CHIRALITY (2012)

Review Article

Stereoselective Interactions between Local Anesthetics and Ion

ChannelsCARMEN VALENZUELA,* CRISTINA MORENO, ALICIA DE LA CRUZ, ÁLVARO MACÍAS, ÁNGELA PRIETO, AND TERESA GONZÁLEZ

Instituto de Investigaciones Biomédicas Alberto Sols, Consejo Superior de Investigaciones Científicas–Universidad Autónoma de Madrid, Madrid, Spain

© 2012 Wiley Perio

ABSTRACT Local anesthetics are useful probes of ion channel function and structure.Stereoselective interactions are especially interesting because they can reveal three-dimensionalrelationships between drugs and channels with otherwise identical biophysical and physicochemi-cal properties. Furthermore, stereoselectivity suggests direct and specific receptor-mediated action,and identification of such stereospecific interactions may have important clinical consequences.The fact that drug targets are able to discriminate between the enantiomers present in a racemicdrug is the consequence of the ordered asymmetric macromolecular units that form living cells.However, almost 25% of the drugs used in the clinical practice are racemic mixtures, and their indi-vidual enantiomers frequently differ in both their pharmacodynamic and pharmacokinetic profiles.Moreover, their effects can be similar to or different from the pharmacological effect of the drug andmay contribute to the undesired effects of the drug. In other cases, the pharmacological effectsinduced by the two enantiomers on the molecular target are opposite. In the present manuscript,we will review the stereoselective effects of bupivacaine-like local anesthetics on cardiac sodiumand potassium channels. Chirality 00:000–000, 2012. © 2012 Wiley Periodicals, Inc.

KEY WORDS: ion channels; enantiomers; stereoselectivity; local anesthetics

Contract grant sponsor: This work has been funded by CICYT SAF2010-14916and Red Cooperativa de Enfermedades Cardiovasculares RECAVA FIS RD06/0014/0006 grants to CV. CM and AP held an FPI grant. AC holds a UAMgrant. AM and TG held a JAE-Predoc and a JAE-Doc grants, respectively.*Correspondence to: Carmen Valenzuela, Instituto de Investigaciones Biomédi-cas ’Alberto Sols, Consejo Superior de Investigaciones Científicas–UniversidadAutónoma de Madrid, C/Arturo Duperier 4, 28029 Madrid, Spain.E-mail: cvalenzuela@iib.uam.esReceived for publication 15 November 2011; Accepted 28 February 2012DOI: 10.1002/chir.22051Published online in Wiley Online Library(wileyonlinelibrary.com).

“All artificial bodies and all minerals have superposableimages. Opposed to these are nearly all organic sub-stances which play an important role in plant and animallife. These are asymmetric, and indeed have the kind ofasymmetry in which the image is not superposable withthe object.”(Louis Pasteur, 1860)

Stereoselective interactions between drugs and channelsare especially interesting because they can reveal three-dimensional relationships with otherwise identical biophysicaland physicochemical properties. Furthermore, stereoselectivitysuggests direct and specific receptor-mediated action, andidentification of such stereospecific interactions may haveimportant clinical consequences.1 The fact that drug molecu-lar targets are able to discriminate between the enantiomerspresent in a racemic drug is the consequence of the orderedasymmetric macromolecular units that form living cells.Indeed, it is this ordered asymmetry that gives the macromo-lecules the necessary information to discriminate betweenthe optical isomers of monomeric substrates such as aminoacids and sugars. However, almost 25% of drugs used in theclinical practice are racemic mixtures, whose enantiomersfrequently differ in both their pharmacodynamic and pharma-cokinetic profiles. Moreover, these different effects inducedby one of the enantiomers of a racemic drug may contributeto the undesired effects that can be similar to or different fromthe pharmacological effect of the drug. In other cases, thepharmacological effects induced by the two enantiomers onthe molecular target are opposite.Bupivacaine is an amide-type local anesthetic widely used

for regional anesthesia that exhibits a high cardiotoxicity,

dicals, Inc.

likely due to its ability to depress the intracardiac conductionvelocity and cardiac contractility. These bupivacaine effectshave been related to its blocking properties of Na+ and Ca2+

channels. Moreover, several cases of long QT syndrome(LQTS), associated to polymorphic ventricular arrhythmias(torsades de pointes) have been reported in animal modelsand in humans after its accidental intravascular injection.Although drug-induced LQTS could result from the inhibitionof any voltage-gated K+ current that contributes to ventricularrepolarization, almost all known drugs exhibiting this effectblock IKr that is generated by the activation of Kv11.1 potas-sium channels. In fact, it is widely accepted that acquiredLQTS is due to an excessive Kv11.1 channel blockade. Thus,sensitivity of Kv11.1K+ channels to drugs has become a majorissue in the development of new and safer drugs.

