JOURNAL HOME CME HOME THIS MONTH PAST ISSUES ETOC COLLECTIONS
AUTHORS REVIEWERS EDITORIAL BOARD FEEDBACK RSS HELP
A&A International Anesthesia Research Society
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a colleague
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (10)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Punke, M. A.
Right arrow Articles by Friederich, P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Punke, M. A.
Right arrow Articles by Friederich, P.
Related Collections
Right arrow Mechanisms
Right arrow Pharmacology

Anesth Analg 2003;96:1665-1673
© 2003 International Anesthesia Research Society

ANESTHETIC PHARMACOLOGY

Inhibition of Human TREK-1 Channels by Bupivacaine

Mark A. Punke, MD*, Thomas Licher, PhD{dagger}, Olaf Pongs, PhD{dagger}, and Patrick Friederich, MD*

*Department of Anesthesiology, University Hospital Hamburg-Eppendorf; and {dagger}Institute of Neural Signal Transduction, University of Hamburg, Hamburg, Germany

Address correspondence and reprint requests to Patrick Friederich, MD, Department of Anesthesiology, University Hospital Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg, Germany. Address e-mail to patrick.friederich{at}zmnh.uni-hamburg.de


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Human TWIK-related K+ channels (TREK-1) stabilize the membrane potential (mp) of neurons and have a major role in the regulation of membrane excitability. In view of their physiological significance, interaction of bupivacaine with TREK-1 channels may be clinically important. Our aim was to characterize with the patch-clamp technique the properties of human TREK-1 channels and the effects of bupivacaine on these channels expressed in Chinese hamster ovary (CHO) cells. Transfection of CHO cells with TREK-1 channels (CHOTREK-1 cells) hyperpolarized the mp from -33 ± 13 to -78 ± 4 mV. The channels were stimulated by intracellular acidosis. Inhibition of TREK-1 channels by bupivacaine was reversible, concentration-dependent, voltage-independent, and increased with intracellular acidosis. Bupivacaine depolarized the mp of CHOTREK-1 cells in a reversible and concentration-dependent manner. Concentrations for channel inhibition and membrane depolarization were not linearly related (50% inhibitory concentration value for channel inhibition 370 ± 20 µM, Hill coefficient 1.8 ± 0.1, n = 51; 50% inhibitory concentration value for membrane depolarization 856 ± 14 µM, Hill coefficient 2.4 ± 0.1, mean ± SEM, n = 27). The results suggest that protonated bupivacaine elicits the observed effects via a site of interaction accessible from the intracellular space. Inhibition of TREK-1 channels and consecutive depolarization of the cell membrane by bupivacaine may contribute to blockade of neuronal signal conduction during regional anesthesia.

IMPLICATIONS: The interaction of bupivacaine with human TREK-1 channels was studied with the patch-clamp technique. Bupivacaine inhibited TREK-1 channels and depolarized the membrane potential of cells expressing TREK-1 channels in a concentration-dependent and reversible manner. Both effects may contribute to conductance block caused by bupivacaine.


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
TREK-1 channels belong to a recently discovered family of potassium channels with a unique structure characterized by two pore domains and four transmembrane segments in each subunit (1–3). The genes encoding these ion channels are called KCNK (2). Channels encoded in KCNK genes are often referred to as two-pore-domain channels. These channels represent the physiological correlate of leak currents or resting conductances of neurons (2,4). TREK-1 channels stabilize the resting membrane potential (mp) of neurons and consequently have a major role in the regulation of membrane excitability (2,5). TREK-1 channels are not voltage-gated and they do not inactivate. They are highly regulated by intracellular pH, temperature, and second messengers (4). Human tissue distribution assays indicate a prominent expression in dorsal root ganglions, the spinal cord, and in the brain (6).

Local anesthetics exhibit their clinical effects not only by binding to voltage-gated sodium channels (7), but also by interacting with other ion channels such as potassium channels (8–11). In view of the physiological significance of TREK-1 channels and their abundant expression in the central nervous system, interaction of bupivacaine with these ion channels may be clinically important. In principle, interaction with TREK-1 channels may contribute to conduction block but also to unwanted side effects such as seizures.

