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(Circulation Research. 1997;81:1053-1064.)
© 1997 American Heart Association, Inc.

Articles

Molecular Determinants of Stereoselective Bupivacaine Block of hKv1.5 Channels

Laura Franqueza, Mónica Longobardo, Javier Vicente, Eva Delpón, Michael M. Tamkun, Juan Tamargo, Dirk J. Snyders, , Carmen Valenzuela

From the Institute of Pharmacology and Toxicology (L.F., M.L., J.V., E.D., J.T., C.V.), CSIC School of Medicine, Universidad Complutense, Madrid, Spain, and the Departments of Pharmacology, Molecular Physiology, and Biophysics (M.M.T.) and the Departments of Medicine and Pharmacology (D.J.S.), School of Medicine, Vanderbilt University, Nashville, Tenn.

Correspondence to Carmen Valenzuela, PhD, Institute of Pharmacology and Toxicology, CSIC, School of Medicine, Universidad Complutense, 28040 Madrid, Spain. E-mail carmenva{at}eucmax.sim.ucm.es

	Abstract

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

Key Words: local anesthetic • K⁺ channel • bupivacaine • drug binding site • enantiomer

	Introduction

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Local anesthetics are useful probes of ion channel function and structure. Stereoselective interactions are especially interesting because they can reveal three-dimensional relationships between drug and channel with otherwise identical biophysical and physicochemical properties. Furthermore, stereoselectivity suggests direct and specific receptor-mediated action,^1–4 and identification of such stereospecific interactions may have important clinical consequences.^1,5 Bupivacaine is a long-acting, potent, and highly cardiotoxic local anesthetic agent widely used for regional anesthesia.^6,7 It contains one chiral carbon atom, and it is clinically used as a racemic mixture of S(-)- and R(+)-bupivacaine (Fig 1). In vivo studies have demonstrated that the potency and duration of local anesthesia are equal or even larger for S(-)-bupivacaine than for the R(+)-enantiomer.^5,8,9 More important, the LD₅₀ was 30% to 40% lower for R(+)-bupivacaine than for S(-)-bupivacaine.^5,8 Although the higher affinity of R(+)-bupivacaine for cardiac Na⁺ channels³ could explain its higher cardiotoxicity over S(-)-bupivacaine, several studies have also shown a prolongation of the QT_c interval of the ECG in anesthetized dogs^10–12 and human volunteers¹³ receiving high doses of bupivacaine. Occasionally, this QT_c prolongation was accompanied by torsades de pointes,¹⁴ suggesting that bupivacaine cardiotoxicity also involves block of K⁺ channels. Furthermore, it has been demonstrated that racemic bupivacaine inhibits guinea pig cardiac delayed rectifier K⁺ current,^15,16 rat atrial transient outward current,¹⁷ hKv1.5-like current,⁴ and delayed rectifier K⁺ current recorded in frog sensory ganglion cells.¹⁸ Inhibition of cardiac transient outward current and neuronal K⁺ current is not stereoselective,^17,18 whereas open-channel block of a human Shaker-related channel, hKv1.5, by bupivacaine is highly stereoselective, with the R(+)-enantiomer being 7-fold more potent than S(-)-bupivacaine.⁴ The intrinsic voltage dependence of hKv1.5 block was consistent with a fractional electrical distance referenced to the inner surface of the membrane () of 0.16 for both bupivacaine enantiomers. This cloned subunit most likely underlies the ultrarapid activating delayed rectifier described in human atrium and may contribute to similar currents in cardiac myocytes of other species.^19–22 This native hKv1.5-like current is involved in the control of the cardiac action potential duration¹⁹ and, therefore, represents a potential molecular target for drugs that prolong the cardiac action potential duration as a therapeutic intervention or a proarrhythmic side effect. Kv1.5 is also expressed in pancreatic beta cells, vascular and visceral smooth muscle, GH₃ cells, and brain,^23–27 indicating that it plays a role in the excitability of a variety of cell types. The hKv1.5 channels consist of four subunits each containing six transmembrane segments (S1 to S6). The segment between S5 and S6 forms the external entrance to and part of the ion conduction pathway (P loop). The flanking S5 and S6 segments are considered to contribute to the presumably wider intracellular mouth of the ion channel,^28,29 which contains binding sites for quaternary ammonium open-channel blockers and similarly acting drugs such as quinidine.^30–33 Both electrostatic and hydrophobic factors are apparently involved in open-channel block by clinically used drugs such as quinidine and bupivacaine.^4,33,34

View larger version (28K): [in this window] [in a new window]   Figure 1. A, Alignment of S6 sequences for members of Kv1 to Kv4 subfamilies and Shaker channels. Sequence is one-letter code; sequence number for the first residue is indicated. B, Chemical structure of bupivacaine. *Asymmetrical carbon.

In the present study, we used site-directed mutagenesis to examine the possible involvement in affinity and stereoselectivity of bupivacaine binding to hKv1.5 channels of the internal TEA binding site (T477 in Kv1.5)^28–30,35 and three residues located in the midsection of S6: (1) T505, which has been implicated in the binding of hydrophobic TEA derivatives in Shaker,³⁰ and quinidine, in Kv1.5,³³ (2) L508, which has been involved in the internal TEA binding in Kv3.1 channels (which contain a methionine at the equivalent position), and (3) V512, which has been implicated in the quinidine binding site in hKv1.5 channels.³³ We found that mutations at these sites affect both affinity and stereoselectivity for bupivacaine binding, and the results suggest that stereoselective binding of bupivacaine to hKv1.5 channels requires a polar interaction at position 505 (T505) combined with hydrophobic interactions at positions 508 and 512 (L508 and V512). Preliminary reports of the present study have been published in abstract form.^36,37

	Materials and Methods

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Site-Directed Mutagenesis
The PCR-based method for site-directed mutagenesis in the S4 to S6 region of hKv1.5 has been described in detail elsewhere.³³ Briefly, PCR-based mutagenesis was used, and all PCR-generated sequences were verified directly by double-stranded sequencing. 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.^19,38 For each mutation, at least six foci were expanded into clonal cell lines. In most cases, at least two cell lines were examined. For the mutations reported in the present study, we found that all Kv1.5 mRNA–positive cell lines were expressing current, albeit at different levels. Of the expressing cell lines, those with currents between 1 and 4 nA at +40 mV were used preferentially to ensure adequate voltage-clamp control. Cell culture and cell preparation for experimental use were as reported previously.^4,19

Solutions
The intracellular pipette filling solution contained (mmol/L) potassium aspartate 80, KCl 50, KH₂PO₄ 10, MgATP 3, HEPES 10, and EGTA 5 and was adjusted to pH 7.25 with KOH. The bath solution contained (mmol/L) NaCl 130, KCl 4, CaCl₂ 1.8, MgCl₂ 1, HEPES 10, and glucose 10 and was adjusted to pH 7.35 with NaOH. Both bupivacaine enantiomers (a gift from Chiroscience, Cambridge, UK) were dissolved in distilled deionized water to yield stock solutions of 10 mmol/L, from which further dilutions were made to obtain the desired final concentration.

Electrical Recording
Experiments were performed in a small volume (0.5-mL) bath mounted on the stage of an inverted microscope (model TMS, Nikon) perfused continuously at a flow rate of 0.5 to 1.0 mL/min. The hKv1.5 currents were recorded at room temperature (20°C to 22°C) using the whole-cell voltage-clamp configuration of the patch-clamp technique³⁹ with an Axopatch-1C patch-clamp amplifier (Axon Instruments). Currents were filtered at 2 kHz (four-pole Bessel filter) and sampled at 4 kHz. Data acquisition and command potentials were controlled by the pClamp software (versions 5.5.1 and 6.01) (Axon Instruments).