BUPIVACAINE-LIKE LOCAL ANESTHETICSLocal anesthetics block the generation and conduction of

nerve impulses by inhibiting the inward current throughvoltage-gated Na+ channels present in the membrane of nervecells.2–4 Bupivacaine is the racemic form of R(+)- and S(�)bupivacaine (Fig. 1). It is a potent amide local anesthetic

Fig. 1. Chemical structure of the two bupivacaine enantiomers.

VALENZUELA ET AL.

widely used for long-lasting epidural anesthesia.5 However,voltage-gated Na+ channels from other tissues whenexposed to sufficient concentrations of these agents will beblocked. This can explain why the accidental intravascularinjection or the use of high concentrations of local anes-thetics can produce profound systemic effects, especially inthe central nervous and cardiovascular systems.5 However,bupivacaine-like local anesthetics are not selective Na+ chan-nel blockers. In fact, at the same range of concentrationsused in the clinical practice to block the generation andpropagation of nerve action potentials, this type of localanesthetics also block K+ channels.6–8 Moreover, at higherconcentrations, they also block the L-type Ca2+ channels,thus decreasing cardiac contractility and conduction velocitythrough the atrioventricular node.5,9 Bupivacaine-inducedK+ channels block has been considered the molecular mech-anism by which this drug induces a prolongation of the QTcinterval of the electrocardiogram in anesthetized dogs10,11

and human volunteers receiving high doses of this anes-thetic.12 It has been reported that this cardiotoxicity ismostly related to the R(+) enantiomer13,14 likely due to itshigher potency for blocking cardiac sodium and potassiumchannels (Fig. 2(A)).6,7,15

Fig. 2. Panel A: differential effects of 10 mM S(�) and R(+) bupivacaines on cardiapart of this panel shows the amino acids involved in the stereoselective bupivacaine

Chirality DOI 10.1002/chir

STEREOSELECTIVE INTERACTIONS BETWEEN LOCALANESTHETIC-LIKE DRUGS AND NA+ CHANNELS

Sodium channels represent the molecular target of localanesthetics, certain anticonvulsants, and class I antiarrhyth-mic drugs used to decrease sodium channel activity in syn-dromes of hyperexcitability such as cardiac arrhythmias andepileptic seizures.3,16,17 Most of these drugs are weak bases,and several lines of evidence indicate that most of their block-ing effects are due to the binding of their charged form to areceptor site located in the internal mouth of the ion pore.5,16

Several studies using alanine-scanning mutagenesis haveidentified the amino acids F1764 and Y1771 in segment S6at domain IV (DIV-S6) in the subunit of the rat brain Nav1.2channel as important molecular determinants of binding oflocal anesthetics.18 These amino acids are also involved inbinding of class I antiarrhythmic and anticonvulsant drugs,which strongly suggest that they share a common receptorsite.18,19 Because these residues are oriented to the same faceof the DIV-S6 helix, it was suggested that they face to thechannel pore and interact with local anesthetics moleculesthrough hydrophobic or cation–p electron interactions.18

Stereoselective blockade of both cardiac and nerve Na+

channels has been better characterized with bupiva-caine.6,20,21 Blockade of the rested or open states of unmodi-fied Na+ channels induced by these therapeutic agents hasbeen reported to be nonstereoselective, whereas a weakstereoselective blockade of the inactivated state of these ionchannels has been observed for bupivacaine. R(+)bupivacainewas about 1.6 times more efficient in blocking Na+ channelsthan the other enantiomer (Fig. 2(A)).6,22

Different amino acid substitutions at position N434 (DI-S6)led to an increase or to a decrease in bupivacaine potency thatdepended on the physicochemical characteristics of theamino acid introduced. Thus, the potency of bupivacaineenantiomers was increased in N434 mutant Nav1.4 channels

c Na+ currents. Panel B: topology of the subunits of the Na+ channel. The rightblockade.

