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Cardiovascular Research 2002 56(1):104-117; doi:10.1016/S0008-6363(02)00509-6
© 2002 by European Society of Cardiology
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Copyright © 2002, European Society of Cardiology

Putative binding sites for benzocaine on a human cardiac cloned channel (Kv1.5)

Ricardo Caballero*, Ignacio Moreno, Teresa González, Carmen Valenzuela, Juan Tamargo and Eva Delpón

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

* Corresponding author. Tel.: +34-91-394-1474; fax: +34-91-394-1470 rcaballero{at}ift.csic.es

Received 31 December 2001; accepted 28 May 2002


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objectives: It has been demonstrated that at nanomolar concentrations benzocaine increased, whereas at micromolar concentrations, it blocked hKv1.5 channels in a voltage-dependent manner and modified the voltage-dependence of channel activation. The present study was undertaken to localize the putative binding sites involved in the ‘agonists’ and blocking effects of benzocaine. Methods: Experiments were carried out on wild-type and site directed mutated hKv1.5 channels stably expressed on Ltk– cells using the whole-cell patch-clamp. Results: At 35 mM [K+]i the voltage-dependent unblock produced by 500 µM benzocaine was preserved at both 4 and 140 mM [K+]o. Mutations located in the inner mouth of the pore (T477S, T505A, L508M and V512M) abolished the agonist but increased the blocking effects of benzocaine. Intracellular application of tetraethylammonium (3 mM) abolished the ‘agonist’ effects whereas the blocking effects of benzocaine remained unaltered. Block induced by benzocaine and intracellular tetraethylammonium was additive. In contrast, the combination of benzocaine and bupivacaine (>25 µM) produced less blockade than bupivacaine alone. However, mutation of the extracellular residue R485Y did not modify the effects of benzocaine. Extracellular application of tetraethylammonium (100 mM) did not modify the agonist effects of benzocaine, but abolished the voltage- and time-dependence of benzocaine-induced block. Conclusions: The results suggested that benzocaine binds with high affinity to an intracellular binding site to produce ‘agonist’ effects and to a low affinity subsite, which is also located in the inner mouth, to produce the blocking effects. Furthermore, benzocaine and extracellular K+ interact to modify the voltage-dependence of channel opening.

KEYWORDS K-channel


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
hKv1.5 channels generate the ultrarapid delayed rectifier current (IKur) in human atrial myocytes, which is involved in the control of action potential duration [1]. It has been proposed that these channels would be the target for drugs that selectively prolong the action potential duration in the atrial but not in the ventricular tissue. Thus, a great effort has been made to characterize the binding site of hKv1.5 blocking agents [2–4]. The blocking effects produced by antiarrhythmic drugs (like quinidine [5]) and local anesthetics (LA) (like bupivacaine [6]) share common features with those exhibited by internal tetraethylammonium (TEA) and its derivatives, collectively called quaternary ammonium compounds (QA). The cationic form of these drugs blocks the current only after channel opening by binding at a site in the internal mouth of the 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 [2]. These results were confirmed when their effects were studied in site-directed mutant channels, indicating that the drug–channel interaction occurs at the internal mouth of the pore, particularly at the pore-lining residues of the S6 segment of the hKv1.5 protein [3,4].

Surprisingly, hKv1.5 block produced by drugs that predominate in their uncharged form at physiological pH, such as loratadine [7], nifedipine [8], benzocaine [9] and rupatadine [10] was voltage-dependent. Moreover, voltage-dependence of the open state block induced by these neutral drugs, which decreased with depolarization, was a mirror image of the voltage-dependent increase in block induced by cationic drugs. Blocking effects induced by micromolar concentrations of benzocaine were accompanied by an acceleration of tail current decline and by a modification of the voltage-dependence of channel activation, which became biphasic. Moreover, the blocking effects of benzocaine resemble those induced by the elevation of extracellular K+ concentration ([K+]o) to 140 mM [9]. This latter result led to the suggestion that the blocking effects of benzocaine could result from its interaction with the proposed extracellular binding site for K+ at the channel level [9].

On the other hand, at nanomolar concentrations, benzocaine increased the peak amplitude of hKv1.5 currents elicited by depolarization, and the peak tail currents upon repolarization, and these ‘agonist’ effects were accompanied by a slowing of the tail current decline. Similar ‘agonist’ effects were produced, at 1000-fold higher concentrations, by a membrane-impermeant quaternary analog of quinidine on Kv1.2 channels, and were explained on the basis of the interaction at an extracellular binding site [11]. Furthermore, recent results suggest the existence of an extracellular and an intracellular binding site for drugs at the hKv1.5 channel level [8,10,12]. Therefore, the present study was undertaken to identify the putative binding site/s of benzocaine on hKv1.5 channels involved in both its blocking and the ‘agonist effects’. For this purpose we analyzed the effects of benzocaine: (a) when the K+ flow through the pore was reduced by decreasing the intracellular [K+] both at 4 and at 140 mM extracellular [K+], (b) on several site-directed mutant channels and (c) in the presence of drugs that bind at the intracellular (i.e. bupivacaine or internally applied TEA) and at the extracellular mouth of the pore (externally applied TEA).


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Cell culture
Wild-type (WT) and site-directed mutated hKv1.5 channels were stably expressed in mouse Ltk– cells using procedures previously described elsewhere [3,4,10,13]. Briefly, point mutations were generated by a method involving the polymerase chain reaction (PCR), and all PCR-generated sequences were verified directly by DNA sequence analysis. Once the desired sequence was confirmed, the complete coding sequence was ligated into the pMSVneo expression vector, used for stable transfection into Ltk– cells as described before [3,4,13]. Cells were cultured in DMEM supplemented with 10% horse serum and 0.25 mg/ml G418 in a 5% CO2 atmosphere.

