lipophilic and stereo specific interactions of amino-amide

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Anesthesiology 2008; 109:895904 Copyright 2008, the American Society of Anesthesiologists, Inc. Lippincott Williams & Wilkins, Inc.

Lipophilic and Stereospecific Interactions of Amino-amideLocal Anesthetics with Human Kv1.1 Channels

Mark A. Punke, M.D.,* Patrick Friederich, M.D.

Background: This studys aim was to investigate the interac-

tion of amino-amide local anesthetics with human Kv1.1 potas-sium channels. These channels were chosen because of their

proven physiologic role. By using a homolog series of local

anesthetics with different lengths of the N-substituent, it was

intended to elucidate the role of lipophilic interactions with

Kv1.1. The use of stereoisomers allowed testing of the role of

polar drug actions.Methods: Human Kv1.1 channels were measured with the

patch clamp technique. Concentrationresponse data were de-

scribed by Hill functions. Gating changes were described by

Boltzmann functions.

Results: Inhibition of Kv1.1 channels by dextrobupivacaine,

bupivacaine, levobupivacaine, dextroropivacaine, levoropiva-

caine, and mepivacaine was concentration dependent, reversible,

stereoselective, voltage dependent, and frequency independent.

The IC50 values were (mean SEM) 41 3M (n 20), 56 3 M

(n 26), 76 8 M (n 24), 135 29 M (n 23), 313 32 M

(n 25), and 1,451 351M (n 23), respectively. The midpoint

of current activation was shifted into the hyperpolarizing direc-

tion. The inhibitory potency as well as the potency to induce

gating changes correlated with the number of CH2 groups in the

side chain of the drugs (r> 0.9, P < 0.05).

Conclusions: Kv1.1 channels constitute an important bio-

physical model for elucidating molecular mechanisms underly-

ing local anesthetic drug effects. Inhibition likely results from

an open statedependent blocking mechanism. Interaction of

local anesthetics with the ion channel protein is determined by

lipophilic drug properties.

LOCAL anesthetics inhibit the conductance of painfulstimuli by blocking action potentials through the inhibi-tion of voltage-gated ion channels.1 They exhibit theireffects not only by binding to sodium channels15 butalso by interacting with other ion channels, such as

voltage-dependent potassium channels.614 In contrastto the effects on sodium channels, the molecular inter-action of local anesthetics with voltage-dependent potas-sium channels is less well defined.6,9,1116

Voltage-dependent potassium channels constitute aheterologous group of membrane-bound proteins with asignificant physiologic and pathophysiologic role.17

Kv1.1 potassium channels are abundantly expressed inthe central nervous system, including myelinated axons,synaptic terminals, and dorsal root ganglia.9,18,19 Kv1.1

channels are important for various cellular functions,

such as action potential generation, repolarization of themembrane potential, spike frequency adaptation, andneurotransmitter release.20 Inhibition of Kv1.1 channelsin dorsal root ganglia neurons offers a possible approachfor the treatment of chronic pain.9 Experimental evi-dence shows that inhibition of Kv1.1 together with en-dogenous sodium channels may contribute to conduc-tion block.9On the other hand, mutated and functionallyimpaired Kv1.1 channels are associated with epilepsy,21

and Kv1.1 knockout mice display an epileptic pheno-type.20 Interaction of amino-amide local anesthetics withKv1.1 channels may, therefore, contribute not only to

analgesic action9

but also to these drugs unwanted neu-rotoxic side effects, such as seizures.8,16

Local anesthetics block potassium channels preferentiallyfrom the open state by interacting with the internal ionchannel pore and the S6 transmembrane segment.6,22,23

Differences regarding the pharmacologic effects of localanesthetics on potassium channels exist8,16,23 and likelyresult from subtype and even species differences in chan-nel structure.23 Information on the effects of local anesthet-ics on Kv1.1 is limited to the interaction of bupivacaine23

and lidocaine with rat Kv1.1 channels.24 It is currentlyunknown whether the effects of local anesthetics on Kv1.1

channels result from lipophilic interactions, and it is un-known to what extent these effects may be influenced bystereospecific properties of the drugs.

