Long QT 1 Mutation KCNQ1A344V Increases Local Anesthetic Sensitivity of the Slowly Activating Delayed Rectifier Potassium Current

Background: Anesthesia in patients with long QT syndrome (LQTS) is a matter of concern. Congenital LQTS is most frequently caused by mutations in KCNQ1 (Kv7.1), whereas drug-induced LQTS is a consequence of HERG (human ether-a-go-go-related gene) channel inhibition. The aim of this study was to investigate whether the LQT1 mutation A344V in the S6 region of KCNQ1, at a position corresponding to the local anesthetic binding site in HERG, may render drug insensitive KCNQ1 channels into a toxicologically relevant target of these pharmacologic agents. This may suggest that LQTS constitutes not only a nonspecific but also a specific pharmacogenetic risk factor for anesthesia.

Methods: The authors examined electrophysiologic and pharmacologic properties of wild-type and mutant KCNQ1 channels. The effects of bupivacaine, ropivacaine, and mepivacaine were investigated using two-electrode voltage clamp and whole cell patch clamp recordings.

Results: The mutation A344V induced voltage-dependent inactivation in homomeric KCNQ1 channels and shifted the voltage dependence of KCNQ1/KCNE1 channel activation by +30 mV. The mutation furthermore increased the sensitivity of KCNQ1/KCNE1 channels for bupivacaine 22-fold (KCNQ1wt/KCNE1: IC50 = 2,431 +/- 582 [mu]m, n = 20; KCNQ1A344V/KCNE1: IC50 = 110 +/- 9 [mu]m, n = 24). Pharmacologic effects of the mutant channels were dominant when mutant and wild-type channels were coexpressed. Simulation of cardiac action potentials with the Luo-Rudy model yielded a prolongation of the cardiac action potential duration and induction of early afterdepolarizations by the mutation A344V that were aggravated by local anesthetic intoxication.     

Local anesthetic interaction with human ether-a-go-go-related gene (HERG) channels: role of aromatic amino acids Y652 and F656

BACKGROUND: Human ether-a-go-go-related gene (HERG) potassium channels constitute a potential target involved in cardiotoxic side effects of amino-amide local anesthetics. The molecular interaction site of these low-affinity blockers with HERG channels is currently unknown. The aim of this study was to determine the effect of the mutations Y652A and F656A in the putative drug binding region of HERG on the inhibition by bupivacaine, ropivacaine, and mepivacaine. METHODS: The authors examined the inhibition of wild-type and mutant HERG channels, transiently expressed in Chinese hamster ovary cells by bupivacaine, ropivacaine, and mepivacaine. Whole cell patch clamp recordings were performed at room temperature. RESULTS: Inhibition of HERG wild-type and mutant channels by the different local anesthetics was concentration dependent, stereoselective, and reversible. The sensitivity decreased in the order bupivacaine > ropivacaine > mepivacaine for wild-type and mutant channels. The mutant channels were approximately 4-30 times less sensitive to the inhibitory action of the different local anesthetics than the wild-type channel. The concentration-response data were described by Hill functions (bupivacaine: wild-type IC50 = 22 +/- 2 microm, n = 38; Y652A IC50 = 95 +/- 5 microm, n = 31). The mutations resulted in a change of the stereoselectivity of HERG channel block by ropivacaine. The potency of the local anesthetics to inhibit wild-type and mutant channels correlated with the lipophilicity of the drug (r > 0.9). CONCLUSIONS: These results indicate that local anesthetics specifically but not exclusively interact with the aromatic residues Y652 and F656 in S6 of HERG channels.     

Local Anesthetic Interaction with Human Ether-a-go-go-related Gene (HERG) Channels: Role of Aromatic Amino Acids Y652 and F656

Background: Human ether-a-go-go-related gene (HERG) potassium channels constitute a potential target involved in cardiotoxic side effects of amino-amide local anesthetics. The molecular interaction site of these low-affinity blockers with HERG channels is currently unknown. The aim of this study was to determine the effect of the mutations Y652A and F656A in the putative drug binding region of HERG on the inhibition by bupivacaine, ropivacaine, and mepivacaine.

Methods: The authors examined the inhibition of wild-type and mutant HERG channels, transiently expressed in Chinese hamster ovary cells by bupivacaine, ropivacaine, and mepivacaine. Whole cell patch clamp recordings were performed at room temperature.