STEREOSELECTIVE DRUG–CHANNEL INTERACTIONS

in which the native asparagine was substituted by aminoacids containing an aromatic group (phenylalanine, trypto-phan, or tyrosine), a polar group (cysteine), or a negativecharge (aspartic) and decreased in a mutation containing apositive charge (lysine).20 In Nav1.5, amino acid residues inpositions F1760, N1765, and N406 may contribute to bindingof bupivacaine enantiomers, whereas the role of Y1767remains unclear (Fig. 2(B)).21 All these results suggest thatsegments DI-S6 and DIV-S6 align adjacently along the poreof the Na+ channel, forming a domain-interface site for bind-ing local anesthetics.23

As stated earlier, stereoselective interactions may haveimportant clinical consequences.1,13 Indeed, weakly stereose-lective interactions such as those described between localanesthetics and Na+ channels can lead to dramatic differencesin cardiac and central nervous toxicity.13,14 A good representa-tive example is bupivacaine with R(+)bupivacaine being only1.6 times more potent in blocking cardiac Na+ channels thanS(�)bupivacaine but with significant differences in cardiotoxi-city; being the S(�)- enantiomer significantly safer than theR(+)- one.24,25Moreover, S(�)bupivacaine is not only less cardi-otoxic than the racemic mixture, but its local anesthetic actionis longer than that of the racemate. These lines of evidencetogether with other findings on other cardiac ion channels haveled to the marketing of S(�)bupivacaine as a less cardiotoxicalternative to racemic bupivacaine and even to ropivacaine(the S(�)- enantiomer of N-propyl-pipecoloxilidide).7,24,26

STEREOSELECTIVE INTERACTIONS BETWEEN LOCALANESTHETICS AND K+ CHANNELS

Unlike Na+ channels, whose activation induces a transientinward current with similar biophysical properties amongdifferent isoforms, voltage-gated K+ channels represent themore diverse family of ion channels. In fact, it is only incardiac tissue where more than 12 K+ currents have beendescribed.27–30K+ currents can be classified in transientoutward, delayed, and inward rectifiers. Voltage-gated K+

channels (including Kv1.5, Kv2.1, Kv4.3, Kv7.1, and Kv11.1)represent the molecular targets of class III antiarrhythmicdrugs, which act by prolonging the cardiac action potential

Fig. 3. Panel A: original Kv1.5 current records obtained in the absence and in tinduced a fast initial decline of the current that indicates an open channel block mechPanel B: concentration–response curve for the Kv1.5 channels blocking effects prod

and, as a consequence, the refractory period.17 However, mostclasses I and IV antiarrhythmic drugs (such as quinidine,verapamil, and nifedipine) also bind to one or more K+

channels.31–39K+ channels are widely expressed in all livingcells, and depending on their location, they play very differentphysiological roles, from excitability to secretory functions. Infact, the involvement of Kv1.3 channels in the immune responsehas even been described following the activation of T-cells.40–42

These channels may thus become molecular targets for thedevelopment of candidate immunosuppressant drugs.41,43

Like Na+ channels, which represent the molecular site ofaction of local anesthetic-like drugs but are also sensitive tovery different compounds, K+ channels are effectivelyblocked by a highly heterogeneous range of agents whichdo not belong to a unique pharmacological family. As a directconsequence of the differences between K+ channels (andcurrents), each type of K+ channel exhibits a higher affinityfor a given drug structure.The ultrarapid activating delayed rectifier K+ current (IKur)

results from the activation of Kv1.5 channels, which are abun-dantly expressed in human atria.44 These K+ channels aresensitive to most class I antiarrhythmic drugs and localanesthetics.31,35,37–39 More importantly, the concentrationsneeded to inhibit these channels are in the same range asthose required to block Na+ channels.6,7,15,31,45–48 All thesecompounds, as well as internal tetraethylammonium (TEA),are open channel blockers that bind to a common internal re-ceptor site at Kv1.5 channels without affinity for the rested orinactivated states of the channel.49 Mutagenesis studies haverevealed that quinidine binds to a receptor site formed byamino acids located in the S6 segment, in particular by T507and V514, competing with tetrapenthylammonium (a hydro-phobic TEA derivative)50,51 in such a way that its binding toKv1.5 channels requires one polar interaction at position507 (T507) and a hydrophobic one at position 514 (V514).Stereoselective interactions between drugs and Kv1.5 chan-

nels have been extensively studied with bupivacaine-likeenantiomers.7,26,46–48,52 Bupivacaine blockade of Kv1.5 chan-nels is stereoselective with R(+)bupivacaine being sevenfoldmore potent than its enantiomer (Fig. 3).7 Bupivacaineblocked the open state of Kv1.5 channels in a state-dependent,

he presence of either S(�) or R(+) bupivacaine at 20 mM. Both enantiomersanism, being the steady-state block higher in the presence of R(+)bupivacaine.uced by bupivacaine enantiomers.