The aim of the study was to systematically investigate thepharmacologic effects on human Kv1.1 channels of a ho-molog series of amino-amide local anesthetics with differ-ent lengths of the N-substituent. Kv1.1 channels were cho-sen because of their physiologic significance. The homologseries of amino-amide local anesthetics was chosen be-cause the length of the N-substituent determines the li-pophilicity of these agents and, therefore, allows elucida-tion of the role and the structural requirements for

lipophilic interactions of local anesthetics with Kv1.1 chan-nels. The use of stereoisomers allowed determination ofthe role of polar interactions of amino-amide local anesthet-ics and Kv1.1 channels. Taken together, this information isan important prerequisite for understanding the molecularmechanisms contributing to local anesthetic drug effects.

Materials and Methods

Cell Culture

Human Kv1.1 channels (accession No. L02750) werecloned by HindIII and KpnI into the eucaryotic expres-

sion vector pcDNA3 (Invitrogen, Karlsruhe, Germany)

* Staff Anesthesiologist, Department of Anesthesiology, University MedicalCenter Hamburg. Chairman, Department of Anesthesiology, Critical CareMedicine, and Pain Therapy, Klinikum Bogenhausen, Munich, Germany.

Received from the Department of Anesthesiology, University Medical CenterHamburg-Eppendorf, Hamburg, Germany. Submitted for publication November13, 2007. Accepted for publication July 16, 2008. Supported by a Research Award from the European Society of Anaesthesiologists, Brussels, Belgium.

Address correspondence to Dr. Friederich: Department of Anesthesiology, CriticalCare Medicine, and Pain Therapy, Klinikum Bogenhausen, Englschalkinger Strae77, 81925 Munich, Germany. [email protected]. Informationonpurchasing reprints may be found at www.anesthesiology.org or on the mastheadpage atthe beginning ofthisissue.ANESTHESIOLOGYs articles aremade freelyaccessibleto all readers, for personal use only, 6 months from the cover date of the issue.

Anesthesiology, V 109, No 5, Nov 2008 895


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and stably expressed in human embryonic kidney 293cells. Human embryonic kidney 293 cells were culturedin 50-ml flasks (NUNC, Roskilde, Denmark) at 37C inDulbecco medium (GIBCO, Invitrogen, Carlsbad, CA)

with 10% fetal calf serum (Biochrom, Berlin, Germany),100 U/l penicillin, and 100 mg/ml streptomycin (GIBCO)

in a humidified atmosphere (5% CO2). The cells weregrown as nonconfluent monolayers and subcultured inmonodishes (35-mm diameter; NUNC) at densities be-tween 2 and 3 104 cells per dish before the electro-physiologic experiments.

Patch Clamp Recordings

Whole cell currents were measured with the voltageclamp method of the patch clamp technique25 using anEPC-9 amplifier (HEKA Elektronik, Lambrecht, Germany)and Pulse software version 8.53 (HEKA Elektronik). Thecurrents were filtered at 1 kHz, and series resistance was

actively compensated by at least 80%. A p/4 leak sub-traction protocol was used in the study, with the excep-tion of the experiments for frequency dependence. Thepatch electrodes were fabricated from borosilicate glasscapillary tubes with filament (World Precision Instru-ments, Saratoga, FL) using a Sutter P-97 puller (SutterInstrument Company, Novato, CA). The pipettes had aresistance of 24 M and were filled with a solutioncontaining the following electrolyte concentrations: 160mM KCl, 0.5 mM MgCl2, 10 mM HEPES, and 2 mM Na2ATP,

with a pH of 7.25 adjusted with KOH. The extracellularsolution consisted of 135 mM NaCl, 5 mM KCl, 2 mM

CaCl2, 2 mM

MgCl2, 5 mM

HEPES, 10 mM

sucrose, and0.01 mg/ml phenol red, with a pH of 7.4 adjusted withNaOH. All experiments were performed at room temper-ature. The local anesthetics were dissolved in the wateryextracellular solution, yielding stock solutions of 1 mM

for dextrobupivacaine, bupivacaine, levobupivacaine,and dextroropivacaine. For levoropivacaine, a stock so-lution of 3 mM was prepared, and for mepivacaine, weused a stock solution of 10 mM (all chemicals werepurchased from Sigma, Deissenhofen, Germany, exceptfor levobupivacaine, dextrobupivacaine, and dextroropi-

vacaine, which were gifts from AstraZeneca, Sodertalje,Sweden). The drugs were applied on the cells by aperfusion system driven by hydrostatic pressure withTeflon tubing (N Research Europe, Guemlingen, Switzer-land). The inflow of the perfusion system did not havesignificant influence of the currents.