2.2 Recording techniques
Cells were superfused with an external solution containing (in mM): NaCl 130, KCl 4, CaCl2 1, MgCl2 1, Hepes 10 and glucose 10; (pH 7.4 with NaOH). To obtain 140 mM extracellular K+ concentration ([K+]o) equimolar substitution of KCl for NaCl was used. Recording pipettes were filled with an ‘internal’ solution containing (in 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 intracellular K+ concentration ([K+]i) was lowered to 25% by the equimolar substitution of K-aspartate by TrisCl. When TEA was added to the external or internal solution, equimolar substitution with NaCl or KCl, respectively, was used. hKv1.5 currents were measured at 24–25 °C using the whole-cell configuration of the patch-clamp technique with 200B Axopatch and pClamp 6.1 software (Axon Instruments). Capacitance and series resistance compensation were optimized and 80% compensation was usually obtained. Maximum outward current amplitudes at +60 mV averaged 1.5±0.1 nA (n=22) mean uncompensated access resistance was 3.2±0.5 M{Omega} and cell capacitance 10.2±0.9 pF. Thus, no significant voltage errors (<5 mV) due to series resistance were expected with the electrodes used (tip resistance <3.5 M{Omega}). The currents were filtered at 1 kHz (four-pole Bessel filter) and sampled at 2 kHz.

Cells were held at –80 mV. The activation curves were constructed by plotting tail current amplitude recorded at –40 mV as a function of the membrane potential and were 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). Under some circumstances, a Boltzmann distribution with two terms was needed to fit the experimental data, the equation being:

Formula (2)
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 and Boltzmann fitting. The results were displayed together with the difference plot. Goodness of the fit was judged by the {chi}2 criterion and by inspection for systematic nonrandom trends in the difference plot.

To determine the voltage-dependence of hKv1.5 currents, the leak-corrected current in the presence of drug was normalized to matching control to yield the fractional block at each voltage. The voltage-dependence of block was fitted to

Formula (3)
where z, F, R and T have their usual meaning, {delta} represents the fractional electrical distance, i.e. the fraction of the transmembrane electrical field sensed by a single charge at the receptor site and KD* represents the apparent dissociation constant at the reference potential (0 mV).

The apparent affinity constant, KD, and Hill coefficient, nH, were obtained from fitting the fractional block, f, at various drug concentrations [D] to the equation:

Formula (4)

2.3 Drugs
Benzocaine and bupivacaine (Sigma) were initially dissolved in dimethyl sulfoxide (DMSO, Sigma) and distilled deionized water, respectively, to yield 0.1 mol stock solutions. Further dilutions were carried out in external solution to obtain the desired final concentrations. Control solutions contained the same DMSO concentrations as the test solution; DMSO did not affect the current at concentrations up to 0.1%.

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 followed by Newman–Keuls test. Results are expressed as mean±S.E.M. A P-value of less than 0.05 was considered significant. More details on each procedure are given in the Results section.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Effects of benzocaine at low [K+]i
The decrease in benzocaine-induced block at membrane potentials at which activation reaches saturation can be explained considering that the drug binds to an extracellular binding site on hKv1.5 channels, in such a way that K+ efflux at positive potentials hinders the binding of benzocaine with the resultant decrease in block. Moreover, an increase in the [K+]o produces similar time- and voltage-dependent effects as does benzocaine, suggesting a common and extracellular binding site for benzocaine and K+ [9]. To explore this possibility we analyzed the effects of benzocaine when the [K+]i was decreased to 25% (35 mM) by the equimolar substitution of the K-aspartate by TrisCl, a procedure that reduced the K+ flow through the channel.

Fig. 1A shows currents elicited by 500-ms pulses to potentials between –80 and +60 mV in the absence and in the presence of benzocaine 500 µM. Under these conditions benzocaine reduced the peak and steady-state currents and increased the amplitude of the inactivating component from 21.7±2.5 to 30.2±2.0% (n=7, P<0.05), so that benzocaine-induced block at the end of pulses to +60 mV averaged 30.9±2.3% (n=7). Cells were then washed with a drug-free solution containing 140 mM [K+]o that decreased the current elicited by pulses to +60 mV by 39.7±5.6% (P<0.05 vs. data at 4 mM [K+]o). In contrast, the tail current amplitude (which became inward) was 16.4±1.2-fold times larger than that elicited at 4 mM [K+]o. When benzocaine was added to this 140 mM [K+]o solution it decreased the current by 38.9±4.1% (P<0.05 vs. data at 140 mM [K+]o). Fig. 1B shows the average values for the I/V curves (500 ms isochronal) obtained under each experimental condition in seven cells. At 4 mM [K+]o, benzocaine decreased currents elicited by pulses more depolarized than –20 mV. At 140 mM [K+]o, hKv1.5 channels display inward currents at potentials between –40 and 20 mV and benzocaine inhibited both the inward and the outward flow of K+ through the channel. To quantify the voltage-dependence of block in Fig. 1C, the relative current (IBZ/ICON) was plotted as a function of the membrane potential. At 4 mM [K+]o, benzocaine-induced block increased in the voltage range for channel opening. Maximum blockade (44.9±2.5%, n=7, P<0.01) was achieved at 5 mV decreasing at more positive potentials. At 140 mM [K+]o, benzocaine-induced block also increased as the channels open reaching a similar amount of block to that obtained at 4 mM [K+]o (47.6±5.7%, at 5 mV, n=6, P>0.05). At 25 mV, when K+ began to flow outward, blockade reached its maximum (65.9±10.6%, P<0.05) decreasing at more positive potentials.


Figure 1
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  		Fig. 1 Effects of benzocaine at low [K+]i (35 mM). (A) Current tracings for 500-ms pulses from –80 to +60 mV at 4 and 140 mM [K+]o in the absence and presence of 500 µM benzocaine (BZ). (B) Current–voltage relationships (500-ms isochronal) in the absence and presence of benzocaine, at 4 and 140 mM [K+]o. (C) Fractional block (f=IBZ/ICON) from data shown in Panel B. (D) Activation curves at 4 mM [K+]o in the absence and presence of benzocaine. Continuous lines illustrate the Boltzmann fit. (E) The dashed line represents the mean activation curve obtained in this group of experiments at 4 mM [K+]o. Tail amplitude was normalized and fitted with a single Boltzmann component. Continuous lines represent the activation curves obtained at 140 mM [K+]o in the absence and presence of benzocaine (fitted with Boltzmann equation with two terms). Each point represents the mean±S.E.M. of seven experiments.