The inhibitory effects of local anesthetics on KCNK channels have previously been studied in oocytes from African clawed frog (Xenopus laevis) (10). In this study, the authors focused on TASK channels (TWIK-related acid-sensitive K+ channel) and also reported about the inhibitory effect of a single concentration of bupivacaine on TREK-1 channels from mice. The effects of local anesthetics on human TREK-1 channels have not been investigated. This would be important for several reasons. Species differences may influence anesthetic drug action (12) and warrant investigation of human ion channels. Furthermore, it is unclear if bupivacaine and levobupivacaine inhibit TREK-1 channels with different potencies. It is unknown if intracellular acidification antagonizes bupivacaine action by stimulation of TREK-1 channels (13), and a quantitative relationship between inhibition of two-pore-domain channels and membrane depolarization has not been provided. The aim of the present study, therefore, was to establish the effects of bupivacaine on human TREK-1 channels. For this purpose, we characterized the basic properties of these ion channels expressed in Chinese hamster ovary (CHO) cells first. This included measuring the influence of channel expression on the mp of CHO cells. We then established concentration-response curves for both the inhibition of TREK-1 channels by bupivacaine as well as the alteration of the mp induced by the local anesthetic. Possible differences in potency between bupivacaine and levobupivacaine were investigated as well as the impact of intracellular acidification on the pharmacological effect of this long acting amide local anesthetic.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Cell Culture
The FLAG-tag sequence (GAC TAC AAA GAC GAC GAC GAC AAA) was C-terminally fused via polymerase chain reaction to the human KCNK2 gene (GenBank no. AF004711). The KCNK2-FLAG construct was cloned by XbaI and ApaI into the eucaryotic expression vector pcDNA6 (Invitrogen, Karlsruhe, Germany) and transfected into CHO cells. CHO cells expressing TREK-1 channels were selected by immunofluorescence using an anti-FLAG antibody (Zytomed, Berlin, Germany) and a Cy3-coupled anti-rabbit antibody (Amersham-Pharmacia, Freiburg, Germany). CHO cells not transfected with TREK-1 channels (CHOwt) as well as CHO cells stably expressing TREK-1 channels (CHOTREK-1) were grown as nonconfluent monolayers in MEM Eagle’s Alpha medium (Gibco Invitrogen, Karlsruhe, Germany) containing 10% fetal calf serum (Biochrom, Berlin, Germany), penicillin (100 IU/mL), streptomycin (100 µg/mL), and L-glutamine (292 µg/mL) (Gibco Invitrogen). The cells were cultured at 37°C in a humidified atmosphere (95% air, 5% CO2). For electrophysiological experiments, the cells were subcultured in monodishes (35-mm diameter; NUNC, Roskilde, Denmark) at least 24 h before recording.

Patch-Clamp Recordings
Whole cell currents and mps were measured with the voltage-clamp and current-clamp methods of the patch-clamp technique (14) using an EPC-9 amplifier (HEKA Elektronik, Lambrecht, Germany) and Pulse software 8.11 (HEKA Elektronik). The patch electrodes were fabricated from borosilicate glass capillary tubes with filament (World Precision Instruments, Saratoga, FL) using a DMZ-Universal puller (Zeitz, Augsburg, Germany). The pipettes had a resistance of 2–4 M{Omega}. They were filled with a solution containing the following electrolyte concentrations: (mM) KCl, 160; MgCl2, 0.5; HEPES, 10; pH 7.2 adjusted with KOH. The external solution consisted of: (mM) NaCl, 135; KCl, 5; CaCl2, 2; MgCl2, 2; HEPES, 5; sucrose, 10; phenol red, 0.01 mg/mL; pH 7.4 adjusted with NaOH. The HCO3- solution used to induce intracellular acidosis was made by substituting 90 mM NaCl with 90 mM NaHCO3 with the pH kept constant at 7.4 by titration with HCl (13,15). Stock solutions (1 mM, 3 mM) of bupivacaine (Sigma, Deissenhofen, Germany) and levobupivacaine (AstraZeneca, Södertalje, Sweden) were prepared with the extracellular solution and stored at -20°C. Test solutions were prepared by diluting the stock solutions and they were superperfused on the cells by a perfusion system with teflon tubing driven by hydrostatic pressure.

Stimulation Protocols, Data Analysis, and Statistics
The holding potential during all experiments was -80 mV. TREK-1 whole-cell currents were elicited by two different protocols. The current-voltage relationship was established by hyperpolarizing the cells to a mp of -100 mV for 500 ms and subsequent depolarizations (500 ms) to a potential of +40 mV in 10-mV steps. For the pharmacological experiments, a ramp protocol was used that increased during 1000 ms from the holding potential to a mp of +60 mV. Inhibition of the currents by bupivacaine and levobupivacaine was measured as the reduction of charge transfer between the mp of -80 mV and 0 mV. For this purpose, the ratio of the charge (Q) under local anesthetic influence and the mean of charge under control and washout conditions was subtracted from one (inhibition = 1 - Qdrug/[(Qcontrol + Qwashout)/2]).