Stimulation Protocols and Data Analysis

The holding potential during all experiments was 80mV. The currentvoltage relation was established bydepolarizing the cell membrane in steps of 10 mV for100 ms between the membrane potentials 70 and60mV. To characterize the concentration-dependent ef-fects of the local anesthetics, outward currents of Kv1.1

channels were measured at 40 mV at the end of the

test potential. The inhibition of the outward currents bylocal anesthetic agents was determined as the inhibitionof the maximum current and calculated by the followingformula: inhibition 1 (Idrug/((Icontrol Iwashout)/2)).The ratio of the maximum current (I) under influence ofthe local anesthetic agents and the mean of maximum

current under control and washout conditions was sub-tracted from 1. The data of the concentrationresponsecurves were mathematically described by Hill functions:f 1/{1 (IC50/c)

h}, where IC50 is the concentration ofthe half-maximal inhibition, c is the drug concentration,and h is the Hill coefficient, using Kaleidagraph software(Synergy-Software, Reading, PA). SEs of calculated Hillparameters were used as defined by Kaleidagraph. The

whole cell conductance was calculated using the follow-ing formula: Gmax Imax/(Vm EK ), where Imax is themaximum current of each test potential, Vm is the mem-brane potential, and EK is the Nernst potential for potas-

sium. The conductancevoltage relation was mathemat-ically described by a Boltzmann equation: I Imax/{1 exp((Vmid Vm)/k)}, where Vmid is the voltage of half-maximal activation, Vm is the membrane potential, and kis the slope factor. The fitting procedure was performed

with Kaleidagraph software. Frequency dependence ofKv1.1 channels was analyzed with repetitive 30-ms pulsesto40 mV with three different interpulse durations (100,300, and 1,000 ms). The remaining current under theinfluence of the local anesthetic agents in the steady statephase at the end of the test potential was normalized tocontrol values. The normalized current values were plotted

against a time axis according to the different interpulsedurations. Time constants of current inactivation were de-termined by fitting current decay with a monoexponentialfunction using Pulsefit software version 8.53.

Statistical Analysis

Statistical significance was tested using analysis of vari-ance and the TukeyKramer multiple comparisons test(Graph Pad; Prism, San Diego, CA) or two-sided pairedand unpaired Student t test as appropriate (Excel; Mi-crosoft, Redmond, WA). Data points are given as meanSD unless stated otherwise; n values indicate the number

of experiments.

Results

Original current recordings of Kv1.1 channels exhib-ited typical delayed rectifier currents devoid of fast inac-tivation (figs. 1AF). The currents activated at membranepotentials more positive than 40 mV, with a midpointof current activation (Vmid) of26 1 mV (n 5) anda slope factor of 7.1 0.5 (n 5).

The inhibitory effects of dextrobupivacaine, racemic bu-pivacaine, levobupivacaine, dextroropivacaine, levoropiva-

caine, and racemic mepivacaine were analyzed at a mem-

896 M. A. PUNKE AND P. FRIEDERICH

Anesthesiology, V 109, No 5, Nov 2008


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brane potential of40 mV and quantified as the reduction

of the steady state outward current at the end of each test

potential. All local anesthetics inhibited currents through

Kv1.1 channels (figs. 1AF). Inhibition of Kv1.1 channels

was concentration dependent, stereoselective, and revers-ible. The concentrationresponse data for inhibition of

Kv1.1 channels were mathematically described by Hill

equations (fig. 2). Dextrobupivacaine inhibited Kv1.1 chan-nels with the highest potency. The IC50 value was 41 3M, and the corresponding Hill coefficient was 1.0 0.1

(mean SEM; n 20). The sensitivity of Kv1.1 channels

for the different local anesthetic agents decreased in the

following order: dextrobupivacaine bupivacaine

levobupivacaine dextroropivacaine levoropivacaine

mepivacaine (fig. 2; for overview, see table 1). Stereose-

lectivity of the effects was analyzed by comparing thecurrent inhibition of Kv1.1 channels by dextrobupiva-

caine and levobupivacaine, as well as by dextroropiva-

caine and levoropivacaine at a membrane potential of

40 mV. Both dextroisomers inhibited Kv1.1 channels

more potently than the respective levoisomers did. Fordextrobupivacaine (30 M) and levobupivacaine (30 M),Kv1.1 channel inhibition was 74 9% (n 5) versus 59

