±2.3 mV) and produced a small hyperpolarizing shift in the Vh of the steeper component (k=4.7±0.2 mV) from –27.9±0.5 mV to –30.9±0.5 mV (n=5, P<0.001).

At 0 mM [K+]o, 10 nM benzocaine decreased the current amplitude at the end of the 500 ms depolarizations to +60 mV by 17.3±2.6% (n=7), and it slowed the decline of tail currents increasing both the {tau}f (from 43.7±4.8 ms to 58.0±9.4 ms, n=6, P<0.05) and the {tau}s (from 139.2±9.9 ms to 247.9±44.2, P<0.05) values. Fig. 5D shows the activation curve obtained at 0 mM [K+]o in the absence and in the presence of 10 nM benzocaine. It can be observed that benzocaine shifted into the negative direction the Vh of the steeper component (k=3.4±0.7 mV) of the activation curve (–22.9±0.2 mV vs. –27.9±1.0 mV, n=6, P<0.001), whereas it did not modify the Vh of the shallow component (Vh=142.6±16.0 and k=47.4±5.2 mV). Moreover, it is evident that the increase in tail current amplitude observed at 4 mM [K+]o at potentials positive to –10 mV was not observed at 0 mM [K+]o.

3.7 Effects of benzocaine at low intracellular [K+]
To test whether the reduction in the intracellular [K+] ([K+]i) modifies the gating properties of the channel as well as the effects of benzocaine, the [K+]i was decreased to 50%. Fig. 6 shows currents in response to 500 ms pulses to +60 mV in the absence and in the presence of benzocaine, 10 nM and 500 µM. Benzocaine reduced both the peak and steady-state currents and increased the amplitude of the inactivating component. Thus, currents inactivated by 21.7±2.5% in the absence and by 33.4±1.6% and 30.2±2.0% (n=5, P<0.05 vs. control), in the presence of benzocaine 10 nM and 500 µM, respectively. Benzocaine at 10 nM and at 500 µM blocked currents elicited by 500 ms pulses to +60 mV by 20.5±1.6% and 24.3±2.3%, respectively (n=5). As can be observed in Fig. 6, block induced by 10 nM benzocaine was accompanied by a slowing of the tail current deactivation ({tau}f=44.6±4.4 ms vs. 21.7±2.9 ms, n=5, P<0.05), but this effect was not observed in the presence of 500 µM benzocaine ({tau}f=28.6±6.7 ms).


Figure 6
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  		Fig. 6 Effects of benzocaine at low intracellular [K+]. Superimposed current traces obtained in the absence and in the presence of benzocaine, 10 nM and 500 µM when applying 500 ms pulses to +60 mV. The dotted line represents the zero current level.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
In the present study, we have analyzed the interaction between the neutral LA benzocaine and human cardiac Kv1.5 channels. The major findings are as follows: (1) at concentrations in the nM range, benzocaine increases hKv1.5 current; (2) at high micromolar concentrations, benzocaine reduces the Kv1.5 currents, changes the voltage-dependence of the activation of channels and induces an apparent acceleration of the time-dependence of channel deactivation; (3) increase in the [K+]o produces similar time- and voltage-dependent effects as does benzocaine. Furthermore, removal of the extracellular K+ also modified the channel gating.

4.1 Effects of extracellular [K+]o on hKv1.5 channels
In Shaker K+ channels there are two different mechanisms of inactivation referred to as N- and C-type inactivation [12]. C-type inactivation is associated with a conformational change involving amino acids located in the outer channel mouth and in the S6 transmembrane region [12,13]. hKv1.5 Channels exhibited a slow and partial inactivation at positive voltages which has been proposed to occur via a C-type mechanism [10]. One of the common features of Shaker K+ channels is that elevation of [K+]o slowed the C-type inactivation [14,15]. This observation has been explained by a "foot-in-the-door" model of gating, in which external cations, or TEA, compete with C-type inactivation by occupancy of a site in the pore that must be emptied before inactivation can proceed [13,14,16]. Our experiments demonstrated that increasing [K+]o to 140 mM slightly increased the amplitude of the inactivating component of hKv1.5 currents without modification of the time constant of this process. This probably reflects that the number of channels from which the inactivation proceeds (O2) depends on the [K+]o. Thus, considering the proposed gating model for hKv1.5 channels (C{rightleftarrows}C{rightleftarrows}···{rightleftarrows}C{rightleftarrows}O1{rightleftarrows}O2{rightleftarrows}I1{rightleftarrows}I2) [17], the effects of high [K+]o can be the consequence of an acceleration and/or of a shift of the voltage-dependence of the transition O1->O2 to more negative potentials. From another point of view, this would indicate that elevation of [K+]o stabilized an open state occupied by K+ (OK), from which the channel can more readily inactivate or close. In fact, the increase in the inactivation observed at high [K+]o was accompanied by a mild acceleration in the tail current decline. The existence of this open state bounded to K+ has been proposed to explain the gating modifications observed in hKv1.5 channels in the absence of permeating ions [18]. Our results suggest that the slow inactivation observed on hKv1.5 channels does not resemble the C-type inactivation described in Shaker channels. Moreover, hKv1.5 channels do not present N-type inactivation because they do not have a long NH2-terminal domain [10]. Thus, it is possible that they inactivate by a "nonN- nonC-type inactivation" molecular mechanism which needs further analysis.