In case of experiments with intracellular acidosis, the inhibition by bupivacaine dissolved in standard extracellular solution (bupst) was compared with the inhibition by bupivacaine dissolved in extracellular solution containing NaHCO3 (bupNaHCO3). We quantified the inhibition as the ratio of Q under the influence of bupst to Q under control conditions (const) or as the ratio of Q under the influence of bupNaHCO3 to Q under washout conditions with extracellular solution containing NaHCO3 (conNaHCO3) and subtracted both ratios from one (inhibition = 1 - [bupst/const]) or (inhibition = 1 - [bupNaHCO3/conNaHCO3]). The effect of bupivacaine on the mp was quantified as the difference ({Delta}) of the mp during the action of bupivacaine and the mean of the mp under control and washout conditions ({Delta} = mpdrug - [mpcontrol + mpwashout]/2). The data of the concentration-response curves were mathematically described by Hill equations (y = M1 x M0M2/M3M2 + M0M2; where y = inhibition, M0 = drug concentration, M1 = maximal inhibition, M2 = Hill coefficient, M3 = half-maximal concentration of the inhibitory effect or IC50 value) using Kaleidagraph software (Synergy-Software, Reading, PA). Standard errors of calculated Hill parameters were used as defined by Kaleidagraph. Statistical significance was tested using two-sided paired or unpaired Student’s t-test as appropriate (Excel; Microsoft, Redmond, WA). Differences between concentration-response curves were tested by the F-test according to Winne (16) and Motulsky (17), using GraphPad software (GraphPad Software, San Diego, CA). Data points were given as mean ± SD unless stated otherwise; n values indicate the number of experiments.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Before performing pharmacological experiments, the basic properties of TREK-1 channels were characterized. The current-voltage relationship was established and the influence of TREK-1 channel expression on the mp of CHO cells was investigated. Original current recordings showed a rapidly activating and noninactivating current (Fig. 1A). The current-voltage relationship demonstrated small inward currents negative and large outward currents positive to the equilibrium potential of potassium. Currents of TREK-1 channels reversed at -89.1 ± 2.6 mV (n = 4) close to the calculated equilibrium potential for potassium (-87.5 mV, Fig. 1B). The average TREK-1 current density measured at an mp of 0 mV was 63 ± 36 pA/pF (n = 5). Currents of untransfected CHOwt cells evoked by a ramp-pulse protocol reversed at -8.4 ± 1.1 mV. The average current density measured at an mp of 0 mV was 0.6 ± 0.2 pA/pF and the charge transfer between the mp of -80 mV and 0 mV was -6.8 ± 9.3 pC (Fig. 1C, n = 5). The mp of CHOwt was -33 ± 13 mV (n = 5). After transfection with TREK-1 channels, the mp of CHO cells (CHOTREK-1) was hyperpolarized by 45 mV to a value of -78 ± 4 mV (n = 8, Fig. 1D). TREK-1 channels are pH sensitive and are opened by internal acidification (13). Consistent with this earlier report, we found that applying on TREK-1 channels an extracellular solution with a pH of 7.4 that contained 90 mM NaHCO3 (13,15) increased the current amplitude (Fig. 2A). The charge transfer through TREK-1 channels increased more than fivefold compared with control conditions (202 ± 31 versus 1114 ± 129 pC, n = 3 paired experiments, P < 0.05, Fig. 2B).



View larger version (16K):
[in this window]
[in a new window]
  		Figure 1. Basic properties of human TREK-1 channels. A, TREK-1 currents were evoked by the pulse protocol depicted below the currents. Shown are original currents ranging from -100 to 0 mV in 10-mV steps. B, The current-voltage relationship demonstrated small inward currents negative and large outward currents positive to a potential of -89.1 ± 2.6 mV (n = 4). C, Original current recordings of Chinese hamster ovary (CHO)TREK-1 and untransfected CHOwt cells. The currents were elicited by the ramp-pulse protocol depicted below the current traces. D, The transfection of CHO cells with TREK-1 channels hyperpolarized the membrane potential from -33 ± 13 mV (CHOwt, n = 5) to -78 ± 4 mV (CHOTREK-1, n = 8). The difference between the membrane potentials was statistically significant (P < 0.05).

 


View larger version (8K):
[in this window]
[in a new window]
  		Figure 2. Influence of intracellular acidosis on TREK-1 channels. A, Original current traces of TREK-1 channels measured under control condition (con), during the application of NaHCO3 (90 mM), and after washout (wash) of NaHCO3. B, Intracellular acidification induced by NaHCO3 increased the transfer of charge across the membrane 5.5 ± 0.3 fold as compared with control conditions (n = 3 paired experiments).