1 nA20 ms

bupivacaine(100 M)

dextrobupivacaine(100 M)

1 nA

20 ms

levobupivacaine(100 M)

20 ms

1 nA

dextroropivacaine(300 M)

1 nA

20 ms

levoropivacaine(300 M)

1 nA

20 ms

mepivacaine(1000 M)

1 nA

20 ms

A

B

D

C

E

F

Fig. 1. Effects of differentamino-amide local anesthetics on human Kv1.1 stablyexpressed in human embryonickidney (HEK) 293 cells.Shownare original current traces of Kv1.1 channels evoked by depolarizing pulses ranging from80 to 60 mV in 10-mV steps, demonstratinginhibition of Kv1.1 channels by dextrobupivacaine (A), levobupivacaine (B), racemic bupivacaine (C), dextroropivacaine (D), levoropivacaine(E), and racemic mepivacaine (F). Shown are current traces under control conditions and under the influence of the different drugs.

Inhibition

0

0.2

0.4

0.6

0.8

1.0

10 100 1000 10000

[M]

dextrobupivacaine

bupivacaine

levobupivacaine

dextroropivacaine

levororopivacaine

mepivacaine

Fig. 2. Current inhibition was quantified as the reduction of thesteady state current at a potential of 40 mV. The concentra-tionresponse data were described by Hill functions. For over-view, see table 1.

897LOCAL ANESTHETIC AND Kv1.1 CHANNELS



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5% (n 6) (P 0.05), and for dextroropivacaine (100 M)and levoropivacaine (100 M ), it was 38 3% (n 6)versus 17 5% (n 5) (P 0.05). All local anestheticagents shifted the voltage dependence of Kv1.1 channelactivation into the hyperpolarizing direction (figs. 3AF).The shift of the activationmidpoint (Vmid) into the hyper-polarizing direction by all local anesthetic agents signifi-

cantly increased with concentration (figs. 4AF). The ef-

fects were dependent on the lipophilic properties of thedrugs.

For the analysis of the voltage dependence of inhibi-tion, the size of the outward current at the end of eachtest potential (Imax ) under the influence of the differentlocal anesthetic agents was related to the mean of con-

trol and washout current. The extent of inhibition wasplotted against the membrane potential. All local anes-thetic agents inhibited Kv1.1 channels in a voltage-de-pendent manner (figs. 5AF; analysis of variance; P

0.05). Inhibition of Kv1.1 channels was not frequencydependent, because the stimulation frequency of thedepolarizing pulse did not significantly influence localanestheticinduced inhibition (figs. 6AF).

Apart from mepivacaine, all local anesthetics inducedinactivation-like behavior of Kv1.1 currents character-ized by an acceleration of macroscopic current decline(figs. 1AF and 7A). Comparing inhibition of the peak

current at the beginning of the depolarizing test pulse

Table 1. Parameters Derived from the Fit of the

ConcentrationResponse Data

SubstanceIC50 Value,

Mean SEM, mHill Coefficient,

Mean SEM n

Dextrobupivacaine 41 3 1.0 0.1 20

Bupivacaine 56 3 1.4 0.1 26

Levobupivacaine 76 8 1.2 0.1 24Dextroropivacaine 135 29 1.2 0.2 23

Levoropivacaine 313 32 1.3 0.1 25

Mepivacaine 1,451 351 1.2 0.2 23

bupivacaine (30 M)dextrobupivacaine (30 M) levobupivacaine (30 M)

dextroropivacaine (300 M) levoropivacaine (300 M) mepivacaine (1000 M)

n = 5 n = 4

n = 6n = 6n = 5

n = 50

0.2

0.4

0.6

0.8

1.0

1.2

0

0.2

0.4

0.6

0.8

1.0

1.2

0

0.2

0.4

0.6

0.8

1.0

1.2

0

0.2

0.4

0.6

0.8

1.0

1.2

0

0.2

0.4

0.6

0.8

1.0

1.2

0

0.2

0.4

0.6

0.8

1.0

1.2

-20 0 20 40

[mV]

-60 -40 -20 0 20 40

[mV]

-60 -40 -20 0 20 40

[mV]

-60 -40

-20 0 20 40

[mV]

-60 -40-20 0 20 40

[mV]

-60 -40-20 0 20 40

[mV]