 
Fig. 1D shows the voltage-dependence of hKv1.5 channels at 4 mM [K+]o. Activation curves were obtained as described in Methods. In control conditions, data were fitted with a single Boltzmann equation (continuous line) the values for Vh and k averaging –19.5±0.8 and 4.1±0.3 mV (n=7), respectively (Table 1). In the presence of 500 µM benzocaine, a single Boltzmann component was adequate to fully describe the voltage-dependence of channel opening but the Vh was shifted in the negative direction, without modifying the slope factor. These results indicated that, when the [K+]i was lowered, the activation curve of hKv1.5 channels in the presence of benzocaine did not exhibit the second shallow component observed in the presence of benzocaine at normal [K+]i [9].


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  		Table 1 Voltage- and time-dependent effects of benzocaine on hKv1.5 channels when the [K+]i was 35 mM both at 4 and 140 mM [K+]o

 
Finally, in order to compare the effects of benzocaine on hKv1.5 channels at normal and low [K+]i, its effects on tail current deactivation were analyzed. According to the model proposed for hKv1.5 channels [14] (C{rightleftarrows}C{rightleftarrows}...{rightleftarrows}C{rightleftarrows}O1{rightleftarrows}O2{rightleftarrows}I1{rightleftarrows}I2) control tail currents were fitted by a biexponential function, and {tau}f and {tau}s values were calculated (Table 1). At 4 mM [K+]o, benzocaine (500 µM) significantly accelerated the initial phase of deactivation ({tau}f=27.2±2.6 ms, n=7, P<0.01), but did not modify the slow phase of tail current decline. Similar results were obtained at normal [K+]i [9]. At 140 mM [K+]o, tail current decline was faster than at 4 mM [K+]o (Table 1), similar to what has been previously described at normal [K+]i [9]. Under these conditions, benzocaine accelerated the initial phase of the deactivation process so that {tau}f decreased to 16.4±0.9 ms (n=6, P<0.01) whereas the slow phase was not modified.

Since it has been previously described that voltage- and time-dependent effects of benzocaine were reproduced by raising the [K+]o to 140 mM, the effects of increasing the [K+]o to 140 mM when the intracellular [K+] was reduced to 25% were also studied. The increase in the [K+]o reduced the outward current at the end of 500-ms pulses to +60 mV by 41.2±2.2% (n=7), and the addition of benzocaine decreased the current by 39.8±3.4% (n=7, P<0.05 vs. current amplitude at 140 mM [K+]o). Fig. 1E shows the activation curves obtained at 4 and at 140 mM [K+]o and after addition of 500 µM benzocaine. The data obtained at 4 mM [K+]o were described with a single Boltzmann equation, average values for Vh and k being –19.9±1.0 and 4.7±0.6 mV (n=7), respectively. The activation curve of hKv1.5 channels at 140 mM [K+]o exhibited two components (Table 1). The solid line illustrates a fit with a sum of two Boltzmann components (Eq. (2) in Methods). At 140 mM [K+]o in the presence of 500 µM benzocaine, the activation curve also exhibited two components, and benzocaine did not modify the Vh or the k values of the steep and the shallow components (Table 1). These results indicated that the second shallow component of the activation curve which appeared when [K+]o was raised to 140 mM at normal [K+]i [9] did not disappear when the [K+]i was lowered.

3.2 Effects of benzocaine on mutant channels
To explore the possible binding site of benzocaine on hKv1.5 channels, the effects of 10 nM and 500 µM benzocaine were analyzed on site-directed mutated channels stably expressed in Ltk– cells. Table 2 summarizes the voltage- and time-dependent characteristics of the mutant channels in the absence and presence of 500 µM benzocaine. The diagram in Fig. 2A indicates the location of the amino acids mutated in our study using a model that illustrates the transmembrane topology of the {alpha}-subunit of hKv1.5 channels. Fig. 2B shows the effects of benzocaine, 10 nM and 500 µM on WT channels. It has been previously described that residue at position 449 on Shaker channels determines important functional properties, such as C-type inactivation rate [15] and external TEA sensitivity [16]. hKv1.5 channels at the equivalent position (485) present arginine, and thus, firstly we studied whether the mutation R485Y affected benzocaine-induced block. Fig. 2C shows current traces recorded in the absence and in the presence of 10 nM and 500 µM benzocaine. R485Y channels inactivated to a lesser extent than WT channels. Indeed, currents elicited by 500-ms and 5-s pulses to +60 mV inactivated by 14.5±0.4 and 31.4±4.3%, respectively, compared to 29.3±1.5 and 52.1±1.3% (n=8, P<0.05) in WT channels (at [K+]o=4 mM; [K+]i=142 mM). The R485Y mutation did not modify either the voltage-dependence of channel activation or the kinetics of tail currents upon repolarization to –40 mV after pulses to +60 mV (Table 2). At 10 nM, benzocaine increased the current amplitude, an effect which was more pronounced at the beginning of the depolarization but decreased progressively during the pulse, so that, at the end of the pulses the current increased by 4.5±1.7% (Table 3). This effect was accompanied by a slowing of the tail current deactivation ({tau}f=60.6±4.3 ms, n=5, P<0.05). At 500 µM, benzocaine reduced the outward current by 28.9±1.2%, and accelerated the tail current decline ({tau}f=17.8±0.9 ms, n=4, P<0.05). Fig. 3A shows activation curves of R485Y channels in the absence and presence of 500 µM benzocaine. As it can be appreciated, the activation curve in the presence of drug exhibited two components (Table 2). Benzocaine-induced block of R485Y channels increased in the voltage-range of channel opening reaching a maximum at 10 mV (37.2±1.4%, n=5, P<0.05) decreasing with a shallow slope at more depolarized potentials. All these results indicated that the characteristics of the benzocaine-induced block of R485Y channels were similar to those previously described for the blockade on WT channels [9].