 
After characterizing the basic properties of TREK-1 channels, the effects of bupivacaine on these channels were analyzed with a ramp-pulse protocol (see Methods). Bupivacaine inhibited TREK-1 channels in a concentration-dependent and reversible manner (Fig. 3A). Channel inhibition was quantified as the reduction of charge transfer (Q) between the mp of -80 mV and 0 mV (see Methods). The data of the concentration-dependent inhibition were mathematically described by a Hill equation (Fig. 3B). The IC50 value was 370 ± 20 µM and the Hill coefficient was 1.8 ± 0.1 (mean ± SEM, n = 51). Voltage dependence of inhibition was analyzed by quantifying the reduction of Q between the mp of -80 and -40 mV, between -40 and 0 mV, and between 0 and +40 mV at a concentration of 300 µM bupivacaine (Fig. 3C). Inhibition of TREK-1 channels was not voltage-dependent (37% ± 9% versus 39% ± 7% versus 39% ± 6%, n = 10, respectively, P > 0.05). To test for possible differences in potency, inhibition of TREK-1 channels by racemic bupivacaine was compared with inhibition of TREK-1 channels by levobupivacaine at a concentration close to the IC50 value of bupivacaine (300 µM) (Fig. 4A). In case of different potency, both drugs would be expected to inhibit TREK-1 channels to a different extent at this concentration. The extent of inhibition was not different between bupivacaine and levobupivacaine (38% ± 8% [n = 10], versus 44% ± 13% [n = 13], P > 0.05, Fig. 4B).



View larger version (14K):
[in this window]
[in a new window]
  		Figure 3. A, Original current traces demonstrating inhibition of TREK-1 channels by bupivacaine. The inhibition of bupivacaine was concentration-dependent and reversible. Shown are current traces under control conditions (con), under the influence of bupivacaine (bup) (300 and 1000 µM), and after washout of the drug (wash). B, The concentration-response data were described by a Hill function. The 50% inhibitory concentration value was 370 ± 20 µM, the Hill coefficient was 1.8 ± 0.1, and the maximal block was 0.93 ± 0.02 (mean ± SEM, n = 51). C, Voltage-dependence of inhibition was analyzed at a bupivacaine concentration of 300 µM by comparing the charge transfer (Q) between the membrane potential of -80 and -40 mV, with Q between -40 and 0 mV, and with Q between 0 and +40 mV, respectively. Inhibition did not depend on voltage (37% ± 9% versus 39% ± 7% versus 39% ± 6%, n = 10, P > 0.05). n.s. = not significant.

 


View larger version (16K):
[in this window]
[in a new window]
  		Figure 4. A, Original current recordings of TREK-1 channels under control conditions (con) during the inhibition by bupivacaine (bup) (300 µM) and levobupivacaine (levobup) (300 µM) and after washout (wash) of the drug effects. B, Both drugs inhibited TREK-1 channels with equal potency (38% ± 8% [n = 10], versus 44% ± 13% [n = 13], P > 0.05). C, Effect of intracellular acidification on inhibition by bupivacaine. Original current traces of TREK-1 channels evoked by a ramp protocol under control condition with standard extracellular solution (const), under the influence of bupivacaine (300 µM) dissolved in standard extracellular solution (bupst), under the influence of bupivacaine (300 µM) dissolved in extracellular solution containing 90 mM NaHCO3 (bupNaHCO3), and after washout of bupivacaine with extracellular solution containing 90 mM NaHCO3 (conNaHCO3). D, The bar graph compares charge transfer (Q) under each condition normalized to the control condition (const). Inhibition of TREK-1 channels by bupivacaine increased approximately twofold by intracellular acidosis (44% ± 4% versus 92% ± 7%, n = 4 paired experiments, P < 0.05). Washout of bupNaHCO3 with extracellular solution containing NaHCO3 increased the charge transfer 6 ± 3 fold when compared with control conditions. n.s. = not significant.

 
The influence of intracellular acidosis on the inhibitory effect of bupivacaine was also investigated (Fig. 4C). For this purpose, TREK-1 currents were recorded under control conditions with standard extracellular solution (const), under the influence of bupivacaine (300 µM) dissolved in standard extracellular solution (bupst), under the influence of bupivacaine (300 µM) dissolved in extracellular solution containing 90 mM NaHCO3 (bupNaHCO3), and after washout of bupivacaine with extracellular solution containing 90 mM NaHCO3 (conNaHCO3). The pH was kept constant at 7.4 under each experimental condition. Q through TREK-1 channels was 131 ± 103 pC during superfusion with const, 72 ± 54 pC during superfusion with bupst, 33 ± 13 pC during superfusion with bupNaHCO3, and 742 ± 529 pC during superfusion with conNaHCO3. Inhibition of Q during application of bupst was quantified in relation to Q under conditions of const. Inhibition of Q during application of bupNaHCO3 was quantified in relation to Q under conditions of conNaHCO3. Inhibition of TREK-1 channels by bupivacaine increased approximately twofold by intracellular acidosis (44% ± 4% versus 92% ± 7%, n = 4 paired experiments, P < 0.05). The inhibition was not voltage-dependent under condition of intracellular acidosis (90% ± 10% versus 93% ± 7% versus 94% ± 5%, n = 4 paired experiments, P > 0.05). After the washout of bupNaHCO3 with the solution containing 90 mM NaHCO3, TREK-1 channels were stimulated by intra- cellular acidosis and the Q increased by 6 ± 3 fold compared with control conditions (n = 4 paired experiments, Fig. 4D). Interaction of bupivacaine with TREK-1 channels prevented stimulation of the channels by intracellular acidification.