-60 -40

G/Gmax G/Gmax G/Gmax

G/Gmax G/Gmax G/Gmax

dextrobupivacaine

mean of control

+ washout

bupivacaine

mean of control

+ washout

levobupivacaine

mean of control

+ washout

dextroropivacaine

mean of control

+ washout

mean of control

+ washout

levoropivacaine mepivacaine

mean of control

+ washout

A B C

D E F

Fig. 3. All local anesthetic agents shifted the voltage dependence of Kv1.1 channel activation into the hyperpolarizing direction.(AC) At a concentration of 30 M, dextrobupivacaine, bupivacaine, and levobupivacaine shifted the voltage of half-maximalactivation (Vmid) by4.5 0.8 mV (n 5), 5.1 1.6 mV (n 5), and5.3 2.2 mV (n 6) into the hyperpolarizing directioncompared with control and washout, respectively. (D and E) At a concentration of 300 M, dextroropivacaine and levoropivacaineshifted Vmidby6.9 2.3 mV(n 5) and4.8 0.7 mV(n 6), respectively. (F) At a concentration of 1,000M, mepivacaine shiftedVmid by4.9 0.5 mV (n 6) to more negative potentials. G conductance; Gmax maximal conductance.




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and inhibition of the outward current at the end of eachtest pulse (fig. 7B) revealed that apart from mepivacaine,inhibition of the peak currents by the local anesthetics

was significantly smaller than inhibition of the steadystate currents (fig. 7B). The time constants of inactiva-tion were determined at concentrations close to the IC50

A B C

D E F

3000 10000

[M]

300 10000

-5

-10

-15

-20

0

-5

-10

-15

0

-5

-10

-15

0

-5

-10

-15

0

-5

-10

-15

0

-5

-10

-15

300 1000

[M]

30 100 300 1000

[M]

30 100

100 300

[M]

10 30 100 300

[M]

10 30 100 300

[M]

10 30

bupivacainedextrobupivacaine levobupivacaine

dextroropivacaine levoropivacaine mepivacaine

Vmid [mV] Vmid [mV] Vmid [mV]

Vmid [mV] Vmid [mV] Vmid [mV]

n = 18 n = 21

n = 23n =22n = 21

n = 21

**

n.s.

n.s.

* n.s.n.s.

*n.s.

n.s. *

*

*

*

n.s.

n.s.

n.s.

n.s.

n.s.

n.s.n.s.

n.s.

*

*

**

*

* **

Fig. 4. The shift of voltage of half-maximal activation (Vmid) into the hyperpolarizing direction by all local anesthetic agents significantlyincreased with concentration (tested withanalysis of variance;P< 0.05). (A) Dextrobupivacaine, (B) levobupivacaine, (C) racemic bupivacaine,(D) dextroropivacaine, (E) levoropivacaine, and (F) racemic mepivacaine. * Significant difference. n.s. no significant difference.

-20 0 20 40 60

[mV]

-20 0 20 40 60

[mV]

-20 0 20 40 60

[mV]

-20 0 20 40 60

[mV]

-20 0 20 40 60

[mV]

-20 0 20 40 60

[mV]

0

0.2

0.4

0.6

0.8

1.0

0

0.2

0.4

0.6

0.8

1.0

0

0.2

0.4

0.6

0.8

1.0

0

0.2

0.4

0.6

0.8

1.0

0

0.2

0.4

0.6

0.8

1.0

0

0.2

0.4

0.6

0.8

1.0

n = 5 n = 5 n = 6

n = 6n = 7n = 5



Inhibition Inhibition Inhibition

InhibitionInhibitionInhibition

A B C

D E F

Fig. 5. All local anesthetic agents inhib-ited Kv1.1 channels in a voltage-depen-dent manner (tested with analysis of vari-ance; P < 0.05). (AC) At a concentrationof 100 M, dextrobupivacaine inhibitedthe maximum current (Imax) at30 mVwith 58 11% versus 76 8% at60 mV(n 5 paired experiments), whereas bu-pivacaine inhibited Imax at30 mV with45 3% versus 71 6% at60 mV (n5 paired experiments), and at the sameconcentration, levobupivacaine inhibitedImax at30 mV with 39 8% versus 61 4% at60 mV (n 6 paired experiments)(P < 0.05). (D and E) Dextroropivacaineand levoropivacaine (300 M) inhibitedImax at30 mV with 59 6% versus 74 4% at60 mV (n 5 paired experiments)and with 29 11% at30 mVversus 537%at60 mV(n 7 paired experiments)(P < 0.05). (F) Finally, mepivacaine in-hibited Imax at 30 mV with 24 13%versus 66 4% at60 mV (n 6 pairedexperiments) (P < 0.05).