Micropipettes were pulled from borosilicate glass capillary tubes (GD-1, Narishige) on a programmable horizontal puller (Sutter Instrument Co) and heat-polished with a microforge (Narishige). When filled with the intracellular solution and immersed into the bath (external) solution, the pipette tip resistance ranged between 1 and 2 M. After gigaohm (16±6 G, n=21) seal formation, the membrane patch was ruptured with brief additional suction. The capacitive transients elicited by symmetrical 10-mV steps from -80 mV were recorded at 50 kHz (filtered at 10 kHz) for subsequent calculation of capacitive surface area, access resistance, and input impedance. Capacitance and series resistance compensation were optimized, and 80% compensation of the effective access resistance was usually obtained.

Pulse Protocol and Analysis
After control data were obtained, bath perfusion was switched to drug-containing solution, and cells were pulsed from -80 to +60 mV every 30 seconds to monitor drug effects. The holding potential was maintained at -80 mV, and the cycle time for any protocol was 0.1 Hz in order to avoid accumulation of block or incomplete recovery from inactivation or slow deactivation. The protocol to obtain I-V relationships and activation curves consisted of 250-millisecond pulses imposed in 10-mV increments between -80 and +60 mV. The "steady-state" I-V relationships were obtained by measuring the current at the end of the 250-millisecond depolarizations. These results were corrected for passive linear leak obtained from fits to the data between -80 and -40 mV, which is below the threshold for channel opening for WT hKv1.5 and most mutations. For T505V, V512M, and V512A mutant channels, passive linear leak was observed between -80 and -60 mV. Deactivating "tail" currents were recorded at -40 mV. The activation curve was obtained from the maximum tail current amplitude after the capacitive transient. Measurements were performed using the clampfit program of pClamp and by a custom-made analysis program. Block after starting the perfusion with each enantiomer reached steady state after 12 to 15 minutes; therefore, 12 to 15 minutes of equilibration was allowed before assessment of drug effects. Since the effects of bupivacaine enantiomers on T477S, T505I, T505V, T505S, T505A, L508M, and V512M mutant channels were voltage dependent and, in the case of T477S, T505S, T505V, T505A, L508M, and V512M, also time dependent, steady-state block of all these mutant channels by bupivacaine enantiomers was measured at the end of 250-millisecond duration depolarizing pulses to +60 mV.

Drug-channel interactions were described by one or two binding curves. The apparent affinity constant, K_d, and the Hill coefficient, n_H, for Kv2.1 and T505V, T505A, L508M, and V512M mutated hKv1.5 channels were obtained from fitting the fractional block (f) at various drug concentrations ([D]) to one Hill curve:

(1)

Experimental data from T505I, T505S, and T477S mutated hKv1.5 channels were fitted to the sum of two Hill equations:

(2)

where I₁ and I₂ are the fractional current of each component (I₁+I₂=1), and K_d1 and K_d2 are the apparent dissociation constants. Apparent rate constants for binding (k) and unbinding (l) were obtained from solving the following equations, in which _B represents the time constant of the fast initial drug-induced current decay after activation from the holding potential to +60 mV:

(3A)

(3B)

In the case of T505I channels, in which block induced by bupivacaine enantiomers was not time dependent, k and l were estimated as follows. If one assumes that the abrupt transition (compared with control) from the rising phase of channel opening into the reduced steady-state level represents fast block, ie, block occurring on the time scale of channel opening, then the time constant for block with 20 µmol/L S(-)- or R(+)-bupivacaine should be <2.5 milliseconds (or a binding rate of >400/s). Combined with Equations 3a, and 3b, this allows an estimate of the rate constants.

Activation curves were fitted with a Boltzmann equation:

(4)

where s represents the slope factor, and E represents the membrane potential. The time course of tail currents and the slow inactivation were fitted with the sum of exponentials. The activation kinetics were determined using the approach of the dominant time constant of activation in which a single exponential was fitted to the latter 50% of the activation time course.^40,41 The 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 the fit was judged by the ² criterion and by inspection for systematic nonrandom trends in the difference plot.

Voltage dependence of block was determined normalizing the leak-corrected current in the presence of drug to matching control to yield the fractional block at each voltage (f=1-I_drug/I_control). The voltage dependence of block was fitted to the following:

(3)

where z, F, R, and T have their usual meaning in thermodynamics, represents the fractional electrical distance measured from the inside of the cell membrane, and K_d* represents the apparent dissociation constant at the reference potential (0 mV).

Statistical Analysis
Results are expressed as mean±SEM. Direct comparisons between mean values in control conditions versus mean values in the presence of drug for a single variable were performed by a paired Student's t test. ANOVA was used to compare more than two groups. Student's t test was also used to compare two regression lines. Differences were considered significant at P<.05.

	Results

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The segment between transmembrane regions S5 and S6 (P loop) of voltage-gated K⁺ channels has been implicated as (part of) the ion-conducting pore; it controls K⁺ selectivity, and the internal part of this segment determines internal TEA binding.^28,29,35,42 Fig 1 illustrates the high degree of conservation in the P loop and S6 sequences of various cloned mammalian Shaker-related channels.^21,43 The functional effects of mutations of several nonconserved sites in Shaker, Kv3.1, and hKv1.5 channels have been interpreted to indicate that these residues line part of the ion-conducting pore.^28,29,44

S(-)-Bupivacaine and R(+)-bupivacaine block WT hKv1.5 channels in a time-dependent manner after channel opening, with R(+)-bupivacaine being 7-fold more potent than the S(-)-enantiomer (Fig 2); the K_d values for block were 4.1 and 27.3 µmol/L, respectively.⁴ Since bupivacaine block of hKv1.5 channels resembled internal TEA and quaternary ammonium binding, we tested whether pore mutation T477S affected affinity and/or stereoselectivity of bupivacaine block (this site is equivalent to the T441S mutation that reduced internal TEA affinity 10-fold in Shaker).³⁵ Moreover, since mutations in the midsection of the S6 sequence of Shaker channels (T469) have been implicated in the binding of hydrophobic TEA derivatives,³⁰ the effects of S(-)-bupivacaine and R(+)-bupivacaine on mutations at T505 (equivalent to T469 in Shaker) were also studied. T505 was replaced with isoleucine (T505I) to increase the hydrophobic character of the side chain. This mutation has been shown to influence alkyl-TEA derivative block in Shaker channels³⁰ and quinidine block in hKv1.5.³³ We also analyzed the effects of mutation T505S, which preserves the polar character of threonine present in the WT channel, and T505V, which retains the geometry but is nonpolar (valine is the corresponding residue in the Kv2 and Kv4 subfamilies; Fig 1). In order to know if the size of the amino acid at position 505 or only its polar character is a potential molecular determinant for stereoselective block, we studied the effects of bupivacaine enantiomers on T505A, which represents a hydrophobic but smaller substitution at this position. The effects of S(-)-bupivacaine and R(+)-bupivacaine on L508M mutant hKv1.5 channels were also studied, since Kv3.1 channels exhibit a methionine at the equivalent position that has been involved in their internal TEA binding.²⁹ Finally, since the valine at position 512 of the hKv1.5 channel is one of the molecular determinants of quinidine binding,³³ the effects of bupivacaine enantiomers on V512M and V512A were studied.