Transfection of CHO cells with TREK-1 channels resulted in a hyperpolarization of the mp of these cells (see above). Inhibition of TREK-1 channels by bupivacaine would thus be expected to depolarize the cell membrane. Superfusing bupivacaine on CHOTREK-1 cells caused a depolarization of the cell membrane that was immediate, concentration-dependent, and reversible (Fig. 5A). Concentration-dependent depolarization was quantified by calculating the difference between the mp under each individual drug condition and the respective mean mp under control and washout conditions (see Methods) (Fig. 5B). Mathematical description of the concentration-dependent effect by a Hill function yielded an IC50 value of 856 ± 14 µM and a Hill coefficient of 2.4 ± 0.1 (mean ± SEM, n = 27). The depolarization at the largest concentration of bupivacaine tested (3000 µM) was 46 ± 4 mV (n = 5). The concentration-response curve for membrane depolarization was significantly different from the concentration-response curve for TREK-1 channel inhibition (P < 0.01).



View larger version (13K):
[in this window]
[in a new window]
  		Figure 5. A, Original recordings of the membrane potential of Chinese hamster ovary (CHO)TREK-1 cells under control condition (con), during application of bupivacaine (bup) (1 mM), and after washout (wash) of the drug. The local anesthetic induced an immediate depolarization of the membrane that was reversible on washout. B, The data of the concentration-dependent depolarization of the cell membrane induced by bupivacaine were mathematically described by a Hill equation. The depolarization induced by each individual concentration was normalized to the mean depolarization induced by 3000 µM bupivacaine. The 50% inhibitory concentration value was 856 ± 14 µM, the Hill coefficient was 2.4 ± 0.1, and the maximal block was 1.04 ± 0.01 (mean ± SEM, n = 27). C, The effect of bupivacaine on the membrane potential of CHOwt cells was not concentration-dependent (n = 14). Linear regression analysis yielded a regression coefficient of r = 0.54 and a negative slope of the regression line of -0.003. The maximal depolarizing effect occurred at 300 µM (24 ± 15 mV, n = 3).

 
To exclude the possibility that the depolarization induced by bupivacaine was not related to inhibition of TREK-1 channels, the effect of bupivacaine on the mp of CHOwt cells was also measured at four different concentrations. Although bupivacaine at these concentrations caused a reversible depolarization of these cells, the effect did not increase with larger concentration as observed with CHOTREK-1 cells (Fig. 5C). Linear regression analysis yielded a regression coefficient of r = 0.54 and a negative slope of the regression line of -0.003. The maximal depolarizing effect occurred at 300 µM (24 ± 15 mV, n = 3).


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
TREK-1 channels and other related members of the KCNK family are the physiological correlate of potassium leak currents or background conductances of excitable cells (1–3). These channels control the excitability of neurons by regulating the resting mp (2,4). The current-voltage relationship of TREK-1 channels in our study exhibited typical features of these channels (5). Under condition of a large internal and small external potassium concentration, small inward currents negative and large outward currents positive to the equilibrium potential of potassium were observed (5). The channels were sensitive to intracellular acidification (13), and they hyperpolarized the mp of CHO cells from -33 mV by 45 mV to a value close to the equilibrium potential of potassium.

Bupivacaine inhibited human TREK-1 channels in a concentration-dependent and reversible manner. The local anesthetic also depolarized the mp of CHOTREK-1 cells in a concentration-dependent manner. However, concentrations necessary for channel inhibition and membrane depolarization were not linearly related. The IC50 values of bupivacaine for channel inhibition and membrane depolarization differed by a factor of 2.3.

Different explanations may be offered for this observation. It may be argued that membrane depolarization did not result from inhibition of TREK-1 channels. However, this seems unlikely. Bupivacaine (3000 µM) had a depolarizing effect of 46 mV on the mp of CHOTREK-1 cells. This effect mirrors the hyperpolarization induced by transfection of CHO cells with TREK-1 channels. Furthermore, control experiments with CHOwt cells demonstrated that the mp of these cells was not influenced by bupivacaine in a concentration-dependent manner. In principle, the observed difference between IC50 values for inhibition and depolarization may also result from a strong voltage-dependence of inhibition. However, inhibition of TREK-1 channels did not depend on voltage.