Furthermore, at 140 mM [K+]o the voltage-dependence of hKv1.5 channels activation was modified, displaying two components. It has been previously described that hKv1.5 channels [18] have a complex activation process, where current voltage relations have a deflection in them and where the relationship between chord conductance and membrane potential appeared best fit by the sum of two Boltzmann components.

4.2 Effects of benzocaine on hKv1.5 channels
At micromolar concentrations, the blocking effects of benzocaine resemble those induced by high [K+]o (140 mM). The drug increased the amplitude of the inactivating component, accelerated the tail currents decline, modified the activation curve of hKv1.5 channels which became biphasic and shifted the voltage dependence of hKv1.5 activation toward more negative potentials. Furthermore, at 140 mM [K+]o, benzocaine inhibited the flow of K+ through the pore but did not modify the deactivation kinetics of hKv1.5 currents. Therefore, it seems that at high [K+]o the time-dependent effects reached saturation and benzocaine cannot induce any further modification. Moreover, in the absence of external K+ the deactivation kinetics of the channel was slower than at 4 mM [K+]o, and under these conditions, benzocaine accelerated the initial phase of current decline in such a way that the time constant of this phase resembled that obtained at 4 mM [K+]o. Thus, it is possible that benzocaine and external K+ act by a similar mechanism stabilizing an open state from which the closing and/or the inactivation can proceed.

Benzocaine-induced block increased in the voltage range of channel opening, suggesting that the channels must enter into an open state to allow drug binding. Thereafter, at potentials positive to 0 mV, the benzocaine-induced block decreased with a shallow voltage dependence. Identical results were previously described with nifedipine and loratadine which also predominate in its uncharged form at the physiological pH [19,20]. This effects of benzocaine can be explained by its selective binding to the first open state (O1) with very fast kinetics of block and unblock accompanied by a modification in the kinetics and voltage-dependence of the transition between O1{rightleftarrows}O2. The state-dependent binding to O1 accounted for the voltage-dependent unblock, which can be considered as an index of the voltage-dependence of the transition O1->O2. This transition has indeed a weak voltage dependence in the proposed gating model [17]. Another possibility is that benzocaine binds to an external site and blocks the channel. Thus, at more positive voltages, K+ permeation increases and hinders the binding of benzocaine to its external site with a resultant relief of block. Such a mechanism has been proposed to explain the nifedipine-induced block of hKv1.5 channels [19]. To examine this possibility the [K+]i was reduced by 50%. The results demonstrated that the blockade and the slope of voltage-dependent unblock were slightly reduced, but not in a consistent manner. The last hypothesis that can account for the voltage-dependent unblock is that neutral drugs may exhibit a binding site coupled to some voltage-dependent process such as conformational changes in the pore with potential or movement of the voltage sensor which can sense the transmembrane voltage change [19]. Recent evidences indicated that the open probability (Po) for the hKv1.5 channel does not saturate at potentials positive to 0 mV, but undergoes further changes at more positive potentials [18]. Thus, it is possible that such changes could alter the conformation of open channels and confer voltage-dependence to block by an uncharged drug.

Benzocaine accelerated the fast initial component of deactivation of tail currents but only after long and strong depolarizations (i.e. >100 ms, +60 mV). Following the proposed scheme for hKv1.5 channels, on return to negative potentials the states sequence will be: O2->O1->->->Cn. Therefore, the acceleration of the initial component of deactivation might be the consequence of the selective binding of benzocaine to a transitional state which appeared early on repolarization (O1). This hypothesis explains why benzocaine did not accelerate the initial component of the tail current when the preceding depolarizing pulse was very short or was applied to a potential below the midpoint of activation of hKv1.5 channels. In these two situations during the test pulse, a larger fraction of channels will be in state O1 allowing the binding of benzocaine (Cn->O1->O1BZ). If binding of benzocaine had reached steady-state during depolarization, on return to negative potentials an increase of block would not be expected. Moreover, when short pulses or pulses to –10 mV were applied, a slowing of the second component of tail current decline was observed which can be interpreted by assuming that benzocaine must unbind from the channel before it can close, similarly to the "foot-in-the-door mechanism".