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  		Table 2 Effects of benzocaine (BZ) on the characteristics of the currents elicited by WT, R485Y, T477S, L508M and V512M channels

 

Figure 2
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  		Fig. 2 Effects of benzocaine on mutant channels. (A) Proposed structure of hKv1.5 channels showing the mutated residues. (B), (C) and (D) Effects of benzocaine, 10 nM and 500 µM, on WT, R485Y and T477S channels, respectively, when applying 500-ms pulses from –80 to +60 mV. (E) Effects of benzocaine, 10 nM (left) and 500 µM (right), on V512M channels when applying 500-ms pulses from –100 to +40 mV.

 

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  		Table 3 Effects of benzocaine on site-directed mutant hKv1.5 channels

 

Figure 3
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  		Fig. 3 Effects of benzocaine on the voltage-dependence of mutant channel activation. (A) Activation curves of R485Y channels in the absence and presence of 500 µM benzocaine ([K+]o=4 mM). (B) Activation curves of R485Y channels at 4 (control), 140 mM [K+]o and 140 mM [K+]o plus benzocaine. (C) Activation curves of T477S channels in the absence and presence of benzocaine ([K+]o=4 mM). Control activation curves were fitted with a single Boltzmann component, whereas at 140 mM or in the presence of benzocaine were better defined by a Boltzmann equation with two components. Each point represents the mean±S.E.M. of five experiments.

 
Since the mutation of the equivalent residue (T449Y) in Shaker channels produces a channel that inactivates very slowly with a time course unaffected by [K+]o [15,17], we studied the effects of benzocaine on R485Y at 140 mM [K+]o. The results indicated that R485Y channels behave similarly to WT channels at 140 mM [K+]o. At 140 mM [K+]o, the amplitude of the inactivating component of R485Y currents elicited by 500-ms and 5-s pulses increased to 21.9±3.2 and 42.5±1.6%, respectively (n=6, P<0.05). Under these conditions, 500 µM benzocaine reduced the steady-state current by 35.5±5.5 and 39.4±4.5% when 500-ms and 5-s pulses to +60 mV were applied, respectively. Activation curves of R485Y channels at 140 mM [K+]o (Fig. 3B) also exhibited a steep (k=3.5±0.3 mV) and a shallow (k=13.6±4.4 mV) component, the Vh averaging –22.7±0.7 and 9.3±5.1 mV, respectively (n=5). Benzocaine did not significantly modify the Vh of these components (–25.1±1.5 and 24.3±3.2 mV, n=5, P>0.05). Benzocaine accelerated the initial phase of tail current decline ({tau}f=16.7±0.8 vs. 33.9±3.4 ms, n=5, P<0.05), whereas it did not modify the slow phase of decay ({tau}s=125.8±16.9 ms vs. 138.7±7.7 ms, P>0.05).

To further determine whether benzocaine binds to the proposed internal binding site for cationic LA, we also examined the effects of the drug on T477S (the internal TEA binding site), T505A, L508M and V512M mutant channels. The mutations of hKv1.5 channels were selected because, under the same experimental conditions, they were found to be critical in determining quinidine affinity [3] and the stereoselectivity of bupivacaine-induced block [4]. T505, L508, and V512 are pore-lining residues located in the midsection of the S6 [3,4]. Table 3 summarizes the percentage of block produced by benzocaine, 10 nM and 500 µM, on each mutant channel. The increase in peak current produced by 10 nM benzocaine in WT disappeared in T477S, T505A, L508M and V521M mutant channels, so that at the end of the pulse, the blockade was significantly increased. The blockade produced by 500 µM benzocaine was also significantly increased with respect to that observed in WT channels with the exception of T505A. Importantly, in these mutants, both in the presence of 10 nM and 500 µM benzocaine, the voltage-dependent unblock was preserved.

Fig. 2D illustrates the effects of benzocaine, 10 nM and 500 µM, in T477S channels. T477S mutation did not modify the voltage-dependence of channel activation (Fig. 3C and Table 2). In contrast, the activation curves of T505A and L508M channels were shifted to more negative and to more positive potentials, respectively (Table 2). As can be observed in Fig. 3C, in the presence of 500 µM benzocaine the activation curve of T477S channels became biphasic, and the voltage-dependent unblock was preserved. Unfortunately, the activation curve of both T505A and L508M channels in the presence of 500 µM benzocaine was difficult to analyze because of the small size of the tail currents. T477S mutation slowed the tail current decline, whereas T505A and L508M mutations did not affect the tail current deactivation (Table 2). Benzocaine at 10 nM did not modify the tail current deactivation in T477S channels ({tau}f=50.2±7.8 ms, {tau}s=222.9±9.2 ms, n=5, P>0.05). At this concentration, benzocaine slowed ({tau}f=66.5±6.5 ms, n=5, P<0.05) and accelerated ({tau}f=15.2±6.5 ms, n=4, P<0.05) the tail current decline in T505A and L508M channels, respectively. Benzocaine at 500 µM, did not modify the time course of current deactivation of either T477S or L508M channels, whereas in the T505A mutant, it significantly accelerated this process (Table 2).

V512M mutation dramatically affected the voltage-dependence of channel activation, shifting the Vh in the hyperpolarizing direction (Table 2). Thus, in this group of experiments, the holding potential was –100 mV and 500-ms pulses to +40 mV were applied. Moreover, the tail current deactivation was extremely slow and only one component was resolved ({tau}=1908±443 ms). In V512M channels, the blockade induced by 10 nM benzocaine was similar to that obtained in the presence of 500 µM benzocaine (Fig. 2E and Table 3). Benzocaine 500 µM did not modify the time course of tail deactivation, while at 10 nM, it accelerated the tail current decline of V512M channels (749±192 ms, n=6, P<0.05).