Results: Inhibition of HERG wild-type and mutant channels by the different local anesthetics was concentration dependent, stereoselective, and reversible. The sensitivity decreased in the order bupivacaine > ropivacaine > mepivacaine for wild-type and mutant channels. The mutant channels were approximately 4-30 times less sensitive to the inhibitory action of the different local anesthetics than the wild-type channel. The concentration-response data were described by Hill functions (bupivacaine: wild-type IC50 = 22 +/- 2 [mu]m, n = 38; Y652A IC50 = 95 +/- 5 [mu]m, n = 31). The mutations resulted in a change of the stereoselectivity of HERG channel block by ropivacaine. The potency of the local anesthetics to inhibit wild-type and mutant channels correlated with the lipophilicity of the drug (r > 0.9).     

Structural requirements of human ether-a-go-go-related gene channels for block by bupivacaine

BACKGROUND: Local anesthetics interact with human ether-a-go-go-related gene (HERG) channels via the aromatic amino acids Y652 and F656 in the S6 region. This study aimed to establish whether the residues T623, S624, and V625 residing deeper within the pore are also involved in HERG channel block by bupivacaine. In addition, the study aimed to further define the role of the aromatic residues Y652 and F656 in bupivacaine inhibition by mutating these residues to threonine. METHODS: Alanine and threonine mutants were generated by site-directed mutagenesis. Electrophysiologic and pharmacologic properties of wild-type and mutant HERG channels were established using two-electrode voltage-clamp recordings of Xenopus laevis oocytes expressing HERG channels. RESULTS: Tail currents at -120 mV through HERG wild-type channels were inhibited with an IC50 value of 132 +/- 22 microm (n = 33). Bupivacaine (300 microm) inhibited wild-type tail currents by 62 +/- 12% (n = 7). Inhibition of HERG tail currents by bupivacaine (300 microm) was reduced by all mutations (P < 0.001). The effect was largest for F656A (inhibition 5 +/- 2%, n = 6) in the lower S6 region and for T623A (inhibition 13 +/- 4%, n = 9) near the selectivity filter. Introducing threonine at positions 656 and 652 significantly reduced inhibition by bupivacaine compared with HERG wild type (P < 0.001). CONCLUSIONS: The authors' results indicate that not only the aromatic residues Y652 and F656 but also residues residing deeper within the pore and close to the selectivity filter of HERG channels are involved in inhibition of HERG channels by the low-affinity blocker bupivacaine.     

Structural Requirements of Human Ether-a-go-go-related Gene Channels for Block by Bupivacaine

Background: Local anesthetics interact with human ether-a-go-go-related gene (HERG) channels via the aromatic amino acids Y652 and F656 in the S6 region. This study aimed to establish whether the residues T623, S624, and V625 residing deeper within the pore are also involved in HERG channel block by bupivacaine. In addition, the study aimed to further define the role of the aromatic residues Y652 and F656 in bupivacaine inhibition by mutating these residues to threonine.

Methods: Alanine and threonine mutants were generated by site-directed mutagenesis. Electrophysiologic and pharmacologic properties of wild-type and mutant HERG channels were established using two-electrode voltage-clamp recordings of Xenopus laevis oocytes expressing HERG channels.

Results: Tail currents at -120 mV through HERG wild-type channels were inhibited with an IC50 value of 132 +/- 22 [mu]m (n = 33). Bupivacaine (300 [mu]m) inhibited wild-type tail currents by 62 +/- 12% (n = 7). Inhibition of HERG tail currents by bupivacaine (300 [mu]m) was reduced by all mutations (P < 0.001). The effect was largest for F656A (inhibition 5 +/- 2%, n = 6) in the lower S6 region and for T623A (inhibition 13 +/- 4%, n = 9) near the selectivity filter. Introducing threonine at positions 656 and 652 significantly reduced inhibition by bupivacaine compared with HERG wild type (P < 0.001).     

Inhibition of Kv4.3/KChIP2.2 Channels by Bupivacaine and Its Modulation by the Pore Mutation Kv4.3V401I

Background: The transient outward current Ito is an important repolarizing K current in human ventricular myocardium mediated by Kv4.3 and KChIP2.2 subunits. Inhibition of Ito by amino-amide local anesthetics may be involved in severe cardiotoxic side effects. This study elucidates the molecular mechanisms of bupivacaine interaction with complexes formed by Kv4.3 and KChIP2.2 as well as the modulatory effect of KChIP2.2. For this purpose, the pharmacologic effects of bupivacaine on Kv4.3wt/KChIP2.2 channels and on the pore mutant Kv4.3V401I were investigated.