At nanomolar concentrations, benzocaine modified the gating properties of hKv1.5 channels without obvious block. The increase in current amplitude ("agonist effect") observed at negative membrane potentials was caused by a parallel shift of the voltage-dependence of hKv1.5 activation in the negative direction. Almokalant, a selective K+ channel blocker, also exerted a dual effect on the delayed rectifier K+ channel in rabbit ventricular myocytes [21]. Similar results were observed with a quaternary analog of quinidine (Q+1C) on Kv1.2 channels but only at 1000-fold higher concentrations than those reported in this paper with benzocaine [22]. Moreover, like Q+1C, benzocaine also induced a marked slowing in the rate of deactivation. Based on the proposed model for hKv1.5 channels, the "agonist effect" of benzocaine could be the result of a decrease in the forward and/or an increase in the backward rate constants of the transition O1{rightleftarrows}O2. The slowing of the tail current decay adds further support to the hypothesis that benzocaine retarded the transition from the last open state.

4.3 Does benzocaine bind to the same receptor as tertiary amine LAs?
The differences in the blocking effects of benzocaine as compared to those described for tertiary amine LAs, as well as the "agonist" effect which appeared at concentrations far from those needed to block the channel, may raise the question as to whether benzocaine binds to the proposed receptor for antiarrhythmics and tertiary amine LAs in hKv1.5 channels [8,9]. It has been demonstrated that K+ modified gating of Shaker channels binding to an external and to several internal binding sites [13,14,16,18,23–25]. Our results confirmed these observations, since both modifications of the extracellular or intracellular [K+] clearly modified the gating properties of hKv1.5 channels. At micromolar concentrations, benzocaine also modified the hKv1.5 channel gating and its effects were reproduced by raising the [K+]o to 140 mM. This result suggests that benzocaine binds to the same or to an overlapping receptor site as K+ at the channel level. Furthermore, the "agonist effect" induced by nanomolar concentrations was abolished either when the permeation of K+ through the pore was reduced (i.e., at low [K+]i), or when the [K+]o was increased to 140 mM or reduced to 0 mM.

Taking all these results together, we proposed that benzocaine and K+ bind to an extracellular and to an intracellular binding site, and that both sites are related to each other. It is possible that binding of benzocaine/K+ to the internal site modifies allosterically the affinity and the binding to the external one ("allosteric mechanism"). Another possibility is that binding of benzocaine to the internal site reduces the permeation of K+ through the pore increasing its binding to the external site or decreasing the binding of K+ to it ("permeation mechanisms"). These two mechanisms, which are not necessarily mutually exclusive and may potentially coexist, have been proposed to explain the interactions of [K+] on C-type inactivation in Shaker channels [15,23]. However, the existence of various sites of drug binding on hKv1.5 channels, their possible location, and the apparent complex relation between them needs further analysis.

Time for primary review 22 days.


   Acknowledgements
 
This study was supported by SAF-96-0042, SAF-98-0058 and CAM (08.4/0016/1998) Grants. The authors thank Dr. M.M. Tamkun for providing us with the cell line transfected with the gene encoding hKv1.5 channels and Dr. J. López-Barneo for stimulating discussions and helpful suggestions. We thank Guadalupe Pablo and Rubén Vara for their technical assistance.