3.3 Effects of bupivacaine and the combination of bupivacaine and benzocaine on hKv1.5 channels
Some effects of benzocaine on channel gating might suggest that the drug does not bind to the internal quaternary ammonium-type receptor. To address this issue, we tested for potential competitive interactions between benzocaine and bupivacaine, a tertiary amine local anesthetic. Fig. 4A shows current traces obtained under control conditions and in the presence of 25 µM bupivacaine when 500-ms pulses to +60 mV were applied. Bupivacaine decreased the peak outward current and induced a subsequent exponential decline of the current during the depolarizing pulse, so that the current amplitude at the end of the pulse decreased by 69.2±4.4% (n=6). The blockade produced by bupivacaine (1–50 µM) at the end of this positive pulse was used as an index of block. A nonlinear least-squares fit of the concentration–response equation (Eq. (4) in Methods) to the individual data points yielded a KD value of 6.6±1.4 µM and a nH value of 0.8±0.1 (inset in Fig. 4F). The time constant of the fast falling phase induced by bupivacaine was considered to represent the time constant of development of bupivacaine-induced block ({tau}Block), whereas the slow time constant reflects the slow and partial inactivation of the channel ({tau}{approx}260 ms) [10]. In the presence of 25 µM bupivacaine, {tau}Block averaged 13.1±1.0 ms and the apparent association (k) and dissociation (L) rate constants calculated for each experiment, averaged (2.8±0.3)x106 M–1 s–1 and 23.4±2.1 s–1, respectively (n=19). Fig. 4B shows the effects of bupivacaine on deactivating tail currents recorded on return to –40 mV. Bupivacaine reduced the initial current amplitude and slowed its time course ({tau}=191.7±11.0 ms using a monoexponential function) and thus, a ‘crossover phenomenon’ was observed. Fig. 4C shows the I–V curve obtained in control conditions and in the presence of 25 µM bupivacaine and panel D the fractional block [IBUPI/ICON)] represented together with the mean activation curve (dotted line) obtained in control conditions for this group of experiments. Bupivacaine-induced block increased in the voltage range for channel opening, whereas at potentials positive to 0 mV, it increased with a more shallow voltage-dependence. Since bupivacaine is a weak base (pKa=8.1) that predominates in its charged form at the intracellular pH, the shallow component could result from the influence of the transmembrane electrical field on the interaction between cationic bupivacaine and the channel receptor. Using a Woodhull formalism (Eq. (3) in Methods) (continuous line in Fig. 4D), the equivalent electrical binding distance {delta} averaged 0.16±0.009 (n=10).


Figure 4
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  		Fig. 4 Effects of bupivacaine and the combination of bupivacaine and benzocaine on hKv1.5 channels. (A) Current tracings obtained in the absence and presence of 25 µM bupivacaine (BUPI) following 500-ms pulses to +60 mV. (B) Tail currents elicited on return to –40 mV after pulses to +60 mV in the absence and presence of bupivacaine. The arrow indicates tail current crossover. (C) Current–voltage relationships (500-ms isochronal) in control conditions and in the presence of bupivacaine. (D) Fractional block (f=IBUPI/ICON) from the data shown in Panel C. The dotted line represents the mean control activation curve for this group of experiments and the continuous line the best fit to data positive to –10 mV to a Woodhull equation that yielded the {delta} value referred to the cytoplasmic side (see Eq. (3) in Methods). (E) Effects of bupivacaine alone or in combination with 500 µM benzocaine. Currents were elicited by 500-ms pulses from –80 to +60 mV. The inset shows the superimposed current traces obtained in the presence of bupivacaine alone or the combination of bupivacaine with benzocaine in an expanded scale to better show the kinetic changes. In panels A, B and E, the dotted line represents the zero current level. (F) Percentage of block produced by bupivacaine (solid bars) or the combination of bupivacaine plus 500 µM measured at the end of 500-ms pulses to +60 mV as a function of the bupivacaine concentration. Each bar represents the mean and vertical lines the S.E.M. of 5–7 experiments. The inset shows the concentration dependence of bupivacaine-induced block of hKv1.5 channels in the absence (continuous line) and presence of 500 µM benzocaine (dashed line). Lines represent the fit of the data to Eq. (4) in Methods.

 
We also studied the effect of 500 µM benzocaine in the presence of increasing concentrations of bupivacaine (1–50 µM). Fig. 4E shows that, following the application of 500-ms pulses at +60 mV, addition of 500 µM benzocaine to 25 µM bupivacaine resulted in a reduction of the peak current, which was followed by a less extensive reduction of the quasi steady-state current at 500 ms. Furthermore, the initial decline was slower ({tau}=17.5±1.2 ms, n=19, P<0.01) compared with that obtained with bupivacaine alone. Moreover, in the presence of the combination, the decline of the tail currents was faster than in the presence of 25 µM bupivacaine alone (141.2±16.7 vs. 203.9±10.5 ms, n=4, P<0.05). Similar results were obtained in the presence of 50 µM bupivacaine. Fig. 4F shows the percentage of block obtained in the presence of increasing concentrations of bupivacaine alone or in the presence of increasing concentrations of bupivacaine plus 500 µM benzocaine. It is important to note that additive blocking effects were observed when both drugs were applied at submaximal concentrations (1 µM bupivacaine+500 µM benzocaine). This result suggests that the slight decrease in block observed when both drugs were present at concentrations of bupivacaine >25 µM was not due to a steric-hindrance and/or allosteric interaction near the binding site. A nonlinear least-squares fit of the concentration–response equation to the data points obtained in the presence of the combination, yielded a KD value of 18.2±6.9 µM (inset in Fig. 4F). These results are consistent with competition between both drugs for a common binding site during strong depolarization.