899LOCAL ANESTHETIC AND Kv1.1 CHANNELS



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value of the respective drug by fitting a monoexponen-tial function to the declining current at a membranepotential of 60 mV. Both dextroisomers induced afaster current decline than the respective levoisomersdid. Dextrobupivacaine (30 M) and levobupivacaine (30M) inactivated Kv1.1 channels with time constants of3.3 1.1 ms (n 6) and 9.3 0.4 ms (n 5),respectively (P 0.05). Racemic bupivacaine (30 M)

yielded a time constant of 7.1 2.2 ms (n 6) (fig. 7C).The time constants for dextroropivacaine (100 M) andlevoropivacaine (100 M) were 4.5 1.1 ms (n 5) and37.9 5.2 ms (n 5), respectively (P 0.05).

Lipophilicity of the different local anesthetic agents26,27

correlated significantly with the number of CH2 groupsin the side chain (y 1.63 0.01x; r 0.90, P 0.05).The IC50 values for Kv1.1 channel inhibition and theshift of Vmid were correlated with the length of theN-substituent of the homolog series of the local anesthet-ics (figs. 8A and B). Both the inhibitory potency and

the potency to shift of the midpoint of current activa-

tion significantly correlated with the number of CH2groups (r 0.96, P 0.05; and r 0.94,

P 0.05, respectively).

Discussion

This study established the effects of different amino-amide local anesthetics on human Kv1.1 channels. Thebiophysical properties of Kv1.1 wild-type channels usedin our study were in accord with biophysical propertiesof these channels reported in previous studies.23,28

Kv1.1 channels exhibited fast activating currents devoidof fast inactivation. The effects of racemic mixtures ofbupivacaine and mepivacaine as well as the R( ) andthe S() enantiomers of bupivacaine and ropivacaine onKv1.1 channels were analyzed. All local anestheticagents studied in this investigation inhibited Kv1.1 cur-rents in a concentration-dependent, reversible, stereose-

lective, voltage-dependent, and frequency-independent

n = 4 n = 4 n = 4

0 5 10 15 20 25 0 5 10 15 20 25 0 5 10 15 20 25

n = 5

0 5 10 15 20 25

0

0.2

0.4

0.6

0.8

1.0

1.2

0

0.2

0.4

0.6

0.8

1.0

1.2

0

0.2

0.4

0.6

0.8

1.0

1.2

0

0.2

0.4

0.6

0.8

1.0

1.2

0 5 10 15 20 25

n = 40

0.2

0.4

0.6

0.8

1.0

1.2



0 5 10 15 20 25

n = 50

0.2

0.4

0.6

0.8

1.0

1.2

Remaining current Remaining current Remaining current

Remaining current Remaining current Remaining current

time [s] time [s] time [s]

time [s] time [s] time [s]

A B C

D E F

30 ms

300 ms

1000 ms

etc.

etc.

etc.

-80 mV

-80 mV

-80 mV

+ 40 mV

+ 40 mV

+ 40 mV

Fig. 6. Frequency dependence of local anesthetic induced block was analyzed at three different interpulse durations (100 ms[triangle], 300 ms [square], and 1,000 ms [circle]). The remaining current under the influence of the local anesthetic agents in thesteady state phase at the end of the test potential was normalized to control values. The normalized current values were plottedagainst a time axis according to the different interpulse durations. Inhibition of Kv1.1 channels was not frequency dependent,because the frequency of the depolarizing pulse did not significantly influence local anestheticinduced inhibition. (A) Dextrobupi-vacaine, (B) levobupivacaine, (C) racemic bupivacaine, (D) dextroropivacaine, (E) levoropivacaine, and (F) racemic mepivacaine.