Alternative explanations will have to account for the observed difference between concentration-response curves for inhibition and depolarization. Transfection of a genetic construct in mammalian cells with a cytomegalovirus promoter such as that used in our study leads to an overexpression of the transfected construct (product information; Invitrogen). As a consequence, more TREK-1 channels will be expressed in CHOTREK-1 cells than necessary to maintain the mp constant at a value close to the equilibrium potential for potassium (18). Overexpression may introduce a margin of safety for keeping the mp stable at -80 mV as known from other biological systems (19). However, because the IC50 values were arrived at in one case by measuring TREK-1 channel inhibition and in the other by measuring the mp of CHOTREK-1 cells, it cannot be excluded that these two different methodical approaches may contribute to the difference in IC50 values.

TREK-1 channels are directly stimulated by intracellular acidosis (13). We, therefore, hypothesized that inhibition of TREK-1 channels by bupivacaine may be antagonized by intracellular acidification. This was not the case. On the contrary, despite stimulation of these channels by intracellular acidosis by a factor >5, inhibition of TREK-1 channels by bupivacaine approximately doubled from 44% to 92%. At a pH of 7.4, nearly 83% of bupivacaine (pKa = 8.1) (20) dissolved in the extracellular solution was protonated and therefore positively charged. This ratio of protonated to unprotonated bupivacaine in the extracellular solution was not altered by the presence of 90 mM NaHCO3 as the pH was kept constant. Extracellular application of NaHCO3 results in intracellular acidosis (13,15) and this in turn increases the fraction of protonated bupivacaine in the intracellular space. The increase of bupivacaine inhibition with intracellular acidosis suggests that bupivacaine exhibits its effect primarily by the protonated molecule. Because the protonated molecule does not diffuse out of the cell it seems plausible to assume that bupivacaine interacts with TREK-1 channels from the intracellular space.

In a previous work, it was shown that two-pore-domain channels are inhibited by local anesthetics (10). In their study, the authors focused on TASK channels and reported that bupivacaine inhibits TASK channels with an IC50 value ninefold less than TREK-1 channels in our study. Furthermore, channel inhibition occurred with a Hill coefficient close to unity and inhibition was voltage-dependent. Because TASK and TREK-1 channels share a sequence identity of only 28% (21), the difference in bupivacaine sensitivity of TASK and TREK-1 channels may be explained by a subtype-specific action of bupivacaine. Although species differences cannot be excluded, the view that bupivacaine action on two-pore-domain channels is subtype specific is further supported by comparable bupivacaine (1000 µM) sensitivities of TREK-1 channels from mice expressed in oocytes from Xenopus laevis (10) and TREK-1 channels from humans expressed in CHO cells (this study). Nonetheless, despite the differences in sensitivity between TASK and TREK-1 channels, bupivacaine (1000 µM) depolarized the mp of CHOTREK-1 cells to nearly the same extent as the mp of oocytes expressing TASK channels (28 versus 31 mV). The cellular environment, therefore, seems to strongly influence the quantitative relationship between channel inhibition and membrane depolarization.

Because of the physiological significance of TREK-1 channels and their abundant expression in dorsal root ganglions, and the spinal cord (6), the inhibition of TREK-1 channels may have implications for clinically observed drug action of bupivacaine. It has to be considered that the concentration of bupivacaine when used for regional anesthesia may be as large as 17 mM (22). During the therapeutic application at a peripheral nerve or at the spinal cord, a concentration of 1 mM will easily be reached. The systemic concentration of bupivacaine in arterial blood during toxic side effects will be smaller than the therapeutic concentration. Because of the pharmacokinetics of bupivacaine and the volume of distribution, toxic plasma concentrations during seizure will be in the range of 35–100 µM (23,24). The relationship of concentrations of in vitro effects and concentrations observed during toxic effects in vivo suggests that the inhibition of TREK-1 channels by bupivacaine may not contribute to toxic side effects of this local anesthetic. TREK-1 channels may thus rather constitute a target for therapeutic than for toxic action of bupivacaine.

The mp of neurons normally rests at a value close to the reversal potential of potassium because of open ion channels such as TREK-1 (2). Action potentials are released after the mp depolarizes to a threshold value that allows opening of voltage-gated sodium channels. Sodium channels inactivate and need to recover from inactivation at the negative resting mp to be able to open again. Bupivacaine-induced TREK-1 channel inhibition will cause the resting mp to be more depolarized. As a consequence, inhibition of TREK-1 channels will impair the generation of action potentials by increasing the number of inactivated sodium channels. Furthermore, local anesthetics preferentially and more rapidly bind to inactivated sodium channels (8,9,25). By increasing the binding of local anesthetics to sodium channels, inhibition of TREK-1 channels will, thus, additionally impair neuronal signal conduction.