Methods: Kv4.3/KChIP2.2 cDNA was transiently expressed in Chinese hamster ovary cells. Site-directed mutagenesis and patch clamp experiments were performed to analyze the effects of bupivacaine on wild-type and mutant channels.

Results: Inhibition of Kv4.3wt/KChIP2.2 channels by bupivacaine was concentration-dependent and reversible. The IC50s for inhibition of the charge conducted by Kv4.3wt/KChIP2.2 channels by bupivacaine and levobupivacaine were 55 +/- 8 and 50 +/- 5 [mu]m, respectively. The local anesthetic accelerated macroscopic current decline of Kv4.3wt/KChIP2.2 and slowed recovery from inactivation without altering steady state inactivation. KChIP2.2 altered the response of Kv4.3wt channels to bupivacaine and bupivacaine modulated KChIP2.2 effects on Kv4.3wt channels. The pore mutation V401I slowed macroscopic current decline of Kv4.3 channels and recovery from inactivation, and it diminished modulation of gating by KChIP2.2. Bupivacaine inhibition of Kv4.3V401I resembled Kv4.3wt and was not changed by coexpression of KChIP2.2.     

Inhibition of Kv4.3/KChIP2.2 channels by bupivacaine and its modulation by the pore mutation Kv4.3V401I

BACKGROUND: The transient outward current Ito is an important repolarizing K current in human ventricular myocardium mediated by Kv4.3 and KChIP2.2 subunits. Inhibition of Ito by amino-amide local anesthetics may be involved in severe cardiotoxic side effects. This study elucidates the molecular mechanisms of bupivacaine interaction with complexes formed by Kv4.3 and KChIP2.2 as well as the modulatory effect of KChIP2.2. For this purpose, the pharmacologic effects of bupivacaine on Kv4.3wt/KChIP2.2 channels and on the pore mutant Kv4.3V401I were investigated. METHODS: Kv4.3/KChIP2.2 cDNA was transiently expressed in Chinese hamster ovary cells. Site-directed mutagenesis and patch clamp experiments were performed to analyze the effects of bupivacaine on wild-type and mutant channels. RESULTS: Inhibition of Kv4.3wt/KChIP2.2 channels by bupivacaine was concentration-dependent and reversible. The IC50s for inhibition of the charge conducted by Kv4.3wt/KChIP2.2 channels by bupivacaine and levobupivacaine were 55 +/- 8 and 50 +/- 5 microm, respectively. The local anesthetic accelerated macroscopic current decline of Kv4.3wt/KChIP2.2 and slowed recovery from inactivation without altering steady state inactivation. KChIP2.2 altered the response of Kv4.3wt channels to bupivacaine and bupivacaine modulated KChIP2.2 effects on Kv4.3wt channels. The pore mutation V401I slowed macroscopic current decline of Kv4.3 channels and recovery from inactivation, and it diminished modulation of gating by KChIP2.2. Bupivacaine inhibition of Kv4.3V401I resembled Kv4.3wt and was not changed by coexpression of KChIP2.2. CONCLUSIONS: These results indicate that bupivacaine blocks Kv4.3/KChIP2.2 channels from the open state. They furthermore give structural evidence that amino-amide local anesthetics interfere with the effects of KChIP2.2 on Kv4.3 by an indirect mechanism.     