3.4 Effects of benzocaine in the presence of internal TEA
In order to analyze whether benzocaine binds to the internal TEA receptor site, in the next group of experiments we studied the effects of benzocaine in the presence of 3 mM TEA added to the internal solution (TEAi). In these experiments, the tip of the pipette was filled with TEA-free internal solution, in order to obtain ‘control’ current records. Fig. 5A shows current traces obtained in the same cell just after seal breaking and when steady-state effects of 3 mM TEAi were achieved, both in the absence and presence of 10 nM benzocaine. TEAi reduced hKv1.5 currents elicited by 500-ms pulses to +60 mV by 39.7±5.7% (n=8) without any time-dependent effect, i.e. it simply scaled down the current. In contrast, TEAi markedly slowed the initial fast phase of tail current decline ({tau}f=19.9±3.2 vs. 39.6±5.5 ms, n=8, P<0.01) (see inset). Addition of benzocaine reduced the current amplitude by 21.4±4.5% without any noticeable effect in its time dependence and further slowed the tail current decline ({tau}f=63.7±9.5 ms, n=8, P<0.05). Fig. 5B shows the I–V curve (500 ms isochronal) obtained in eight cells after steady-state effects of TEAi were achieved in the absence and presence of benzocaine. Benzocaine reduced hKv1.5 currents at all the membrane potentials. As it was derived from the ratio IBZ/ICON (panel C), the blockade increased in the voltage range of channel opening, but it remained constant at more depolarized membrane potentials. TEAi did not modify the activation curve of hKv1.5 channels (Vh=–16.7±1.5 mV; k=4.2±0.3 mV) (panel D), but benzocaine shifted the Vh to more negative potentials (Vh=–20.7±1.3 mV, n=8, P<0.01).


Figure 5
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  		Fig. 5 Effects of benzocaine on the presence of internal TEA. Effects of benzocaine 10 nM (Panels A–D) and 500 µM (panels E–H) in cells dialyzed with 3 mM TEA. (A and E) Current traces for 500-ms pulses from –80 to +60 mV in the absence and presence of benzocaine. The inset shows tail currents recorded on return to –40 mV after 500-ms pulses to +60 mV obtained before and after the dialysis with 3 mM TEA and after addition of benzocaine, 10 nM. Tail amplitudes were normalized in order to better show the kinetic modifications. (B and F) Current–voltage relationships (500-ms isochronal) in the absence and presence of benzocaine. (C and G) Fractional block (f=IBZ/ICON) from data shown in panels B and F, respectively. (D and H) Activation curves in the absence and presence of benzocaine. Continuous lines represent the fit to a Boltzmann equation. Each point represents the mean±S.E.M. of 6–8 experiments.

 
Fig. 5E–H shows the effects of 500 µM benzocaine in cells dialyzed with 3 mM TEAi. Under these conditions, addition of benzocaine produced a further decrease in current amplitude (37.0±3.1% at +60 mV, n=6, P<0.05). However, benzocaine did not accelerate the time course of tail current decline ({tau}f=30.4±9.9 ms, n=6, P>0.05). Panels F and G show the I–V relationship in the absence and presence of benzocaine, and fractional block (IBZ/ICON) as a function of the voltage of the pulse test. In the presence of TEAi, the voltage-dependent unblock observed with benzocaine at potentials at which activation of channels reached saturation was abolished. Moreover, in the presence of benzocaine the activation curve did not display two components (panel H), but the Vh was shifted to more negative potentials (Vh=–21.3±2.5 vs. –15.2±1.2 mV, n=6, P<0.01) without modification of the slope factor (3.9±0.4 vs. 4.0±0.7 mV, P>0.05).

3.5 Effects of benzocaine in the presence of external TEA
To analyze the possible binding of benzocaine to the external TEA binding site in this group of experiments, the effects of 10 nM and 500 µM benzocaine on cells perfused with 100 mM TEA (TEAo) were studied. Our results confirmed the low sensitivity to TEAo of hKv1.5 channels. Fig. 6A shows current traces recorded when applying 500-ms pulses to +60 mV under control conditions, and in the presence of TEAo (100 mM) alone or combined with 10 nM benzocaine. TEAo decreased hKv1.5 currents by 24.4±2.8% and slightly decreased the amplitude of the inactivating component (16.2±0.8 vs. 21.1±1.5%, n=14, P<0.05). TEAo also slowed the tail current decline, thus, {tau}f and {tau}s increased from 22.8±1.7 to 35.5±4.2 ms and from 80.9±10.4 to 138.8±19.9 ms (n=6, P<0.05), respectively. Under these conditions, 10 nM benzocaine increased the peak current elicited at the early beginning of the pulse (9.7±4.8%, n=6), and the current decline during the pulse. Thus, the steady-state current at the end of the pulse decreased by 1.6±2.9% (n=6), whereas tail current decline was unchanged ({tau}f=45.7±9.1, n=6, P>0.05). Fig. 6B shows the I–V curves (500 ms isochronal) obtained in this group of experiments. TEAo decreased the current amplitude at all the membrane potentials at which the current is activated, but this effect was independent of the voltage of the test pulse at potentials between 0 and +60 mV. However, in the presence of TEAo, benzocaine did not modify the current amplitude. TEAo shifted the Vh from –15.9±1.8 to –21.1±1.7 mV (n=6, P<0.05) (Fig. 6C), without modifying the slope factor (4.8±0.4 mV), whereas benzocaine, 10 nM, did not modify the Vh nor the slope of the activation curve (Vh –22.5±1.7 mV, k=4.5±0.7 mV, n=6, P>0.05).


Figure 6
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  		Fig. 6 Effects of benzocaine in the presence of external TEA. Effects of TEAo (100 mM) and benzocaine, 10 nM (Panels A–C) and 500 µM (panels D–F), on hKv1.5 channels. (A and D) Current tracings for 500-ms pulses from –80 to +60 mV in control conditions and in the presence of TEAo before and after the addition of benzocaine. The inset in panel D shows normalized tail currents on return to –40 mV in the presence of TEAo before and after the addition of 500 µM benzocaine. (B) Current–voltage relationships (500-ms isochronal) in control conditions and in the presence of TEAo before and after the addition of 10 nM benzocaine, together with the fractional block (f=ITEAo/ICON) induced by TEAo. (C and F) Activation curves in the absence and presence of TEAo before and after the addition of benzocaine. Continuous lines represent the fit to a Boltzmann equation. (E) Fractional block (f=IBZ/ITEA) induced by 500 µM benzocaine in cells perfused with TEAo. Dotted line represents the mean activation curve for this group of experiments. Each point represents the mean±S.E.M. of six experiments.