Chirality DOI 10.1002/chir

VALENZUELA ET AL.

time-dependent, and voltage-dependent manner similar toquinidine. Mutagenesis studies have identified the amino acidsT507, L510, and V514 located at the S6 segment as moleculardeterminants of stereoselective blockade induced by bupiva-caine and Kv1.5 channels.52 In fact, stereoselective interactionsbetween bupivacaine and Kv1.5 channels require a polar inter-action at position 507 and two hydrophobic interactions atpositions 510 and 514 (Fig. 4(A)). When the threonine presentin wild-type channels at position 507 was substituted by hydro-phobic amino acids (alanine, valine, or isoleucine), stereoselec-tive blockade was completely abolished, whereas a substitutionthat maintained the polar character of the native threonine(T507S) altered the potency of both enantiomers but preservedstereoselectivity,52 thus indicating that stereoselective block-ade of wild-type Kv1.5 channels induced by bupivacaineinvolves a specific interaction between the -OH moiety of theresidue at position 507 and the tertiary amine group of thedrug. Replacement of L510 or V514 by a more polar residue(methionine) abolished stereoselectivity. In contrast, replace-ment of V514 by an alanine that preserved the hydrophobicnature of the amino acid maintained stereoselectivity.52

Interestingly, blockade of Kv2.1 and Kv4.3 channels that carrya valine at the T507 equivalent position is not stereoselective,thus indicating that bupivacaine stereoselectivity displays sub-family selectivity (Fig. 4).47,52

Mutation of L510 was previously reported to decrease chan-nel block induced by quinidine, bupivacaine, and tetraethy-lammonium.51,52 Although leucine at position 510 is predictedto face away from the central cavity, it has been demonstratedthat mutation of this residue to alanine prevents the interac-tion with Kvb1.3 and thus its ability to induce N-type inactiva-tion and alter Kv1.5 channel gating.53 If L510 determinesbupivacaine stereoselectivity and is also required for Kvb1.3-induced inactivation, then it could be hypothesized thatthe inactivating ball of the b subunit and bupivacaine maycompete for the same internal receptor site, as it has been

Fig. 4. Panel A: amino acid sequence of Kv1.1, Kv1.5, Kv2.1, Kv3.1, and Kv4.3. Ishown. Panel B: effects of R(+) and S(�)bupivacaine on Kv2.1 and Kv4.3 that carrtwo channels is not stereoselective.

Chirality DOI 10.1002/chir

suggested for quaternary ammonium derivatives.54 It hasbeen demonstrated that bupivacaine and Kvb1.3 competefor a binding site at the inner cavity of the Kv1.5 channel,thus suggesting an overlapping binding site for bupivacaineand Kvb1.3 subunit.55

Which are the structural determinants of local anestheticpotency and stereoselective blockade of Kv1.5 channels?The potency of blockade of Kv1.5 channels induced byalkyl-TEA derivatives in Shaker channels is related to thelength of the side chain.56,57 Because bupivacaine-like localanesthetics can be considered hydrophobic TEA derivatives,potency of blockade and degree of stereoselectivity of differ-ent bupivacaine-like local anesthetics (bupivacaine, IQB-9302,ropivacaine, and mepivacaine) on Kv1.5 channels were ana-lyzed.46,48 These drugs exhibit the same chemical structure,and they only differ in the length of the alkyl substituentof the tertiary nitrogen (position 1), which is a butyl, cycle-propyl-methyl, propyl, or methyl group, respectively (Fig. 5).These studies demonstrate that the length of the N-substituentat position 1 in bupivacaine-related local anesthetics, such as inalkyl TEA-derivatives, determines the potency of these drugsto block Kv1.5 channels. The observed decrease in the dissoci-ation rate constants with the number of -CH2 groups in the sidechain could be explained if the stability of the drug–channelcomplex is mainly due to a hydrophobic interaction betweenthe alkyl chain of the local anesthetic and some amino acidresidue of the channel protein (Fig. 6(A)).46,48 Moreover, ithas been shown that the length of the alkyl substituent alsodetermines the degree of stereoselective blockade of Kv1.5channels in such a way that drugs with longer side chainsdisplay a higher potency and also a higher degree of stereose-lectivity (Fig. 6(B)). The faster association rate constantsobserved for R enantiomers suggest that stereoselectivity ismainly determined by the conformation of these enantiomersthat better permits a hydrophobic interaction between theN-substituent and the channel at the S6 segment level. Further

n red, residues involved in stereoselective bupivacaine blockade of Kv1.5 arey a valine at the Kv1.5 T507 equivalent position. Note that blockade of these

Fig. 5. Chemical structure of bupivacaine-like local anesthetics.