Inhibition of HERG channels by the local anaesthetic articaine

BACKGROUND AND OBJECTIVE: Articaine is an amide local anaesthetic widely used in dentistry. Human ether-a-go-go-related gene (HERG) potassium channels constitute potential targets involved in cardiotoxic side-effects of various pharmacological agents including amide local anaesthetics. The aim of this study was to determine the sensitivity of HERG channels to the inhibitory action of articaine and to further evaluate the effect of the mutations Y652A and F656A in the putative drug-binding region of HERG on the sensitivity for articaine. METHODS: We examined the inhibition of wild-type and mutant HERG channels, transiently expressed in Chinese hamster ovary cells by articaine. Whole cell patch-clamp recordings were performed at room temperature. RESULTS: Inhibition of HERG wild-type and HERG Y652A channels by articaine was concentration dependent and reversible. The concentration-response data were described by Hill functions (wild type: IC50 = 224 +/- 6 micromol L-1, Hill coefficient h = 1.17 +/- 0.03, n = 23; Y652A: IC50 = 360 +/- 48 micromol L-1, h = 0.93 +/- 0.08, n = 26). The mutation Y5652A decreased the sensitivity by factor 1.6. The mutation F656A decreased inhibition of inward tail currents by 300 micromol L-1 articaine in 100 mmol extracellular K+ 3-fold. CONCLUSIONS: Our results indicate that the local anaesthetic articaine does not inhibit HERG channels at clinically relevant concentrations. Articaine may therefore constitute a safer alternative for local and regional anaesthesia. The aromatic amino acid F656 rather than Y652 in the S6 region might play a role in interaction of the drug with the channel.     

Retigabine Stimulates Human KCNQ2/Q3 Channels in the Presence of Bupivacaine

Background: Inhibition of KCNQ2/Q3 channels may cause convulsion in humans. The interaction of bupivacaine with these channels is unknown. The anticonvulsant retigabine activates KCNQ2/Q3 channels and may reverse inhibitory actions of bupivacaine. Potassium channel stimulation may thus constitute a novel approach to treat local anesthetic-induced seizures. The aim of this study was to characterize bupivacaine effects on KCNQ2/Q3 channels and to investigate whether retigabine reverses the effects of the local anesthetic.

Methods: KCNQ2/Q3 channels were transiently expressed in Chinese hamster ovary cells. The effects of bupivacaine and retigabine were studied with the patch-clamp technique.

Results: Bupivacaine inhibited KCNQ2/Q3 channels in a concentration-dependent and reversible manner. The concentration-response curve was described by a Hill equation (IC50 = 173 +/- 7 [mu]m, Hill coefficient = 1.4 +/- 0.1, mean +/- SEM, n = 37). The inhibitory effect did not differ between bupivacaine and levobupivacaine (42 +/- 4%, n = 7, versus 42 +/- 5%, n = 10; P > 0.05). Ropivacaine was four times less potent than bupivacaine. The inhibition of KCNQ2/Q3 channels by bupivacaine resulted in a significant and reversible depolarization of the membrane potential. Retigabine (300 nm-10 [mu]m) reversed the inhibitory action of bupivacaine on KCNQ2/Q3 channels as well as the depolarization of the membrane potential.     

Retigabine stimulates human KCNQ2/Q3 channels in the presence of bupivacaine

BACKGROUND: Inhibition of KCNQ2/Q3 channels may cause convulsion in humans. The interaction of bupivacaine with these channels is unknown. The anticonvulsant retigabine activates KCNQ2/Q3 channels and may reverse inhibitory actions of bupivacaine. Potassium channel stimulation may thus constitute a novel approach to treat local anesthetic-induced seizures. The aim of this study was to characterize bupivacaine effects on KCNQ2/Q3 channels and to investigate whether retigabine reverses the effects of the local anesthetic. METHODS: KCNQ2/Q3 channels were transiently expressed in Chinese hamster ovary cells. The effects of bupivacaine and retigabine were studied with the patch-clamp technique. RESULTS: Bupivacaine inhibited KCNQ2/Q3 channels in a concentration-dependent and reversible manner. The concentration-response curve was described by a Hill equation (IC50 = 173 +/- 7 microm, Hill coefficient = 1.4 +/- 0.1, mean +/- SEM, n = 37). The inhibitory effect did not differ between bupivacaine and levobupivacaine (42 +/- 4%, n = 7, versus 42 +/- 5%, n = 10; P > 0.05). Ropivacaine was four times less potent than bupivacaine. The inhibition of KCNQ2/Q3 channels by bupivacaine resulted in a significant and reversible depolarization of the membrane potential. Retigabine (300 nm-10 microm) reversed the inhibitory action of bupivacaine on KCNQ2/Q3 channels as well as the depolarization of the membrane potential. CONCLUSIONS: The anticonvulsant retigabine at nanomolar concentrations reverses the inhibitory effect of micromolar concentrations of bupivacaine. Our results allow the hypothesis that activation of KCNQ2/Q3 channels by retigabine may offer a novel therapeutic approach for the treatment of bupivacaine-induced seizures.     