It cannot be excluded that inhibition of TREK-1 channels may also account for neurotoxic side effects of bupivacaine such as seizures. In humans, first signs of central nervous system toxicity such as perioral numbness occur at free plasma concentrations of 1 µM bupivacaine (26). Seizures are observed at concentrations of 32–126 µM in dogs and have been estimated to occur at concentrations of bupivacaine of at least 35 µM in humans (23,24). However, because of the difference between concentrations of in vitro effects and concentrations of toxic effects in vivo (23,24), this seems less likely. Basically, we did not observe significant in vitro effects at concentrations measured systemically during toxic side effects of bupivacaine (23,24). Furthermore, despite an equivalent analgesic potency of bupivacaine and levobupivacaine (27), both drugs are supposed to differ in their neurotoxic reaction profile (28). Drugs with a specific drug-receptor interaction would be expected to exhibit different potencies at a receptor responsible for a given drug effect (29). The identical potencies of bupivacaine and levobupivacaine to inhibit TREK-1 channels and the relatively large concentrations needed to block these ion channels, suggest that TREK-1 channels rather constitute a target for therapeutic than for toxic action of bupivacaine and levobupivacaine.

In summary, transfection of CHO cells with TREK-1 channels hyperpolarized the mp from -33 to -78 mV. TREK-1 currents were small inward currents negative and large outward currents positive to the reversal potential of potassium. The channels were stimulated by intracellular acidosis. Human TREK-1 channels were inhibited by bupivacaine in a concentration-dependent and reversible manner. Inhibition was neither voltage-dependent nor different between bupivacaine and levobupivacaine. Inhibition of TREK-1 channels by bupivacaine increased with intracellular acidosis indicating that the local anesthetic primarily acted from the intracellular space. Inhibition of TREK-1 channels depolarized the mp although channel inhibition and membrane depolarization were not linearly related. Because of the physiological role of TREK-1 channels and the relationship of concentrations for in vitro effects and concentrations for in vivo action, as well as identical potencies of bupivacaine and levobupivacaine to inhibit these channels, inhibition of TREK-1 channels likely contributes to neuronal conductance block caused by these local anesthetics.


   Acknowledgments
 
This study was supported by the Deutsche Forschungsgemeinschaft (Graduierten Kolleg 255, Neurale Signaltransduktion und deren pathologische Störungen) and the Bundesministerium für Bildung und Forschung (BMBF 03F0261C).