View larger version (21K): [in this window] [in a new window]   Figure 2. Effects of S(-)- and R(+)-bupivacaine [S(-)- and R(+)-Bupi, respectively] on WT hKv1.5 channels (WT), T505I, and T505V (left panel) and T505A and T505S (right panel). Current records were elicited by 250-millisecond depolarization pulses from -80 to +60 mV. For T505V, the holding potential was -100 mV. Beneath the current records, the concentration-response curves for both bupivacaine enantiomers are shown. Tail currents were recorded after repolarization to -40 mV, with the exception of T505V channels, in which they were recorded at -60 mV.

Mutations at Position 505: T505I, T505V, T505A, and T505S
Fig 2 shows currents elicited by the activation of T505I mutant hKv1.5 channels in the absence and in the presence of 20 µmol/L S(-)- and R(+)-bupivacaine. The basic hKv1.5 phenotype (fast activating delayed rectifier with partial slow C-type inactivation) was largely preserved in this mutation.⁴⁴ As shown in Table 1, the voltage dependence for channel opening was shifted to slightly more negative membrane potentials, and deactivation kinetics were much slower than in WT channels when measured at -40 mV (2 seconds) (Table 1).^19,33,36 The T505V mutant hKv1.5 channels produced outward K⁺ currents with an E_h shifted to more negative potentials than WT channels and a much faster activation at 0 mV; the latter was due to the shift in the activation curve. As for T505I, the T505V channels closed much slower than did WT channels (Table 1). T505S mutant channels induced outward K⁺ currents that exhibited a voltage dependence and an activation time course that were similar to those of WT channels but a faster deactivation process (Table 1). Activation of T505A mutant channels generated outward K⁺ currents with an E_h shifted toward more negative potentials, similar activation kinetics at +60 mV, but a faster activation time constant at 0 mV. These channels deactivated slower compared with WT channels but faster compared with T505I and T505V (Table 1). Detailed analysis of the gating properties of these mutants was beyond the scope of the present study, but most important for the present study was that the drug-channel interactions (affinity, voltage dependence) were analyzed at positive potentials (>+10 mV), where intrinsic channel properties were minimally affected by the mutations.

View this table: [in this window] [in a new window]   Table 1. Characteristics of the Currents Elicited by Eight hKv1.5 Mutants (T477S, T505I, T505V, T505A, T505S, L508M, V512M, and V512A) Compared With Those Observed in hKv1.5 WT Channels

In contrast to the stereoselective block of the WT hKv1.5 channel, block of T505I mutant channels by bupivacaine enantiomers did not display stereoselectivity: both enantiomers were equipotent in blocking these channels (Fig 2). These data suggested that threonine at position 505 could be a basic requirement for the stereoselective block observed in WT channels. Fig 2 shows the concentration dependence for block induced by S(-)- and R(+)-bupivacaine of T505I mutant channels. Although the concentration dependence for block of WT hKv1.5 was adequately described by a single binding site model,⁴ block induced by either enantiomer in the T505I mutant was better fit assuming two binding sites, with the fraction of channels blocked with high affinity being <30%. This lack of stereoselectivity was observed over the whole concentration range studied, with K_d values for the predominant binding site of 20.0±1.4 µmol/L (n=31) and 22.0±6.0 µmol/L (n=26) for S(-)- and R(+)-bupivacaine, respectively. The n_H values obtained for both enantiomers were close to unity (Table 2); when the experimental data were fit to a Hill curve with n_H fixed to 1, the K_d values were similar to those obtained without any constriction of the Hill coefficient. Similar to block of WT channels, block of T505I mutant channels by bupivacaine enantiomers was voltage dependent at all concentrations tested. Using the Woodhull formalism, the value was 0.160±0.005 (n=9) and 0.150±0.007 (n=6) for S(-)-bupivacaine and R(+)-bupivacaine, respectively (Fig 3) (Table 3). This result is consistent with the proposal that the cationic form of bupivacaine enantiomers acts as an internal pore blocker.⁴

View this table: [in this window] [in a new window]   Table 2. Apparent Kd and Association (k) and Dissociation (l) Rate Constants for S(-)-Bupivacaine and R(+)-Bupivacaine Block of WT, T477S, T505I, T505V, T505A, T505S, L508M, V512M, and V512A Channels

View larger version (35K): [in this window] [in a new window]   Figure 3. Voltage dependence of block of S(-)-bupivacaine and R(+)-bupivacaine [S(-)- and R(+)-Bupi, respectively] of mutant channels at position 505: T505I (A), T505V (B), T505A (C), and T505S (D). For each mutant, the I-V relation and the relative current (Idrug/Icontrol) for each membrane potential are represented. Block steeply increased in the range of activation of the channel. At membrane potentials between saturation of activation curve and +60 mV, block still increased with a shallow voltage dependence, which was fitted to a Woodhull equation (Equation 5) (continuous line) that yielded the value referred to the cytoplasmic side. Dashed lines represent the fits of the activation curves for each mutant in the particular experiment shown.

View this table: [in this window] [in a new window]   Table 3. Values Obtained for the Block Induced by S(-)-Bupivacaine and R(+)-Bupivacaine of hKv1.5 WT Channels, Point Mutations, and Kv2.1 Channels

To further characterize the molecular requirements of the stereoselective bupivacaine-induced hKv1.5 block, we studied the effects of both bupivacaine enantiomers in two additional substitutions at the T505 position: T505V and T505S. If the stereoselective bupivacaine block of hKv1.5 requires a polar interaction, we would expect that block of T505V mutant channels would not be stereoselective, since in this mutation a -CH₃ group replaces the threonine -OH group. This would not be in the case of T505S, since serine, like threonine, contains an -OH group. The mutation T505V decreased the affinity for block by R(+)-bupivacaine (K_d=13.7±3.5 µmol/L [n=12] compared with 4.1 µmol/L for WT) but increased the affinity for S(-)-bupivacaine (the K_d dropped to 18.2±1.8 µmol/L [n=12] compared with 27.3 µmol/L in WT). The Hill coefficients were 0.76±0.07 and 0.78±0.18 for S(-)-bupivacaine and R(+)-bupivacaine, respectively. Again, when the n_H values were fixed to unity, the K_d values obtained for S(-)- and R(+)-bupivacaine were 17.2±2.2 and 13.1±3.0 µmol/L, respectively (P>.05 versus the K_d values obtained without the latter constriction of the n_H). These results suggest that binding of a single bupivacaine molecule is sufficient to block the T505V mutant channel. This opposite effect on the affinity for each enantiomer effectively eliminated the stereoselective block (P>.05 for K_ds for both enantiomers in T505V). As shown in Fig 2, the concentration-dependent block of T505V channels induced by bupivacaine enantiomers was well described by a single site model, as in the case of WT hKv1.5 channels.⁴ Fig 2 also illustrates that the T505S mutation preserved the stereoselectivity for block induced by bupivacaine enantiomers: the R(+)-enantiomer was 8-fold more potent than was S(-)-bupivacaine. The low-affinity K_d values obtained for S(-)- and R(+)-bupivacaine were 60.1±10.5 µmol/L (n=30) and 7.4±1.6 µmol/L (n=21), respectively. As in the case of T505I, the concentration-dependent block of both bupivacaine enantiomers of T505S channels was best fit by assuming two binding processes with different affinities, the amplitude of each component being 50%. The effects of bupivacaine enantiomers on all these mutant channels were reversible: the amplitude of the current was restored to 90±2% (n=142) after 20 minutes of perfusion of the cells with drug-free solution.