Molecular interaction of droperidol with human ether-a-go-go-related gene channels: prolongation of action potential duration without inducing early afterdepolarization

BACKGROUND: The cardiac safety of droperidol given at antiemetic doses is a matter of debate. Although droperidol potently inhibits human ether-a-go-go-related gene (HERG) channels, the molecular mode of this interaction is unknown. The role of amino acid residues typically mediating high-affinity block of HERG channels is unclear. It is furthermore unresolved whether droperidol at antiemetic concentrations induces action potential prolongation and arrhythmogenic early afterdepolarizations in cardiac myocytes. METHODS: Molecular mechanisms of HERG current inhibition by droperidol were established using two-electrode voltage clamp recordings of Xenopus laevis oocytes expressing wild-type and mutant channels. The mutants T623A, S624A, V625A, Y652A, and F656A were generated by site-directed mutagenesis. The effect of droperidol on action potentials was investigated in cardiac myocytes isolated from guinea pig hearts using the patch clamp technique. RESULTS: Droperidol inhibited currents through HERG wild-type channels with a concentration of half-maximal inhibition of 0.6-0.9 microM. Droperidol shifted the channel activation and the steady state inactivation toward negative potentials while channel deactivation was not affected. Current inhibition increased with membrane potential and with increasing duration of current activation. Inhibition of HERG channels was similarly reduced by all mutations. Droperidol at concentrations between 5 and 100 nM prolonged whereas concentrations greater than 300 nm shortened action potentials. Early afterdepolarizations were not observed. CONCLUSIONS: Droperidol is a high-affinity blocker of HERG channels. Amino acid residues typically involved in high-affinity block mediate droperidol effects. Patch clamp results and computational modeling allow the hypothesis that interaction with calcium currents may explain why droperidol at antiemetic concentrations prolongs the action potential without inducing early afterdepolarizations.     

Kvbeta1.3 reduces the degree of stereoselective bupivacaine block of Kv1.5 channels

BACKGROUND: Kvbeta1.3 subunit modifies the gating and the pharmacology of Kv1.5 channels, decreasing their sensitivity to block induced by drugs, suggesting that Kvbeta1.3 competes with them for a binding site at Kv1.5 channels. METHODS: Currents generated by the activation of Kv1.5 and Kv1.5 + Kvbeta1.3 channels expressed in HEK293 cells and Xenopus oocytes were recorded by using whole cell patch clamp and voltage clamp techniques. RESULTS: Block of Kv1.5, but not that produced on Kv1.5 + Kvbeta1.3 channels, was voltage dependent. In both channels, bupivacaine block was time dependent. R(+)- and S(-)-bupivacaine blocked Kv1.5 with IC50 4.4 +/- 0.5 microM (n = 15) and 39.8 +/- 8.2 microM (n = 16; P < 0.05), respectively. These values increased fourfold for R(+)-bupivacaine (17.2 +/- 2.2 microM) and twofold for S(-)-bupivacaine (71.9 +/- 11.5 microM) in Kv1.5 + Kvbeta1.3 channels. Therefore, the degree of stereoselectivity (theta) decreased from 9 to 4 in the presence of Kvbeta1.3. The decrease in potency to block Kv1.5 + Kvbeta1.3 channels was the result of a less stable interaction between bupivacaine enantiomers and channels. Differences in stereoselectivity in each situation were due to a more favorable interaction between the channel and R(+)-bupivacaine. In the presence of Kvbeta1.3, stereoselectivity was abolished for V514A mutant channels (involved in bupivacaine binding but not in Kvbeta1.3 binding) but not for L510A (part of Kvbeta1.3 binding site). CONCLUSIONS: The degree of stereoselective block of Kv1.5 decreases from 9 to 4 when Kvbeta1.3 is present. L510 is determinant for the modulation of bupivacaine block, because it is the only residue of the S6 segment that binds to both bupivacaine and Kvbeta1.3. These findings support an overlapping binding site for drugs and Kvbeta1.3.     

Ketamine Blockade of Voltage-gated Sodium Channels: Evidence for a Shared Receptor Site with Local Anesthetics

Background : The general anesthetic ketamine is known to be an N-methyl-d-aspartate receptor blocker. Although ketamine also blocks voltage-gated sodium channels in a local anesthetic-like fashion, little information exists on the molecular pharmacology of this interaction. We measured the effects of ketamine on sodium channels.