We thank Andrea Zaisser for technical help. Levobupivacaine was a kind gift of AstraZeneca, Södertalje, Sweden.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Lesage F, Lazdunski M. Molecular and functional properties of two-pore-domain potassium channels. Am J Physiol Renal Physiol 2000; 279: F793–801.[Abstract/Free Full Text]
  2. Goldstein SA, Bockenhauer D, O’Kelly I, Zilberberg N. Potassium leak channels and the KCNK family of two-P-domain subunits. Nat Rev Neurosci 2001; 2: 175–84.[ISI][Medline]
  3. Patel AJ, Honore E. Anesthetic-sensitive 2P domain K+ channels. Anesthesiology 2001; 95: 1013–21.[ISI][Medline]
  4. Patel AJ, Honore E. Properties and modulation of mammalian 2P domain K+ channels. Trends Neurosci 2001; 24: 339–46.[ISI][Medline]
  5. Meadows HJ, Benham CD, Cairns W, et al. Cloning, localisation and functional expression of the human orthologue of the TREK-1 potassium channel. Pflugers Arch 2000; 439: 714–22.[ISI][Medline]
  6. Medhurst AD, Rennie G, Chapman CG, et al. Distribution analysis of human two pore domain potassium channels in tissues of the central nervous system and periphery. Brain Res Mol Brain Res 2001; 86: 101–14.[Medline]
  7. Butterworth JFIV, Strichartz GR. Molecular mechanisms of local anesthesia: a review. Anesthesiology 1990; 72: 711–34.[ISI][Medline]
  8. Drachman D, Strichartz G. Potassium channel blockers potentiate impulse inhibition by local anesthetics. Anesthesiology 1991; 75: 1051–61.[ISI][Medline]
  9. Brau ME, Nau C, Hempelmann G, Vogel W. Local anesthetics potently block a potential insensitive potassium channel in myelinated nerve. J Gen Physiol 1995; 105: 485–505.[Abstract/Free Full Text]
  10. Kindler CH, Yost CS, Gray AT. Local anesthetic inhibition of baseline potassium channels with two pore domains in tandem. Anesthesiology 1999; 90: 1092–1102.[ISI][Medline]
  11. Friederich P, Benzenberg D, Urban BW. Bupivacaine inhibits human neuronal Kv3 channels in SH-SY5Y human neuroblastoma cells. Br J Anaesth 2002; 88: 864–6.[Abstract/Free Full Text]
  12. Dilger JP. Basic pharmacology of inhalational anesthetics agents. In: Bowdle TA, Horita A, Kharasch ED, eds. The pharmacological basis of anesthesiology. New York: Churchill Livingstone, 1994: 497–521.
  13. Maingret F, Patel A, Lesage F, et al. Mechano- or acid stimulation, two interactive modes of activation of the TREK-1 potassium channel. J Biol Chem 1999; 274: 26691–6.[Abstract/Free Full Text]
  14. Hamill OP, Marty A, Neher E, et al. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch 1981; 391: 85–100.[ISI][Medline]
  15. Fakler B, Schultz JH, Yang J, et al. Identification of a titratable lysine residue that determines sensitivity of kidney potassium channels (ROMK) to intracellular pH. EMBO J 1996; 15: 4093–9.[ISI][Medline]
  16. Winne D. Sequential statistical trials in medicine. Arzneimittelforschung 1965; 15: 1088–91.[Medline]
  17. Motulsky H. Graph pad guide to nonlinear regression. San Diego, CA: GraphPad Software, 1996.
  18. Hille B. Introduction. In: Hille B, ed. Ion channels of excitable membranes. 3rd ed. Sunderland, MA: Sinauer Associates, 2001: 1–22.
  19. Paton WD, Waud DR. The margin of safety of neuromuscular transmission. J Physiol 1967; 191: 59–90.[ISI][Medline]
  20. Merck index: an encyclopedia of chemicals, drugs and biologicals. 12th ed. Whitehouse Station, NJ: Merck & Co., 1996.
  21. Patel A, Honore E, Maingret F, et al. A mammalian two pore domain mechano-gated S-like K+ channel. EMBO J 1998; 17: 4283–90.[ISI][Medline]
  22. Urban BW, Friederich P. Local anaesthetics-toxicity. In: Schulte am Esch J, Scholz J, Tonner P, eds. Molecular pharmacology of anaesthesia. Lengerich: Pabst Science Publishers, 2000: 151–63.
  23. Feldman HS, Arthur GR, Covino BG. Comparative systemic toxicity of convulsant and supraconvulsant doses of intravenous ropivacaine, bupivacaine, and lidocaine in the conscious dog. Anesth Analg 1989; 69: 794–801.[Abstract/Free Full Text]
  24. Tucker GT. Pharmacokinetics of local anaesthetics. Br J Anaesth 1986; 58: 717–31.[Free Full Text]
  25. Hille B. Classical mechanisms of block. In: Hille B, ed. Ion channels of excitable membranes. 3rd ed. Sunderland, MA: Sinauer Associates, 2001: 503–37.
  26. Knudsen K, Beckman Suurkula M, Blomberg S, et al. Central nervous and cardiovascular effects of IV infusions of ropivacaine, bupivacaine and placebo in volunteers. Br J Anaesth 1997; 78: 507–14.[Abstract/Free Full Text]
  27. Cox CR, Faccenda KA, Gilhooly C, et al. Extradural S(-)-bupivacaine: comparison with racemic RS-bupivacaine. Br J Anaesth 1998; 80: 289–93.[Abstract/Free Full Text]
  28. Huang YF, Pryor ME, Mather LE, Veering BT. Cardiovascular and central nervous system effects of intravenous levobupivacaine and bupivacaine in sheep. Anesth Analg 1998; 86: 797–804.[Abstract]
  29. Nau C, Vogel W, Hempelmann G, Brau ME. Stereoselectivity of bupivacaine in local anesthetic-sensitive ion channels of peripheral nerve. Anesthesiology 1999; 91: 786–95.[ISI][Medline]
Accepted for publication February 3, 2003.




This article has been cited by other articles:


Home page
 Am. J. Physiol. Cell Physiol.Home page
M. Magra, S. Hughes, A. J. El Haj, and N. Maffulli
VOCCs and TREK-1 ion channel expression in human tenocytes
Am J Physiol Cell Physiol, March 1, 2007; 292(3): C1053 - C1060.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
J. Chemin, A. Patel, F. Duprat, M. Zanzouri, M. Lazdunski, and E. Honore
Lysophosphatidic Acid-operated K+ Channels
J. Biol. Chem., February 11, 2005; 280(6): 4415 - 4421.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a colleague
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (10)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Punke, M. A.
Right arrow Articles by Friederich, P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Punke, M. A.
Right arrow Articles by Friederich, P.
Related Collections
Right arrow Mechanisms
Right arrow Pharmacology

Lippincott, Williams & Wilkins Anesthesia & Analgesia® is published for the International Anesthesia Research Society® by Lippincott Williams & Wilkins with the assistance of Stanford University Libraries' HighWire Press®. Copyright 2006 by the International Anesthesia Research Society. Online ISSN: 1526-7598   Print ISSN: 0003-2999 HighWire Press