 
In the presence of 100 mM TEAo, 500 µM benzocaine further decreased the current amplitude by 33.7±6.8% (n=6) (Fig. 6D). The inset in panel D shows the normalized tail currents demonstrating that benzocaine did not modify the kinetics of tail current decay ({tau}f=31.7±5.8 ms, P>0.05). Fig. 6E shows the ratio IBZ/ITEAas a function of the voltage of the test pulse. Benzocaine-induced block steeply increased in the voltage range of channel activation but, at potentials ranging between 0 and +60 mV the blockade remained constant, i.e. the voltage-dependent unblock was abolished in the presence of TEAo. In the presence of TEAo, benzocaine shifted the Vh from –19.4±2.0 to –22.6±3.1 mV (n=7, P<0.01), but the activation curve did not exhibit two components (Fig. 6F).


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
In the present study, we analyzed the agonist and blocking effects of benzocaine on site-directed mutated and WT hKv1.5 channels at different [K+]i/[K+]o and in the presence of TEAo and TEAi. Since the current carried by hKv1.5 channels corresponds to the human atrial IKur, it is important to elucidate the structural determinants of the blocking and the ‘agonist’ effects of benzocaine, which in turn, are different from those produced by cationic open channel blockers. Our results can be summarized as follows: (a) when the [K+]i was reduced, the voltage-dependence of block was preserved both at 4 and 140 mM [K+]o, (b) mutations in residues located at the inner mouth of the pore and TEAi abolished the ‘agonist’ but not the blocking effects of benzocaine, (c) combination of benzocaine and bupivacaine produced less blockade than bupivacaine alone, and (d) TEAo did not modify the ‘agonist’ and blocking effects of benzocaine but suppressed the voltage-dependence.

4.1 Interactions of extracellular and intracellular [K+] on the benzocaine effects
hKv1.5-induced block produced by neutral drugs like loratadine [7], benzocaine [9], rupatadine [10] and nifedipine [8] as well as by drugs that predominate in their anionic form, like losartan [18], candesartan and eprosartan [19], decreased in a voltage-dependent manner. One possible explanation that can account for this effect is that these drugs bind to an external site and block hKv1.5 channels. Thus, at positive membrane potentials, K+ efflux hinders the binding of the drug 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 [8]. In fact, nifedipine is a neutral drug (pKa{approx}1) that induces an open-state block followed by a voltage-dependent unblock at positive potentials identical to that reported previously with benzocaine [9]. Moreover, a similar mechanism was proposed to account for the Na+ blockade in K+ channels of the squid giant axon [20]. To examine the possibility that, at positive voltages, K+ permeation increases and hinders the binding of benzocaine to its external site with a resultant relief of block, the [K+]i was reduced to 25%. In fact, decreasing the [K+]i to 25% abolished the voltage-dependent unblock observed in the presence of rupatadine, candesartan and eprosartan [10,19]. However, the present results demonstrated that under these conditions, the voltage-dependent unblock induced by benzocaine was not abolished. This finding suggests that benzocaine did not bind to an extracellular binding site or that the reduction in K+ flow was insufficient to prevent the unbinding of benzocaine.

Another piece of evidence suggesting a putative binding site of benzocaine located at the external entryway of the pore was that increasing the [K+]o to 140 mM produces identical time- (acceleration of tail current decline) and voltage-dependent (activation curves became biphasic) effects to benzocaine. Moreover, both benzocaine and external K+ increase the amplitude of the inactivating component. Thus, it has been proposed that extracellular K+ and benzocaine, by binding at a common external site at the channel level, would stabilize an open state from which the closing and/or the inactivation can proceed [9]. The present results demonstrated that at low [K+]i the activation curve in the presence of benzocaine did not exhibit two components. In contrast, the activation curve of hKv1.5 channels at 140 [K+]o exhibited two components even when the [K+]i was reduced to 25%. Therefore, our results suggest that the gating effects of benzocaine were only apparent when the K+ occupancy of the pore is high, particularly at the extracellular side of the entryway of the pore and that benzocaine and extracellular K+ interact at related but not the same site.

4.2 Effects of benzocaine on mutant channels
Since open state block induced by cationic drugs resembled the blockade produced by internal QA derivatives, we tested whether the pore mutation T477S affected benzocaine binding. This mutation is equivalent to the T441S mutation that reduced internal TEA affinity 10-fold in Shaker channels [21]. We also tested the effects of mutations in residues T505, L508 and V512 on the effects of nanomolar and micromolar concentrations of benzocaine. T505 has been implicated in the binding of hydrophobic TEA derivatives in Shaker (T469), of quinidine in hKv1.5, and in determining the stereoselective block of bupivacaine in hKv1.5 channels [3,4,22]. L508 has been implicated in the differential affinity of Kv2.1 and Kv3.1 for internal TEA [21]. V512M seems to be critical in determining the bupivacaine and quinidine block of hKv1.5 channels [3,4]. Moreover, if the S6 segment adopts an {alpha}-helical secondary structure in this region, then the V512 should align with L508 as proposed by Yeola and colleagues [3]. Our results demonstrated that the blockade induced by micromolar concentrations of benzocaine in T477S, T505A, L508M and V512M channels was not abolished but significantly increased, suggesting that all these residues were involved in the binding site of benzocaine on hKv1.5 channels or, alternatively, that the single critical residue has not yet been identified. Similarly, affinity for quinidine and the enantiomers of bupivacaine was not completely abolished in any of the mutants in several residues of the S6 region of hKv1.5 channels studied [3,4]. Thus, at least apparently the binding sites for benzocaine, bupivacaine and internal TEA are the same or overlapping. Further support of this hypothesis came from the results obtained in the presence of the combination of bupivacaine plus benzocaine. If both drugs bind independently, the addition of benzocaine should further reduce the current. Our results indicated that the combination produced a smaller amount of block than the more potent drug alone, which suggests that both drugs bind to the same or to an overlapping receptor site although their access and exit pathways are probably different. On the other hand, this result did not discard the possibility of a negative allosteric interaction of two separate receptors or, alternatively, the existence of steric hindrance interactions when bupivacaine and benzocaine were present near the binding site. However, the additive blocking effects observed at submaximal concentrations adds further support to the hypothesis of a common or overlapping binding site.