Methods : Wild-type and mutant (F1579A) recombinant rat skeletal muscle sodium channels were expressed in Xenopus oocytes. The F1579A amino acid substitution site is part of the intrapore local anesthetic receptor. The effect of ketamine was measured in oocytes expressing wild-type or mutant sodium channels using two-electrode voltage clamp.

Results : Ketamine blocked sodium channels in a local anesthetic-like fashion, exhibiting tonic blockade (concentration for half-maximal inhibition [IC50] = 0.8 mm), phasic blockade (IC50 = 2.3 mm), and leftward shift of the steady-state inactivation; the parameters of these actions were strongly modified by alteration of the intrapore local anesthetic binding site (IC50 = 2.1 mm and IC50 = 10.3 mm for tonic and phasic blockade, respectively). Compared with lidocaine, ketamine showed greater tonic inhibition but less phasic blockade.     

Molecular Interaction of Droperidol with Human Ether-a-go-go-related Gene Channels: Prolongation of Action Potential Duration without Inducing Early Afterdepolarization

Background: The cardiac safety of droperidol given at antiemetic doses is a matter of debate. Although droperidol potently inhibits human ether-a-go-go-related gene (HERG) channels, the molecular mode of this interaction is unknown. The role of amino acid residues typically mediating high-affinity block of HERG channels is unclear. It is furthermore unresolved whether droperidol at antiemetic concentrations induces action potential prolongation and arrhythmogenic early afterdepolarizations in cardiac myocytes.

Methods: Molecular mechanisms of HERG current inhibition by droperidol were established using two-electrode voltage clamp recordings of Xenopus laevis oocytes expressing wild-type and mutant channels. The mutants T623A, S624A, V625A, Y652A, and F656A were generated by site-directed mutagenesis. The effect of droperidol on action potentials was investigated in cardiac myocytes isolated from guinea pig hearts using the patch clamp technique.

Results: Droperidol inhibited currents through HERG wild-type channels with a concentration of half-maximal inhibition of 0.6-0.9 [mu]m. Droperidol shifted the channel activation and the steady state inactivation toward negative potentials while channel deactivation was not affected. Current inhibition increased with membrane potential and with increasing duration of current activation. Inhibition of HERG channels was similarly reduced by all mutations. Droperidol at concentrations between 5 and 100 nm prolonged whereas concentrations greater than 300 nm shortened action potentials. Early afterdepolarizations were not observed.     

Ketamine blockade of voltage-gated sodium channels: evidence for a shared receptor site with local anesthetics.

BACKGROUND: The general anesthetic ketamine is known to be an N-methyl-D-aspartate receptor blocker. Although ketamine also blocks voltage-gated sodium channels in a local anesthetic-like fashion, little information exists on the molecular pharmacology of this interaction. We measured the effects of ketamine on sodium channels. METHODS: Wild-type and mutant (F1579A) recombinant rat skeletal muscle sodium channels were expressed in Xenopus oocytes. The F1579A amino acid substitution site is part of the intrapore local anesthetic receptor. The effect of ketamine was measured in oocytes expressing wild-type or mutant sodium channels using two-electrode voltage clamp. RESULTS: Ketamine blocked sodium channels in a local anesthetic-like fashion, exhibiting tonic blockade (concentration for half-maximal inhibition [IC50] = 0.8 mm), phasic blockade (IC50 = 2.3 mm), and leftward shift of the steady-state inactivation; the parameters of these actions were strongly modified by alteration of the intrapore local anesthetic binding site (IC50 = 2.1 mm and IC50 = 10.3 mm for tonic and phasic blockade, respectively). Compared with lidocaine, ketamine showed greater tonic inhibition but less phasic blockade. CONCLUSIONS: Ketamine interacts with sodium channels in a local anesthetic-like fashion, including sharing a binding site with commonly used clinical local anesthetics.     