To determine if the stereoselective block was determined by a polar amino acid at position 505 or if, on the contrary, it was a consequence of the presence of a small amino acid at this position, we studied the effects of S(-)-bupivacaine and R(+)-bupivacaine on T505A mutant channels. The mutation T505A decreased 1.6-fold the affinity for block by R(+)-bupivacaine (K_d= 6.4±0.4 µmol/L [n=12]) but increased 2.6-fold the affinity for S(-)-bupivacaine (the K_d dropped to 10.3±1.9 µmol/L [n=12]) (P>.05 for K_ds for both enantiomers in T505A). The Hill coefficients were 0.84±0.13 and 0.85±0.05 for S(-)-bupivacaine and R(+)-bupivacaine. When the n_H values were fixed to unity, the K_d values obtained for S(-)- and R(+)-bupivacaine were 10.5±1.9 µmol/L and 6.6±0.7 µmol/L, respectively (P>.05 versus the K_d values obtained without the latter constriction of the n_H). These results suggest that binding of a single bupivacaine molecule is sufficient to block the T505A mutant channel. This opposite effect on the affinity for each enantiomer effectively eliminated the stereoselective block. As shown in Fig 2, the concentration-dependent block of T505A channels induced by bupivacaine enantiomers was well described by a single-site model, as in the case of WT hKv1.5 channels.

Interestingly, a valine residue is found at the T505 equivalent position in Kv2 and Kv4 families (in Fig 1). To further test whether a polar interaction at this position is an important determinant for stereoselective block of this class of voltage-gated K⁺ channels, we studied the effects of S(-)-bupivacaine and R(+)-bupivacaine on Kv2.1 channels. As shown in Fig 4, bupivacaine-induced block of Kv2.1 channels was not stereoselective, with S(-)- and R(+)-bupivacaine exhibiting K_d values of 8.0±0.2 µmol/L (n=15) and 12.4±0.8 µmol/L (n=11) (P>.05), respectively. The Hill coefficients (n_H) were 1.06±0.03 and 1.01±0.06 for S(-)-bupivacaine and R(+)-bupivacaine, respectively (Fig 4). The apparent K_d values obtained for both bupivacaine enantiomers were similar when the same data were fitted constraining the Hill coefficients to unity. The effects of bupivacaine enantiomers on Kv2.1 channels were reversible: the amplitude of the current was restored to 89±3% (n=26) after 20 minutes of perfusion of the cells with drug-free solution. Other aspects of block by either enantiomer were qualitatively and quantitatively similar [ values of 0.172±0.010 and 0.184±0.009 for S(-)-bupivacaine and R(+)-bupivacaine, respectively; Table 3]. Taken together, these results suggest that stereoselective block of WT hKv1.5 channels induced by bupivacaine involves a specific interaction between the -OH moiety of the residue at position 505.

View larger version (13K): [in this window] [in a new window]   Figure 4. A, Effects of S(-)- and R(+)-bupivacaine [S(-)- and R(+)-Bupi, respectively] on Kv2.1 channels. Current records were obtained after applying depolarizing pulses from -80 to +60 mV for 250 milliseconds. Tail currents were obtained after repolarization to -40 mV. B, Concentration-response curve for block induced by S(-)-Bupi and R(+)-Bupi on Kv2.1 channels.

Mutations at Positions 508 and 512: L508M, V512M, and V512A
Fig 5A shows currents for the L508M substitution in hKv1.5 channels before and after application of 20 µmol/L S(-)- and R(+)-bupivacaine. The basic hKv1.5 properties were mostly preserved in this mutation (Table 1).⁴⁴ The voltage dependence for channel opening (E_h) was shifted to more positive membrane potentials. Consequently, the activation kinetics were slower compared with WT channels, without significant changes in deactivation kinetics measured at -40 mV (Table 1). In contrast, V512M mutant channels exhibit an activation curve shifted to more negative membrane potentials, a similar activation time constant at +60 mV, and a faster time constant at 0 mV, presumably due to the negative shift in the activation curve. Somewhat similar results were obtained for V512A (Table 1). Both V512M and V512A channels deactivated slower than WT channels (Table 1).

View larger version (20K): [in this window] [in a new window]   Figure 5. A, Effects of S(-)- and R(+)-bupivacaine [S(-)Bupi and R(+)Bupi, respectively] on WT hKv1.5 channels (WT) and indicated L508M and V512M mutations. Current records were elicited by 250-millisecond depolarization pulses from –80 to +60 mV. For V512M, the holding potential was -100 mV. Beneath the current records, the concentration-response curves for both bupivacaine enantiomers are shown. Tail currents were recorded at -40 mV for L508M and at -60 mV for V512M. B, Time-dependent block induced by 100 µmol/L S(-)Bupi and R(+)Bupi on L508M and V512M hKv1.5 mutant channels. B indicates time constant of the fast initial drug-induced current decay.

The methionine substitution of the hydrophobic residues L508 and V512 abolished the stereoselectivity for block by bupivacaine enantiomers (Fig 5A). In both cases, the experimental data could be fit to a single concentration-response curve. The K_d values obtained in L508M channels were 35.7±1.5 µmol/L (n=15) and 24.0±1.4 µmol/L (n=15) for S(-)- and R(+)-bupivacaine, respectively. The n_H values obtained for both enantiomers were close to unity [1.14±0.06 and 1.14±0.08 for S(-)-bupivacaine and R(+)-bupivacaine, respectively] (Table 2), and the K_d values obtained from fits with the Hill coefficient constrained to unity were similar. Block induced by bupivacaine enantiomers of L508M and V512M channels was time dependent, although this time dependence was only visible at high concentrations (>100 µmol/L) (Fig 5B). Similar to block of WT channels, block of L508M mutant channels induced by bupivacaine enantiomers was voltage dependent at all concentrations tested, and it was described with values of 0.165±0.002 (n=4) and 0.160±0.007 (n=6) for S(-)-bupivacaine and R(+)-bupivacaine, respectively (Table 3). The K_d values obtained for V512M channels were 29.7±2.6 µmol/L (n=10) and 28.0±5.2 µmol/L (n=11) for S(-)- and R(+)-bupivacaine, respectively. Again, the n_H values were close to unity (Table 2). Block induced by bupivacaine enantiomers of V512M channels was also voltage dependent, consistent with values of 0.162±0.005 (n=5) and 0.157±0.008 (n=5) for S(-)-bupivacaine and R(+)-bupivacaine, respectively (Table 3). For V512A mutant hKv1.5 channels, we obtained K_d values for each enantiomer that averaged 24.4±0.1 (n=2) and 3.2± 0.6 µmol/L (n=2) for S(-)-bupivacaine and R(+)-bupivacaine, respectively. These results suggest that stereoselective bupivacaine block of hKv1.5 channels requires at least two hydrophobic interactions at positions 508 and 512. The decreased hydrophobia of methionine would act to disrupt this interaction.

Mutation Affecting the Internal TEA Binding Site (T477S)
The threonine at the cytoplasmic turn of the P loop has been implicated in binding of internally applied TEA.^19,35 Since bupivacaine action resembles internal quaternary ammonium block, we sought to determine the possible involvement of this site in bupivacaine binding. Therefore, we analyzed the effect of the T477S mutations on affinity and stereoselectivity of bupivacaine block. Outward K⁺ currents through T477S mutant channels exhibited a voltage dependence for channel opening similar to that described previously for WT hKv1.5 channels (Table 1).^19,36,44 The activation kinetics were also similar, although the deactivation kinetics at -40 mV were slower (Table 1).