STEREOSELECTIVE DRUG–CHANNEL INTERACTIONS

studies using site-directed Kv1.5 mutant channels are neededto determine the amino acids involved in this interaction.Unlike Na+ channels, Kv1.5 channels are tetrameric supramo-lecular ensembles. This fact can modify the stereoselectivebinding of bupivacaine-like local anesthetics, and indeed, ithas been demonstrated that bupivacaine and the inactivatingparticle of the Kvb1.3 subunit share an overlapping binding siteat the inner part of the pore of Kv1.5 channel.55

The rapid activating delayed rectifier K+ current (IKr)results from the activation of Kv11.1 channels.58,59 However,the contribution of KCNE2 to the native IKr is an issue underdebate.60,61 This current is characterized by its inward rectifi-cation, its sensitivity to micromolar concentrations of La3+,and by its specific blockade caused by methanesulfonanilide

Fig. 6. Panel A: relation between KD (left) and dissociation rate constants (k�1) (bupivacaine, ropivacaine, and mepivacaine. Note that KD and k�1 decrease when thpotency observed in the presence of bupivacaine enantiomers than in the presencePanelB: Relation among the degree of stereoselective block (), the association rate constaThe degree of stereoselective blockade increases when the number of -CH2 groups is higenantiomers did not changewith the number of -CH2 groups, suggesting that the degreeis reflected by a faster k+1 value.

drugs such as E4031 and dofetilide.58,59,62 These drugs bindto Kv11.1 channels near the internal mouth of the channelpore; a serine at the S5-S6 linker (S620) as well as an intactC-type inactivation are crucial for binding dofetilide-like drugsat Kv11.1 channels.63–65 In fact, the mutation S620T thatabolishes high-affinity dofetilide blockade of Kv11.1 channelshas been correlated with a loss of C-type inactivation. Becauseit is widely accepted that acquired LQTS is due to an excessiveKv11.1 channel blockade, the knowledge of the possiblestereoselective blockade of Kv11.1 channels by bupivacainerepresents a very important issue to know the safer bupiva-caine enantiomer. Gonzalez et al. demonstrated that blockadeof Kv11.1 channels was also, such as Kv1.5 and Nav1.5 chan-nels, stereoselective.66 However, and in contrast to Kv1.5 orNav1.5 channels, S(�)bupivacaine was twofold more potentthan R(+)bupivacaine to block Kv11.1 channels (Fig. 7).66 Bothracemic bupivacaine, S(�) and R(+), bind to the inactivatedstate of Kv11.1 channels with much less affinity for the openand the rested states of the channel.66

Which are the molecular determinants of Kv11.1 stereose-lective bupivacaine block? Stereoselective interactions be-tween bupivacaine and Kv11.1 channels require interactionsat positions Y652 and F656. When the tyrosine present inwild-type channels at position 652 was substituted by ala-nine, stereoselective blockade was completely abolished,and similar effects were observed after replacing phenylala-nine at position 656 by an alanine,67 thus indicating thatstereoselective blockade of wild-type Kv11.1 channels inducedby bupivacaine involves specific interactions between the drugand these two residues at the S6 segment. These results couldexplain recent studies in which the incidence of ventriculararrhythmias was similar with these three anesthetics.68

However, caution should be exerted before extrapolatingthe present results to the clinics because many factors(temperature, b subunits, etc.) are not similar under theseexperimental conditions.66

right) and number of-CH2 groups in the molecule for the two enantiomers ofe number of -CH2 groups increase in the molecule, indicating that the higherof mepivacaine enantiomers is due to a more stable drug–channel interaction.nt (k+1), and the number of-CH2 groups present in theR(+)- and S(�)enantiomers.her, as do the k+1 values for these enantiomers, whereas the k+1 values for theS(�)of stereoselective blockade is determined by amore favored conformation, which

Chirality DOI 10.1002/chir

Fig. 7. Original Kv11.1 current records obtained in the absence and in thepresence of either S(�) or R(+)bupivacaine at 20mM.

VALENZUELA ET AL.

CONCLUSIONSLocal anesthetics are state-dependent pore blockers of Na+

channels. Analysis of their receptor-binding site by site-directedmutagenesis has identified three amino acid residues intransmembrane segment DIV-S6 and another one in segmentDI-S6, which are required for high-affinity binding and effectivefrequency-dependent blockade of the channel.18,21 Therefore, allmajor drugs that blockNa+ andK+ channels in a state-dependentmanner interact with the IVS6 segments of these channels.This is a remarkable confluence of binding sites for drugs witha wide range of structures. Further elucidation of the detailedmechanisms of interaction of these drugs with their receptorsites may reveal the common way through which they affectNa+ and K+ channel functions.

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Chirality DOI 10.1002/chir