Amitriptyline versus bupivacaine in rat sciatic nerve blockade

BACKGROUND: Amitriptyline, a tricyclic antidepressant, is frequently used orally for the management of chronic pain. To date there is no report of amitriptyline producing peripheral nerve blockade. The authors therefore investigated the local anesthetic properties of amitriptyline in rats and in vitro. METHODS: Sciatic nerve blockade was performed with 0.2 ml amitriptyline or bupivacaine at selected concentrations, and the motor, proprioceptive, and nociceptive blockade was evaluated. Cultured rat GH3 cells were externally perfused with amitriptyline or bupivacaine, and the drug affinity toward inactivated and resting Na+ channels was assessed under whole-cell voltage clamp conditions. In addition, use-dependent blockade of these drugs at 5 Hz was evaluated. RESULTS: Complete sciatic nerve blockade for nociception was obtained with amitriptyline for 217 +/- 19 min (5 mM, n = 8, mean +/- SEM) and for 454 +/- 38 min (10 mM, n = 7) versus bupivacaine for 90 +/- 13 min (15.4 mM, n = 6). The time to full recovery of nociception for amitriptyline was 353 +/- 12 min (5 mM) and 656 +/- 27 min (10 mM) versus 155 +/- 9 min for bupivacaine (15.4 mM). Amitriptyline was approximately 4.7-10.6 times more potent than bupivacaine in binding to the resting channels (50% inhibitory concentration [IC50] of 39.8 +/- 2.7 vs. 189.6 +/- 22.3 microM) at - 150 mV, and to the inactivated Na+ channels (IC50 of 0.9 +/- 0.1 vs. 9.6 +/- 0.9 microM) at -60 mV. High-frequency stimulation at 3 microM caused an additional approximately 14% blockade for bupivacaine, but approximately 50% for amitriptyline. CONCLUSION: Amitriptyline is a more potent blocker of neuronal Na+ channels than bupivacaine in vivo and in vitro. These findings suggest that amitriptyline could extend its clinical usefulness for peripheral nerve blockade.     

Molecular Mechanisms of the Inhibitory Effects of Bupivacaine, Levobupivacaine, and Ropivacaine on Sarcolemmal Adenosine Triphosphate-sensitive Potassium Channels in the Cardiovascular System

Background: Sarcolemmal adenosine triphosphate-sensitive potassium (KATP) channels in the cardiovascular system may be involved in bupivacaine-induced cardiovascular toxicity. The authors investigated the effects of local anesthetics on the activity of reconstituted KATP channels encoded by inwardly rectifying potassium channel (Kir6.0) and sulfonylurea receptor (SUR) subunits.

Methods: The authors used an inside-out patch clamp configuration to investigate the effects of bupivacaine, levobupivacaine, and ropivacaine on the activity of reconstituted KATP channels expressed in COS-7 cells and containing wild-type, mutant, or chimeric SURs.

Results: Bupivacaine inhibited the activities of cardiac KATP channels (IC50 = 52 [mu]m) stereoselectively (levobupivacaine, IC50 = 168 [mu]m; ropivacaine, IC50 = 249 [mu]m). Local anesthetics also inhibited the activities of channels formed by the truncated isoform of Kir6.2 (Kir6.2[DELTA]C36) stereoselectively. Mutations in the cytosolic end of the second transmembrane domain of Kir6.2 markedly decreased both the local anesthetics' affinity and stereoselectivity. The local anesthetics blocked cardiac KATP channels with approximately eightfold higher potency than vascular KATP channels; the potency depended on the SUR subtype. The 42 amino acid residues at the C-terminal tail of SUR2A, but not SUR1 or SUR2B, enhanced the inhibitory effect of bupivacaine on the Kir6.0 subunit.     

Molecular mechanisms of the inhibitory effects of bupivacaine, levobupivacaine, and ropivacaine on sarcolemmal adenosine triphosphate-sensitive potassium channels in the cardiovascular system

BACKGROUND: Sarcolemmal adenosine triphosphate-sensitive potassium (KATP) channels in the cardiovascular system may be involved in bupivacaine-induced cardiovascular toxicity. The authors investigated the effects of local anesthetics on the activity of reconstituted KATP channels encoded by inwardly rectifying potassium channel (Kir6.0) and sulfonylurea receptor (SUR) subunits. METHODS: The authors used an inside-out patch clamp configuration to investigate the effects of bupivacaine, levobupivacaine, and ropivacaine on the activity of reconstituted KATP channels expressed in COS-7 cells and containing wild-type, mutant, or chimeric SURs. RESULTS: Bupivacaine inhibited the activities of cardiac KATP channels (IC50 = 52 microm) stereoselectively (levobupivacaine, IC50 = 168 microm; ropivacaine, IC50 = 249 microm). Local anesthetics also inhibited the activities of channels formed by the truncated isoform of Kir6.2 (Kir6.2 delta C36) stereoselectively. Mutations in the cytosolic end of the second transmembrane domain of Kir6.2 markedly decreased both the local anesthetics' affinity and stereoselectivity. The local anesthetics blocked cardiac KATP channels with approximately eightfold higher potency than vascular KATP channels; the potency depended on the SUR subtype. The 42 amino acid residues at the C-terminal tail of SUR2A, but not SUR1 or SUR2B, enhanced the inhibitory effect of bupivacaine on the Kir6.0 subunit. CONCLUSIONS: Inhibitory effects of local anesthetics on KATP channels in the cardiovascular system are (1) stereoselective: bupivacaine was more potent than levobupivacaine and ropivacaine; and (2) tissue specific: local anesthetics blocked cardiac KATP channels more potently than vascular KATP channels, via the intracellular pore mouth of the Kir6.0 subunit and the 42 amino acids at the C-terminal tail of the SUR2A subunit, respectively.     