Fig 6 shows K⁺ currents from T477S mutant channels in the absence and in the presence of 20 µmol/L S(-)-bupivacaine and R(+)-bupivacaine. In these mutant channels, R(+)-bupivacaine was again more potent than S(-)-bupivacaine [K_d values of 45.5±4.4 µmol/L (n=35) and 7.8±1.6 µmol/L (n=46) for S(-)- and R(+)-bupivacaine, respectively], indicating that the WT stereoselectivity was preserved in this mutation (Table 2). Block of T477S mutant channels induced by either enantiomer was reversible: the amplitude of the current was restored to 85±1% (n=81) after a 20-minute perfusion of the cells with drug-free solution. Fig 6 shows the dose-response curve for the block induced by S(-)-bupivacaine and R(+)-bupivacaine of T477S mutant channels. Contrary to the WT channel, the concentration-dependence curve could not be fit using a single binding site model. A good fit was obtained using a two-site model, in which each component accounts for 50% of the binding. Importantly, the stereoselectivity was preserved over the full concentration range; ie, R(+)-bupivacaine was the more potent enantiomer for both sites.

View larger version (24K): [in this window] [in a new window]   Figure 6. Stereoselective block by bupivacaine enantiomers of T477S mutant hKv1.5 channels. Top panels show original records obtained in the absence and in the presence of S(-)-bupivacaine and R(+)-bupivacaine [S(-)Bupi and R(+)Bupi, respectively]. Bottom panel shows the concentration-response curve for both enantiomers.

Voltage Dependence of Block
To interpret stereoselective effects in a meaningful way, it is necessary to test whether other aspects of drug binding were preserved. The voltage dependence of hKv1.5 block by S(-)- and R(+)-bupivacaine displays a biphasic voltage dependence with a steep phase coincident with the voltage dependence of channel opening and a shallow phase at potentials positive to 0 mV.⁴ Using the Woodhull formalism,⁴⁵ the latter can be used to gauge the equivalent value, ie, the fraction of the transmembrane electrical field sensed at the binding site. We routinely determined this parameter for each mutation by using the fractional reduction of current during depolarizing steps between 0 and +60 mV. The values for the apparent value clustered in a narrow range (=0.15 to 0.20) similar to that of WT channels (Table 3) (Fig 3). This was of particular interest in the case of T505I, T505V, T505A and V512M mutations, which displayed slower deactivation kinetics and variable negative shifts in the activation curve with respect to WT channels (Table 1). Despite these kinetic changes, it is important to note that we determined the affinity for block at strong depolarizations (+60 mV), where the differences in gating were minimal.

Time Dependence of Block
Block induced by either bupivacaine enantiomer of T477S, T505V, T505S, T505A, L508M, and V512M channels was time dependent, as was the case for WT channels.⁴ In those cases in which block was best fit assuming two binding processes with different affinities (T477S, T505S, and T505I), the rate constants for the lower affinity process, which accounted for 50% to 70% of block, were analyzed. As shown in Table 2, there were no major changes in the association rate constants for block of T477S by bupivacaine enantiomers versus WT channels. However, the dissociation rate constants for these channels were faster than those observed for WT channels.⁴ These faster dissociation kinetics suggest that the lower affinity of S(-)-bupivacaine and R(+)-bupivacaine reflects a decreased stability of the drug-channel complex.

The dissociation rate constants of bupivacaine enantiomer–induced block of T505S channels were increased to the same extent [2.2-fold and 2.6-fold for S(-)-bupivacaine and R(+)-bupivacaine, respectively] (Table 2). Similarly, the association rate constant was increased 1.05-fold for the S(-)-enantiomer and 2.2-fold for the R(+)-enantiomer. This similar effect on both dissociation and association rate constants explains the decreased affinity observed for both enantiomers as well as the similar stereoselective block of these mutant channels versus WT channels by bupivacaine enantiomers.

Compared with that for WT channels, the dissociation rate constant for the interaction with T505V channels was increased 2-fold for both enantiomers. On the other hand, the association rate constant was increased 3-fold for S(-)-bupivacaine, whereas it decreased 1.3-fold for R(+)-bupivacaine. This opposite effect on the association rate constant for both bupivacaine enantiomers is sufficient to explain the lack of stereoselective bupivacaine block observed in the present experiments on these mutant channels (Table 2).

The dissociation rate constants for the interaction between bupivacaine enantiomers and T505A channels remained similar to those values calculated for WT channels (Table 2). However, the association rate constant increased 2.5-fold for S(-)-bupivacaine and remained unaltered for R(+)-bupivacaine (Table 2). The modification of these values explains the lack of stereoselective block of bupivacaine of these channels.

The time dependence of block of T505I channels by bupivacaine enantiomers could not be resolved experimentally. The following experimental observations support an open-channel block mechanism: biphasic voltage dependence with a value similar to that described in WT channels and a slower time constant of deactivation versus control conditions. Therefore, lower limits for the association and dissociation rate constants were extracted assuming a very fast transition (compared with WT) from the rising phase of channel opening into the reduced steady-state level representing a fast block, ie, block occurring on the time scale of channel opening (_B<1.5 milliseconds). Under these assumptions, mutation T505I increased the dissociation rate constant for both enantiomers 10-fold (from 24 to 250 s^-1) and the association rate constant for S(-)-bupivacaine 11-fold. However, it only increased this parameter 2.65-fold for R(+)-bupivacaine, which explains the lack of stereoselective bupivacaine block of these mutated channels.

Compared with that for WT channels, the dissociation rate constant for the interaction between S(-)-bupivacaine and L508M channels was not modified, whereas it decreased 2-fold for the interaction with R(+)-bupivacaine. On the other hand, the association rate constant between S(-)-bupivacaine and R(+)-bupivacaine and L508M channels decreased 1.8-fold and 9.6-fold, respectively. As in the case of T505V, the opposite effect on the association rate constants for both bupivacaine enantiomers is enough to explain the lack of stereoselective bupivacaine block observed in these experiments (Table 2).

Similarly, in V512M mutant channels, the association rate constant for the interaction between S(-)-bupivacaine and the channel was similar to that obtained in WT channels, whereas it decreased 3.5-fold for R(+)-bupivacaine. The dissociation rate constants for both enantiomers were increased 2-fold. Therefore, the opposite effect on the association rate constants, together with the similar effects on the dissociation kinetics, can explain the lack of stereoselective block induced by bupivacaine enantiomers on V512M channels.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Stereoselective block of hKv1.5 channels by bupivacaine strongly indicates that it results from binding to a specific binding site, which in turn suggests that the three-dimensional configuration of the drug can be an important determinant of drug binding to its receptor (in addition to other factors, such as molecular size and hydrophobia). In the present study, we have localized putative amino acids in hKv1.5 responsible for stereoselective interactions between enantiomers of the local anesthetic bupivacaine. Our results suggest that (1) a polar interaction at position 505 of the channel protein and (2) hydrophobic interactions at positions 508 and 512 are required for stereoselective interactions between bupivacaine enantiomers and hKv1.5 channels.