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

  1. Hille B. Local anesthetics: hydrophilic and hydrophobic pathways for the drug receptor reaction. J Gen Physiol (1977) 69:497–515.[Abstract/Free Full Text]
  2. Valenzuela C, Delpón E, Tamkun M.M, Tamargo J, Snyders D.J. Stereoselective block of a human cardiac potassium channel (Kv1.5) by bupivacaine enantiomers. Biophys J (1995) 69:418–427.[Web of Science][Medline]
  3. Valenzuela C, Delpón E, Franqueza L, et al. Effects of ropivacaine on a potassium channel (hKv1.5) cloned from human ventricle. Anesthesiology (1997) 86:718–728.[Web of Science][Medline]
  4. Longobardo M, Delpón E, Caballero R, Tamargo J, Valenzuela C. Structural determinants of potency and stereoselective block of hKv1.5 channels induced by local anesthetics. Mol Pharmacol (1998) 54:162–169.[Abstract/Free Full Text]
  5. 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]
  6. Armstrong C.M. Interaction of tetraethylammonium ion derivatives with the potassium channels of giant axons. J Gen Physiol (1971) 58:413–437.[Abstract/Free Full Text]
  7. Snyders D.J, Yeola S.W. Determinants of antiarrhythmic drug action: electrostatic and hydrophobic components of block of the human cardiac hKv1.5 channel. Circ Res (1995) 77:575–583.[Abstract/Free Full Text]
  8. Yeola S.W, Rich T.C, Uebele V.N, Tamkun M.M, Snyders D.J. Molecular analysis of a binding site for quinidine in a human cardiac delayed rectifier K+ channel. Role of S6 in antiarrhythmic drug binding. Circ Res (1996) 78:1105–1114.[Abstract/Free Full Text]
  9. Franqueza L, Longobardo M, Vicente J, et al. Molecular determinants of stereoselective bupivacaine block of hKv1.5 channels. Circ Res (1997) 81:1053–1064.[Abstract/Free Full Text]
  10. Snyders D, Tamkun M.M, Bennett P.B. A rapidly activating and slowly inactivating potassium channel cloned from human heart. J Gen Physiol (1993) 101:513–543.[Abstract/Free Full Text]
  11. Wang G.K, Wang S.-Y. Binding of benzocaine in batrachotoxin-modified Na+ channels: state-dependent interactions. J Gen Physiol (1994) 103:501–518.[Abstract/Free Full Text]
  12. Hoshi T, Zagotta W.N, Aldrich R.W. Biophysical and molecular mechanisms of Shaker potassium channel inactivation. Science (1990) 250:533–538.[Abstract/Free Full Text]
  13. Choi K.L, Aldrich R.W, Yellen G. Tetraethylammonium blockade distinguishes two inactivation mechanisms in voltage-activated K+ channels. Proc Natl Acad Sci USA (1991) 88:5092–5095.[Abstract/Free Full Text]
  14. López-Barneo J, Hoshi T, Heinemann S.H, Aldrich R.W. Effects of external cations and mutations in the pore region on C-type inactivation of Shaker potassium channels. Recept Channels (1993) 1:61–71.[Web of Science][Medline]
  15. Rasmusson R.L, Morales M.J, Wang S, et al. Inactivation of voltage-gated cardiac K+ channels. Circ Res (1998) 82:739–750.[Abstract/Free Full Text]
  16. Baukrowitz T, Yellen G. Two functionally distinct subsites for the binding of internal blockers to the pore of voltage-activated K+ channels. Proc Natl Acad Sci USA (1996) 93:13357–13361.[Abstract/Free Full Text]
  17. Rich T.C, Snyders D.J. Evidence for multiple open and inactivated states of the hKv1.5 delayed rectifier. Biophys J (1998) 75:183–195.[Web of Science][Medline]
  18. Chen F.S.P, Steel D, Fedida D. Allosteric effects of permeating cations of gating currents during K+ channel deactivation. J Gen Physiol (1997) 110:87–100.[Abstract/Free Full Text]
  19. 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]
  20. Delpón E, Valenzuela C, Gay P, et al. Block of human cardiac Kv1.5 channels by loratadine: voltage-, time- and use-dependent block at concentrations above therapeutic levels. J Cardiovasc Pharmacol (1997) 35:341–350.[CrossRef]
  21. Carmeliet E. Use-dependent block and use-dependent unblock of the delayed rectifier K+ current by almokalant in rabbit ventricular myocytes. Circ Res (1993) 73:857–868.[Abstract/Free Full Text]
  22. Tseng G.-N, Zhu B, Ling S, Yao J.-A. Quinidine enhances and suppresses Kv1.2 from outside and inside the cell, respectively. J Pharmacol Exp Ther (1996) 279:844–855.[Abstract/Free Full Text]
  23. Baukrowitz T, Yellen G. Modulation of K+ current by frequency and external [K+]: a tale of two inactivation mechanisms. Neuron (1995) 15:951–960.[CrossRef][Web of Science][Medline]
  24. Kiss L, Korn S.J. Modulation of C-type inactivation by K+ at the potassium channel selectivity filter. Biophys J (1998) 74:1840–1849.[Web of Science][Medline]
  25. Harris R.E, Larsson H.P, Isacoff E.Y. A permeant ion binding site located between two gates of the Shaker K+ channel. Biophys J (1998) 74:1808–1820.[Web of Science][Medline]

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