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lipophilic interaction of local anesthetics with this groupof ion channels, thus, seems to constitute a commonpharmacologic mechanism. In parallel to clinical knowl-

edge, inhibition of Kv1.1 channels follows the samecorrelation of lipophilicity and analgetic potency.30 Toobtain equipotent analgesia in patients, higher concen-trations of mepivacaine and ropivacaine have to be ad-ministered compared with bupivacaine.30

All local anesthetics induced a concentration-dependentshift of the activation midpoint into the hyperpolarizingdirection. This shift correlated with lipophilicity as well andmay be explained by interference of local anesthetics withthe coupling of voltage sensing and channel opening ofKv1.1 channels.31 Interaction of local anesthetics withKv1.1 channels is, therefore, unlikely to be restricted to

single amino acids in the pore region of the channels.Rather, it seems to result from a more complex interactionwith the channel protein. The interaction may likely in- volve regions of the S6 segment that are necessary forcoupling the activation gate of these potassium channels totheir voltage sensor residing in the S4 region.23,31 Recently,a concerted contribution of the S4S5 linker and the S6segment to the modulation of voltage-dependent potassiumchannels by n-alkanols has been described.31 A similar in-teraction of local anesthetics with the coupling of theactivation gate to the voltage sensor of Kv1.1 channelsrather than a direct interaction with the voltage sensormay, therefore, offer a possible explanation for the shift ofthe activation midpoint. Taken together, the effects oncurrent inhibition and activation gating allow hypothesiz-ing that local anesthetics, by binding to lipophilic aminoacids L402 and V406 in the pore region, alter channelconductance and modulate gating by interference with theactivation gate.

Stereoselective drug binding implies that the drug re-ceptor is able to discriminate between the stereoisomersof a drug molecule.32 Franqueza et al.15 reported thatbupivacaine block of Kv1.5 channels is stereoselective,

with the R() enantiomer being sevenfold more potentthan the S( ) enantiomer. In Kv1.5 channels, the ste-

reoselective effects of bupivacaine enantiomers were

reduced by substituting the amino acid threonine atposition 505 by valine.15 Threonine at the correspondingposition 399 of Kv1.1 channels may suggest stereospe-

cific interaction with Kv1.1 channels as well. Althoughthis was the case, inhibition of Kv1.1 channels by amino-amide local anesthetics displayed a four- to five-times-

weaker stereoselectivity than Kv1.5 channels. This quan-titative difference in stereoselectivity between Kv1.1and Kv1.5 channels supports the hypothesis that differ-ences regarding the pharmacologic effects of local anes-thetic action on potassium channels exist.8,16,23 Thesedifference likely result from subtype and even speciesdifferences in channel structure.23 The difference in ste-reoselectivity between Kv1.1 and Kv1.5 channels may,furthermore, imply that despite structural similarities,

the degree of lipophilic and polar interactions differbetween different voltage-dependent potassium channelsubtypes. Because the pore region is well conservedamong different voltage-dependent potassium channels,such as Kv1.1 and Kv1.5, other regions of the protein orthe lipidprotein interface may be involved in drug chan-nel interaction. In this context, it is interesting to notethat the three-dimensional orientation of R() as com-pared with S() ropivacaine reduces the difference inpotency between S( ) ropivacaine and S( ) bupiva-caine introduced by an additional CH2 group in the latterby a factor of two. Furthermore, Kv1.1 is the only po-tassium channel that has been systematically studied

with the same pattern of stereospecificity as sodiumchannels.33

In view of the physiologic significance of Kv1.1 chan-nels, interaction with amino-amide local anesthetics maycause increased neuronal excitability.9 Because Kv1.1channels are predominately expressed in myelinatednerve fibers and large dorsal root ganglia neurons,19

binding of local anesthetics to Kv1.1 channels may im-pair and delay the repolarization of the resting mem-brane potential of neurons.9 This would slow the recov-ery of inactivated sodium channels and thereforedecrease the availability of these channels, which would

consequently increase the refractory period for action

0 2 41 3 5

mepivacaine

bupivacaine

ropivacaine

log IC50(M)

0

2

3

4

1

number of CH2 groups

A B

0 2 41 3

mepivacaine (300 M)

bupivacaine (300 M)

ropivacaine (300 M)

Vmid [mV]

0

-5

-10

-155

number of CH2 groups

Fig. 8. The IC50 values for Kv1.1 channelinhibition and the shift of voltage of half-maximal activation (Vmid) were corre-lated with the length of the N-substituentof the homolog series of local anesthetics

(figs. 7A and B). (A) The inhibitory po-tency and the Vmid shift of the local anes-thetic agents (300 M) correlated with thenumber of CH2 groups (r 0.94, P

lipophilic and stereo specific interactions of amino-amide

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