Interaction of ropivacaine with cloned cardiac Kv4.3/KChIP2.2 complexes

BACKGROUND: Inhibition of cardiac K channels by local anesthetic may contribute to QTc interval prolongation of the electrocardiogram and induction of ventricular arrhythmia. The transient outward current Ito has been identified as a toxicologically relevant target of bupivacaine. S(-)-ropivacaine has been developed as a safer alternative to bupivacaine. The effects of S(-)-ropivacaine on Ito have not been investigated. In human ventricular myocardium, Ito is formed by Kv4.3 and KChIP2.2 subunits. Therefore, the aim of this study was to establish the effects of S(-)-ropivacaine on human Kv4.3/KChIP2.2 channels. METHODS: Kv4.3/KChIP2.2 complementary DNA cloned from human heart was transiently transfected in Chinese hamster ovary cells. The pharmacologic effects of S(-)-ropivacaine were investigated with the patch clamp method. RESULTS: Ropivacaine inhibited Kv4.3/KChIP2.2 channels in a concentration-dependent, stereospecific, and reversible manner. The IC50 value of S(-)-ropivacaine for inhibition of the charge conducted by Kv4.3/KChIP2.2 channel was 117 +/- 21 microm (n = 30). The local anesthetic accelerated macroscopic current decline with an IC50 value of 77 +/- 11 microm (n = 30). It shifted the midpoint of channel activation into the depolarizing direction, and it slowed recovery from inactivation without altering steady state inactivation. Kv4.3 channels are more sensitive to the inhibitory effect than Kv4.3/KChIP2.2 channels. CONCLUSIONS:: The results are consistent with the idea that ropivacaine, by blocking Kv4.3/KChIP2.2 from the open state, interferes with the gating modifying effects of KChIP2.2 on Kv4.3 channels. Inhibition of Kv4.3/KChIP2.2 channels by the local anesthetic may contribute to the deterioration of cardiac function during events of intoxication.     

Interaction of Ropivacaine with Cloned Cardiac Kv4.3/KChIP2.2 Complexes

Background: Inhibition of cardiac K channels by local anesthetic may contribute to QTc interval prolongation of the electrocardiogram and induction of ventricular arrhythmia. The transient outward current Ito has been identified as a toxicologically relevant target of bupivacaine. S(-)-ropivacaine has been developed as a safer alternative to bupivacaine. The effects of S(-)-ropivacaine on Ito have not been investigated. In human ventricular myocardium, Ito is formed by Kv4.3 and KChIP2.2 subunits. Therefore, the aim of this study was to establish the effects of S(-)-ropivacaine on human Kv4.3/KChIP2.2 channels.

Methods: Kv4.3/KChIP2.2 complementary DNA cloned from human heart was transiently transfected in Chinese hamster ovary cells. The pharmacologic effects of S(-)-ropivacaine were investigated with the patch clamp method.

Results: Ropivacaine inhibited Kv4.3/KChIP2.2 channels in a concentration-dependent, stereospecific, and reversible manner. The IC50 value of S(-)-ropivacaine for inhibition of the charge conducted by Kv4.3/KChIP2.2 channel was 117 +/- 21 [mu]m (n = 30). The local anesthetic accelerated macroscopic current decline with an IC50 value of 77 +/- 11 [mu]m (n = 30). It shifted the midpoint of channel activation into the depolarizing direction, and it slowed recovery from inactivation without altering steady state inactivation. Kv4.3 channels are more sensitive to the inhibitory effect than Kv4.3/KChIP2.2 channels.