Surprisingly, nanomolar concentrations of benzocaine induced a marked blocking effect on T477S, T505A, L508M and V512M channels, which was particularly pronounced on T477S and V512M. Using the reduction of current induced by 10 nM benzocaine as an index of block, the apparent affinity (KD) for benzocaine blockade of T477S and V512M (calculated assuming nH=1) ranged between 20 and 30 nM. These values are far away from those obtained when the reduction of current induced by 500 µM benzocaine in WT, T477S and V512M channels was used as an index of block (KD=560–900 µM). Several hypotheses may account for these effects. First, if the ‘agonist effect’ induced by benzocaine is a state-dependent effect, it is possible that the interaction of the drug with this particular state was influenced by the modified gating properties of the mutant channels rather than by the role of the residues involved in the binding of benzocaine. However, it is important to note that the voltage-dependent activation of T477S channels is identical to that of WT channels, whereas activation curves of L508M and V512M channels were shifted toward more depolarized and hyperpolarized membrane potentials, respectively. Thus, even when the voltage-dependence of channel activation is affected in a different manner in all these mutants, the ‘agonist effect’ disappeared. Although we cannot rule out the possibility that these mutations might ‘create’ a new binding site for benzocaine, the present results strongly suggest that: (a) these mutations affected in a different way the blocking and the ‘agonist’ effects of benzocaine, and (b) the gating effects of benzocaine responsible for its ‘agonist’ actions were exerted from the intracellular side of the pore.

Furthermore, the effects of nanomolar and micromolar concentrations of benzocaine, as well as the effects of increasing the [K+]o were studied on R485Y channels. The R485 residue is located at the external entryway of the pore and has been implicated in the C-type inactivation [15] and external TEA sensitivity [16]. Our results demonstrated that both the ‘agonist’ and the blocking effects of benzocaine were not affected by this mutation suggesting that R485 is not implicated in the binding site of benzocaine. Furthermore, the gating effects observed on WT channels at 140 mM [K+]o were reproduced on R485Y channels. Thus, [K+]o modulates the gating properties of hKv1.5 channels in a different manner than in other Shaker channels, and the R485Y residue which is the equivalent to 449, is not involved in this effect.

4.3 Effects of benzocaine in the presence of TEA
Our results demonstrated that TEA- and benzocaine-induced block were additive, independent of whether TEA was applied at the extracellular or at the intracellular side of the channel pore, indicating that TEA and benzocaine bind to different sites to block hKv1.5 channels. TEAo slightly decreases the inactivation in hKv1.5 channels, an effect that has been previously described in Shaker channels and it has been explained by a ‘foot-in-the-door’ model in which external TEA competes with C-type inactivation by occupancy of a site in the pore that must empty before inactivation can proceed [23].

TEAo does not affect the agonist effect observed at nanomolar concentrations of benzocaine, whereas in the presence of TEAi the increase in the peak current was abolished and only blocking effects were apparent. These results are in agreement with those obtained in mutant channels and add further support to the hypothesis that benzocaine acts from the intracellular side to produce its ‘agonist’ effects, whereas its blocking effects are produced on a separated subsite also located in the inner mouth of the pore. Furthermore, it was a consistent finding that in the presence of TEA the voltage- and the time-dependence of benzocaine-induced block was abolished. Moreover, activation curves of hKv1.5 channels did not become biphasic. Probably, the mechanism for this effect was different for intracellular and for extracellular TEA. Since these gating effects of benzocaine were only apparent when K+ is present at some extracellular site, TEAi might suppress the voltage-dependent effects of benzocaine by decreasing the K+ flow through the channel. In contrast, under this assumption, TEAo would interfere with the K+ binding [24].

In view of all these results, we proposed that benzocaine binds to an intracellular binding site on hKv1.5 channels to both increase and decrease the hKv1.5 current. Probably, partially separated subsites exist; one of them, for which benzocaine exhibited a high affinity, is responsible for the ‘agonist’ actions. However, these actions exhibited saturation, so that at higher concentrations, the blocking effects produced by the binding of benzocaine to a low affinity subsite, which is the same or overlapping that of bupivacaine, became evident. Indeed, sequential binding of two small neutral molecules to Kv1 channels has recently been proposed to account for the voltage-dependent block produced by S-nitrosodithiothreitol [25]. Furthermore, S-nitrosodithiothreitol, despite a lack of fixed charge, blocked channels with a voltage dependence of comparable magnitude, but opposite sign, to that of cationic open channel blockers. Therefore, in the light of the results obtained with drugs that predominate in their uncharged form at the physiological pH, it can be hypothesized that the voltage-dependence of cationic open-channels blockers does not represent the only fraction of the transmembrane electrical field sensed at the binding site.

On the other hand, both benzocaine and extracellular K+ interact to modify the voltage-dependence of channel opening. The voltage-dependent unblock produced by benzocaine can be explained considering that the binding site of benzocaine is coupled to voltage-dependent transitions that occur when channels are open, thus conferring voltage-dependence to the open state block. In fact, it has been demonstrated that the open probability for the hKv1.5 channel does not saturate at potentials positive to 0 mV, but undergoes further changes at more positive potentials [26].

Time for primary review 29 days.


   Acknowledgements
 
This study was supported by CICYT (SAF99-0069) and FIS (01/1130) grants. We thank Drs D.J. Snyders and M.M. Tamkun for providing WT and mutated hKv1.5 channels.


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

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