Mutation at the Internal TEA Binding Site (T477S)
The fact that the affinity for bupivacaine block was modified in the T477S mutation suggests a possible overlap of the binding site for bupivacaine with that for the internal TEA, as has been suggested for Shaker channels.⁴⁶ This is in contrast with the lack of effect of the same mutation regarding quinidine block.³³ The reduced affinity due to an increase in the apparent dissociation rate constant is compatible with a reduced stabilization of binding, which may reflect the lack of a CH₃ group in the serine residue. However, the results obtained with this mutant suggest that this residue is not involved in determining stereoselective block of hKv1.5 channels by bupivacaine. Indeed, block induced by both bupivacaine enantiomers was stereoselective, with R(+)-bupivacaine being 6-fold more potent than S(-)-bupivacaine, similar to the 7-fold ratio previously described for WT hKv1.5 channels.⁴ This could reflect a more stable interaction of the R(+)-enantiomer with the receptor. However, difference in potency between both enantiomers was explained largely by their different association rate constants, which was faster for R(+)-bupivacaine. Since these drugs are enantiomers, the difference in association rates suggests that the S(-)-enantiomer needs to adopt a less favored conformation than R(+)-bupivacaine, similar to that observed in WT channels.⁴

Mutations at Position 505 (T505I, T505V, T505A, and T505S)
The replacement of threonine at position 505 by the hydrophobic residues, valine, alanine, and isoleucine, completely abolished the stereoselective block observed in WT hKv1.5 channels. In contrast, stereoselective block was retained and even enhanced with the conservative replacement of threonine 505 by serine (T505S), which preserved the polar hydroxyl side chain. Taken together, these results suggest that bupivacaine stereoselective block of hKv1.5 channels is derived from a stereoselective interaction between the drug and the channel, irrespective of the size of the side chain at this position. Furthermore, we propose that the hydroxyl group of this threonine is a basic requirement, since stereoselectivity was only preserved with the serine substitution but abolished with all hydrophobic substitutions (T505V, T505A, and T505I). These results could be due to the following: (1) the association rate constants of block changed in opposite directions compared with those observed in WT channels (for T505V), (2) the increase in the association rate constant was more pronounced for S(-)-bupivacaine than for R(+)-bupivacaine (for T505I), or (3) the association rate constant remained unaltered for one enantiomer and increased for the other one (for T505A) (Table 2). It is interesting to note that hydrophobic substitutions at 505 enhanced the affinity for quinidine in this channel³³ but had opposing effects on the bupivacaine affinity. This further indicates drug-specific interactions at the internal local anesthetic binding site.

Interestingly, the equivalent 505 residue of Kv2.1 channels (position 389) and Kv4.3 channels is a valine, in contrast to the threonine in hKv1.5 and Shaker (Fig 1). Moreover, bupivacaine-induced block of these two K⁺ channels is not stereoselective. These results suggest the existence of a similar bupivacaine receptor in Kv1, Kv2, and Kv4 channels with the same stereoselective determinants. In fact, we have observed that block of Kv4.3 channels by bupivacaine enantiomers is not stereoselective, with a K_d for both enantiomers of 31 µmol/L (L. Franqueza, J. Eck, C. Valenzuela, M.M. Tamkun, and D.J. Snyders, unpublished data, 1997). On the basis of these results, one would expect that block of Kv1.3 (carrying a serine at the T505 equivalent position) would also be stereoselective, but this remains to be determined. Moreover, the inhibition induced by bupivacaine enantiomers of the delayed rectifier K⁺ current recorded in frog sensory ganglion cells was not stereoselective. This K⁺ current is very similar in voltage- and time-dependent properties to Kv2.1 current; therefore, the results shown in the present study could explain the molecular basis of the lack of stereoselectivity observed in native neuronal cells previously described.¹⁸

Mutations at Positions 508 and 512 (L508M, V512M, and V512A)
Replacement of L508 or V512 by a methionine abolished bupivacaine stereoselective block. However, substitution of V512 by an alanine preserved it, with K_d values very similar to those observed in WT channels.⁴ These results suggest that stereoselective block induced by bupivacaine involves hydrophobic interactions at positions 508 and 512, in addition to the polar interaction with T505. Leucine, valine, and alanine are all hydrophobic amino acids, whereas methionine is a less hydrophobic residue. Moreover, the sulfur atom in methionine can act as an electron donor or acceptor; therefore, it could establish electrostatic interactions either with the partially positively charged amide or with the partially negatively charged carbons of the aromatic ring (Fig 7). The establishment of these electrostatic interactions would redirect the position of the entire molecule; thus, it would abolish the stereoselective bupivacaine block observed in WT channels.⁴ An alternate explanation would be that a decrease of the local hydrophobicity or steric hindrance from the bulkier methionine side chain disrupts the interaction between bupivacaine and its receptor site. Although these hypotheses could theoretically explain the experimental results, further structure-analysis studies at the chemical level are required in order to discern the exact mechanism by which these mutations abolish the bupivacaine stereoselective block.

View larger version (18K): [in this window] [in a new window]   Figure 7. Model of stereoselective interaction between bupivacaine enantiomers and the ion-conducting pore of hKv1.5 channels. Amino acids at positions 505, 508, and 512 () of the channel protein are involved in the molecular binding site of bupivacaine enantiomers. The -helix represents the S6 segment of the hKv1.5 channel. The dashed line in the -helix represents uncertainty about the structure but does not negate the fact of alignment on the same side of the helix. This could be due to the fact that proline 509 may cause a 30° bend in the backbone, which could be reverted by proline 511 located on the other side of the helix.

Biphasic Dose-Response Curves Suggest Two Different Open States With Different Affinities
A surprising finding of the present study is that dose-response curves obtained for some mutations (T505I, T505S, and T477S) were better fit assuming two binding processes with different affinities. This could be explained by the existence of two different populations of channels with different affinities for bupivacaine enantiomers. This seems unlikely to occur with a cloned channel that forms homomultimers. Heteromultimer formation with endogenous subunits is unlikely, since the Ltk^- cells used do not contain endogenous voltage-gated ion currents or detectable K⁺ channel mRNA.¹⁹ The Ltk^- cells do contain the Kvß2.1 subunit, which resembles the heterologously expressed hKv1.5, but this ß subunit does not affect antiarrhythmic drug action.⁴⁷ In addition, we did not obtain evidence for nonhomogeneous channel population when examining quinidine pharmacology and channel gating.^33,48 A second possible explanation is that these mutations are introducing a high-affinity binding site for bupivacaine in hKv1.5 channels that was not present in WT channels. However, if there were two separate drug binding sites, then all the current would be blocked by the high-affinity binding site before the low-affinity site could be occupied, thus masking low-affinity binding. A third explanation would be that mutated residues in each subunit of the homotetramer permits the access of more than one bupivacaine molecule in the pore. This would then lead to negative cooperative interactions between them (electrostatic or hindrance interactions) as has recently been proposed for block of IRK1 channels by polyamines.⁴⁹ Finally, the hypothesis we favor is that multiple open states exist with different bupivacaine affinities, even in WT hKv1.5 channels. Bupivacaine binding to the high-affinity open state could represent an intermediate transition state in WT channels, as has been proposed previously to explain the blockade of cardiac Na⁺ channels by QX-314.⁵⁰ Under this framework, T477S, T505I, and T505S mutant channels would be stabilizing an ultrafast (higher affinity) interaction between bupivacaine and hKv1.5 channels. In fact, we have preliminary evidence that multiple open states exist in hKv1.5 channels and that conversion between them can be influenced by drug concentration.⁴⁸ Thus, as bupivacaine concentration increases, an open state with low affinity is favored. This hypothesis requires a drug-induced shift in gating that is independent of open-channel block, and indeed, we have observed that concentrations of racemic bupivacaine below the K_d for block of hKv1.5 actually modify channel activity in a manner consistent with the transition from one open state to another (J. Vicente, M. Longobardo, L. Franqueza, E. Delpón, D.J. Snyders, and C. Valenzuela, unpublished data, 1996). Mutations such as T505S could enhance the drug-induced shift to the low-affinity state and thus unmask bupivacaine-induced transitions from one open state to the next. The mechanism responsible for the biphasic dose-response curves requires further investigation.

Limitations of the Present Study
A potential problem of site-directed mutagenesis is that the observed results may be derived from more generalized perturbations of the protein. In fact, we observed differences in the deactivation kinetics of several mutants used in the present study (T505I, T505V, V512A, V512M, and L508M), which in part resulted from shifts in the voltage dependence of channel opening (Table 1). However, we could not discern a relationship between altered deactivation gating and the affinity for open-channel block at depolarized potentials; ie, T505V slowed the deactivation gating, whereas T505A did not, and both suppressed stereoselective bupivacaine block. Moreover, we analyzed not only the apparent affinity but also the association and dissociation kinetics and the value and checked for drug-modified tail current kinetics as a qualitative indicator for open-channel block. The preserved open-channel block mechanism and the similarity of the values suggest that no major conformational changes were introduced to the binding site.

Conclusions
The results shown in the present study suggest a model of stereoselective interactions between bupivacaine enantiomers and hKv1.5 channels that involves the interaction between bupivacaine and the -OH group of threonine at position 505, combined with hydrophobic interactions at positions 508 and 512 of the channel protein. The enhanced stereoselectivity in the T505S mutant and the lack of stereoselectivity in T505I, T505A, and T505V mutants as well as in Kv2.1 and Kv4.3 channels are consistent with this hypothesis. T505 and V512 are separated by two turns of the S6 helix, so that they are 11 Å apart. Bupivacaine is 11 Å in length⁵¹ with positively charged and hydrophobic moieties at either end. Therefore, the positively charged end could interact with the T505, and the hydrophobic aromatic ring could establish a hydrophobic interaction with the valine residue at position 512, which is perturbed by a substitution with methionine, whose sulfur atom decreases its hydrophobicity (Fig 7). We have previously reported that a decrease in the length of the N-substituent decreases the potency of the molecule to block hKv1.5 channels.⁵² One possible explanation would be that this alkyl chain interacts with a hydrophobic amino acid (ie, L508) in the S6 of the channel (Fig 7). Our results further suggest that threonine at position 477 is involved in the binding of bupivacaine, although it does not determine the stereoselective block. The unusual dose-response curve observed in T477S, T505I, and T505S mutant channels may reflect a transition-intermediate state between bupivacaine and the channel.

	Selected Abbreviations and Acronyms

 

= fractional electrical distance Eh = voltage at which 50% of the channels are open I-V = current-voltage PCR = polymerase chain reaction TEA = tetraethylammonium WT = wild type

	Acknowledgments

 
This study was supported by FIS 95/0318 (Dr Valenzuela), CICYT SAF96–0042 (Dr Tamargo), and National Institute of Health grants HL-47599 (Dr Snyders), HL-46681 (Drs Snyders and Tamkun), and HL-49330 (Dr Tamkun). The authors want to express their thanks to Dr Ricardo Caballero for his constructive criticism of the manuscript. We also thank Guadalupe Pablo and Ruben Vara for their excellent technical assistance. We thank Chiroscience (Cambridge, UK) for supplying us with S(-)-bupivacaine and R(+)-bupivacaine.

	Footnotes

 
Previously published as preliminary reports in abstract form (Biophys J. 1996;70:A400; Biophys J. 1997;72:A141).

Received February 10, 1997; accepted September 19, 1997.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
1. Ariëns E. Nonchiral, homochiral and composite chiral drugs. Trends Pharmacol Sci. 1993;14:68–75.[Medline] [Order article via Infotrieve]

2. Franks N, Lieb W. Stereospecific effects of inhalational general anesthetic optical isomers on nerve ion channels. Science. 1994;254:427–430.

3. Valenzuela C, Snyders D, Bennett P, Tamargo J, Hondeghem L. Stereoselective block of cardiac sodium channels by bupivacaine in guinea pig ventricular myocytes. Circulation. 1995;92:3014–3024.[Abstract/Free Full Text]

4. Valenzuela C, Delpón E, Tamkun M, Tamargo J, Snyders D. Stereoselective block of a human cardiac potassium channel (Kv1.5) by bupivacaine enantiomers. Biophys J. 1995;69:418–427.[Medline] [Order article via Infotrieve]

5. Åberg G. Toxicological and local anaesthetic effects of optically active isomers of two local anaesthetic compounds. Acta Pharmacol Toxicol. 1972;31:273–286.[Medline] [Order article via Infotrieve]

6. Strichartz G, Ritchie J. The action of local anesthetics on ion channels of excitable tissues. In: Strichartz G, ed. Local Anesthetics Handbook of Experimental Pharmacology. Heidelberg, Germany: Springer-Verlag; 1987;81:21–52.

7. Covino B. Toxicity and systemic effects of local anesthetic agents. In: Strichartz G, ed. Local Anesthetics Handbook of Experimental Pharmacology. Heidelberg, Germany: Springer-Verlag; 1987;81:187–212.

8. Luduena F, Bogado E, Tullar B. Optical isomers of mepivacaine and bupivacaine. Arch Int Pharmacodyn. 1972;200:359–369.

9. Aps C, Reynolds F. An intradermal study of the local anaesthetic and vascular effects of the isomers of bupivacaine. Br J Clin Pharmacol. 1978;6:63–68.[Medline] [Order article via Infotrieve]

10. Avery P, Redon D, Schaenzer G, Rusy B. The influence of serum potassium on the cerebral and cardiac toxicity of bupivacaine and lidocaine. Anesthesiology. 1984;61:134–138.[Medline] [Order article via Infotrieve]

11. Wheeler D, Bradley E, Woods W. The electrophysiologic actions of lidocaine and bupivacaine in the isolated, perfused canine heart. Anesthesiology. 1988;68:201–212.[Medline] [Order article via Infotrieve]

12. Solomon D, Bunegin L, Albin M. The effect of magnesium sulfate administration on cerebral and cardiac toxicity of bupivacaine in dogs. Anesthesiology. 1990;72:341–346.[Medline] [Order article via Infotrieve]

13. Scott B, Lee A, Fagan D, Bowler G, Bloomfield P, Lundh R. Acute toxicity of ropivacaine compared with that of bupivacaine. Anesth Analg. 1989;69:563–569.[Abstract/Free Full Text]

14. Kasten G, Martin S. Bupivacaine cardiovascular toxicity: comparison of treatment with bretylium and lidocaine. Anesth Analg. 1985;64:911–916.[Abstract/Free Full Text]

15. Courtney K, Kendig J. Bupivacaine is an effective potassium channel blocker in heart. Biochim Biophys Acta. 1988;939:163–166.[Medline] [Order article via Infotrieve]

16. Sánchez-Chapula J. Effects of bupivacaine on membrane currents of guinea-pig ventricular myocytes. Eur J Pharmacol. 1988;156:303–308.[Medline] [Order article via Infotrieve]

17. Castle N. Bupivacaine inhibits the transient outward K⁺ current but not the inward rectifier in rat ventricular myocytes. J Pharmacol Exp Ther. 1990;255:1038–1046.[Abstract/Free Full Text]

18. Guo X, Castle N, Chernoff D, Strichartz G. Comparative inhibition of voltage-gated cation channels by local anesthetics. Ann N Y Acad Sci. 1991;625:181–199.[Medline] [Order article via Infotrieve]

19. Snyders D, Tamkun M, Bennett P. A rapidly activating and slowly inactivating potassium channel cloned from human heart. J Gen Physiol. 1993;101:513–543.[Abstract/Free Full Text]

20. Wang Z, Fermini B, Nattel S. Sustained depolarization-induced outward current in human atrial myocytes: evidence for a novel delayed K⁺ current similar to Kv1.5 cloned channel currents. Circ Res. 1993;73:1061–1076.[Abstract/Free Full Text]

21. Deal K, England S, Tamkun M. Molecular physiology of cardiac potassium channels. Physiol Rev. 1996;76:49–67.[Abstract/Free Full Text]

22. Feng J, Wible B, Li G, Wang Z, Nattel S. Antisense oligodeoxynucleotides directed against Kv1.5 mRNA specifically inhibit ultrarapid delayed rectifier K⁺ current in cultured adult human atrial myocytes. Circ Res. 1997;80:572–579.[Abstract/Free Full Text]

23. Overturf K, Russell S, Carl A, Vogalis F, Hart P, Hume J, Sanders K, Horowitz B. Cloning and characterization of a Kv1.5 delayed rectifier K⁺ channel from vascular and visceral smooth muscles. Am J Physiol. 1994;267:C1231–C1238.[Abstract/Free Full Text]

24. Takimoto K, Gealy R, Levitan E. Multiple protein kinases are required for basal Kv1.5 K⁺ channel gene expression in GH₃ clonal pituitary cells. Biochim Biophys Acta. 1995;1265:22–28.[Medline] [Order article via Infotrieve]

25. García-Guzmán M, Sala F, Criado M, Sala S. A delayed rectifier potassium channel cloned from bovine adrenal medulla: functional analysis after expression in Xenopus oocytes and in a neuroblastoma cell line. FEBS Lett. 1994;354:173–176.[Medline] [Order article via Infotrieve]

26. Mi H, Deerinck T, Ellisman M, Schwarz T. Differential distribution of closely related potassium channels in rat Schwann cells. J Neurosci. 1995;15:3761–3774.[Abstract]

27. Maletic-Savatic M, Lenn N, Trimmer J. Differential spatiotemporal expression of K⁺ channel polypeptides in rat hippocampal neurons developing in situ and in vitro. J Neurosci. 1995;15:3840–3851.[Abstract]

28. López G, Jan Y, Jan L. Evidence that the S6 segment of the Shaker voltage-gated K⁺ channel comprises part of the pore. Nature. 1994;367:179–182.[Medline] [Order article via Infotrieve]

29. Aiyar J, Nguyen A, Chandy K, Grissmer S. The P-region and S6 of Kv3.1 contribute to the formation of the ion conduction pathway. Biophys J. 1994;67:2261–2264.[Medline] [Order article via Infotrieve]

30. Choi K, Mossman C, Aubé J, Yellen G. The internal quaternary ammonium receptor site of Shaker potassium channels. Neuron. 1993;10:533–541.[Medline] [Order article via Infotrieve]

31. Rampe D, Wible B, Brown A, Dage R. Effects of terfenadine and its metabolites on a delayed rectifier K⁺ channel cloned from human heart. Mol Pharmacol. 1993;44:1240–1245.[Abstract]

32. Malayev A, Nelson D, Philipson L. Mechanism of clofilium block of the human Kv1.5 delayed rectifier potassium channel. Mol Pharmacol. 1995;47:198–205.[Abstract]

33. Yeola S, Rich T, Uebele V, Tamkun M, Snyders D. 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]

34. Snyders D, Yeola S. 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]

35. Yellen G, Jurman M, Abramson T, MacKinnon R. Mutations affecting internal TEA blockade identify the probable pore-forming region of a K⁺ channel. Science. 1991;251:939–942.[Abstract/Free Full Text]

36. Franqueza L, Delpón E, Gay P, Snyders D, Tamargo J, Valenzuela C. Molecular determinants for stereoselective bupivacaine binding in hKv1.5 channels. Biophys J. 1996;70:A400. Abstract.

37. Vicente J, Franqueza L, Delpón E, Tamkun M, Tamargo J, Snyders D, Valenzuela C. Threonine 505 determines the stereoselective bupivacaine block of hKv1.5 channels. Biophys J. 1997;72:A141. Abstract.

38. Snyders D, Knoth K, Roberds S, Tamkun M. Time-, state- and voltage-dependent block by quinidine of a cloned human cardiac channel. Mol Pharmacol. 1992;41:332–339.

39. Hamill O, Marty A, Neher E, Sakmann B, Sigworth F. Improved patch-clamp techniques for high resolution current recording from cells and cell-free membrane patches. Pflugers Arch. 1981;391:85–100.[Medline] [Order article via Infotrieve]

40. White M, Bezanilla F. Activation of squid axon K⁺ channels: ionic and gating current studies. J Gen Physiol. 1985;85:539–554.[Abstract/Free Full Text]

41. Valenzuela C, Sánchez-Chapula J, Delpón E, Elizalde A, Pérez O, Tamargo J. Imipramine blocks rapidly activating and delays slowly activating K⁺ current activation in guinea pig ventricular myocytes. Circ Res. 1994;74:687–699.[Abstract/Free Full Text]

42. MacKinnon R, Yellen G. Mutations affecting TEA blockade and ion permeation in voltage-activated K⁺ channels. Science. 1990;250:276–279.[Abstract/Free Full Text]

43. Pongs O. Molecular biology of voltage-dependent potassium channels. Physiol Rev. 1992;72:S69–S88.

44. Snyders D, Rich T, Mays D, Tamkun M, Yeola S. Involvement of S6 in voltage-dependent deactivation gating of hKv1.5 potassium channels. Biophys J. 1996;70:A144. Abstract.

45. Woodhull A. Ionic blockade of sodium channels in nerve. J Gen Physiol. 1973;61:687–708.[Abstract/Free Full Text]

46. Baukrowitz T, Yellen G. Two functionally subsites for the binding of internal blockers to the pore of voltage-activated K⁺ channels. Proc Natl Acad Sci U S A. 1996;93:13357–13361.[Abstract/Free Full Text]

47. Uebele V, England S, Chaundary A, Tamkun M, Snyders D. Functional differences in Kv1.5 currents expressed in mammalian cell lines are due to the presence of endogenous Kvß2.1 subunits. J Biol Chem. 1996;271:2406–2412.[Abstract/Free Full Text]

48. Rich T. Gating of the hKv1.5 Delayed Rectifier: The Implications of Multiple Conducting States. Vanderbilt University. UMI, Ann Arbor, Mich (Microfilm #9714424). 1996:161. PhD thesis.

49. Yang J, Nung Y, Jan L. Determination of the subunit stoichiometry of an inwardly rectifying potassium channel. Neuron. 1995;15:1441–1447.[Medline] [Order article via Infotrieve]

50. Gingrich K, Beardsley D, Yue D. Ultra-deep blockade of Na⁺ channels by a quaternary ammonium ion: catalysis by a transition-intermediate state? J Physiol (Lond). 1993;471:319–341.[Abstract/Free Full Text]

51. Singh B, Courtney K. The classification of antiarrhythmic mechanisms of drug action: experimental and clinical considerations. In: Zipes D, Jalife J, eds. Cardiac Electrophysiology: From Cell to Bedside. Philadelphia, Pa: WB Saunders; 1990:882–897.

52. Valenzuela C, Delpón E, Franqueza L, Gay P, Snyders DJ, Tamargo J. Effects of ropivacaine on a cardiac potassium channel (hKv1.5) cloned from human ventricle. Anesthesiology. 1997;86:718–728.[Medline] [Order article via Infotrieve]

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