Bupivacaine inhibits human neuronal Kv3 channels in SH-SY5Y human neuroblastoma cells

P. Friederich*,1, D. Benzenberg2 and B. W. Urban3

1Clinic of Anaesthesiology, University Hospital Eppendorf and Institute of Neural Signal Transduction, Centre for Molecular Neurobiology, University of Hamburg, Germany. 2Department of Anaesthesiology and Intensive Care Medicine, University of Bonn, Germany. 3Department of Anaesthesiology and Intensive Care Medicine, University of Bonn, Germany, and Departments of Anaesthesiology and Physiology, Weill Medical College of Cornell University, New York, NY, USA.*Corresponding author: Klinik für Anästhesiologie, Universitätsklinik Eppendorf, Martinistrasse 52, D-20246 Hamburg, Germany

Accepted for publication: January 28, 2002


    Abstract
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 Abstract
 Introduction
 Methods and results
 Comment
 References
 
Background. Information on molecular targets that may be involved in the neurotoxicity of bupivacaine is limited. Suppression of Kv3 channels has been demonstrated to result in abnormal patterns in the electroencephalogram and in seizures. Inhibition of Kv3 channels by bupivacaine may consequently contribute to its neuroexcitatory side-effects. Data on the effects of bupivacaine on these potassium channels are lacking. We therefore characterized the effects of bupivacaine on human Kv3 channels natively expressed in SH-SY5Y cells.

Methods. Kv3 channels natively expressed in human SH-SY5Y cells were studied using a standard whole-cell patch-clamp protocol.

Results. Bupivacaine reversibly inhibited Kv3 channels in a concentration-dependent manner. The half-maximal inhibitory concentration (IC50) for conductance block was 57 µM and the Hill coefficient was close to unity. Bupivacaine accelerated macroscopic current decline by inducing inactivation-like behaviour. The midpoint of current activation was shifted to depolarized potentials in a concentration-dependent and reversible manner by a maximum of 26 mV. The IC50 was 47 µM and the Hill coefficient was 2.4. The free arterial plasma concentrations of bupivacaine that have been estimated to occur during convulsions in man would inhibit the Kv3 channels by at least 40% and would shift the midpoint of current activation by a minimum of 9 mV.

Conclusions. Both inhibition of potassium channels and a depolarizing shift of their activation midpoint would increase neuronal excitability. The effects of bupivacaine on human Kv3 channels are thus compatible with a contributory role of Kv channel alteration in bupivacaine-induced neuronal excitation.

Br J Anaesth 2002; 88: 864–6

Keywords: anaesthetics local; complications, side-effects; complications, seizure


    Introduction
 Top
 Abstract
 Introduction
 Methods and results
 Comment
 References
 
Excitatory actions in the human nervous system are well-known side-effects of local anaesthetic agents.1 These side-effects may occur in up to one in 145 patients during regional anaesthesia.1 Seizures induced by local anaesthetics may be accompanied by cardiovascular changes, acidosis and severe hypoxia.2

Information on the anaesthetic targets that may be involved in these neurotoxic effects is limited. Recent molecular evidence emphasizes the importance of human voltage-dependent potassium channels in the pathogenesis of epileptic disorders.3 Mutations in the genes encoding voltage-dependent potassium channels of the Kv and KCNQ channel families cause seizures in animal and man through a decrease in neuronal potassium currents.3 Local anaesthetic-induced potassium current suppression may thus contribute significantly to the excitatory side-effects of these anaesthetic agents.

The aim of this study was to characterize the effects of bupivacaine on human neuronal Kv3 channels. Kv3 channels are crucial for spike frequency adaptation in central neurones.4 Evidence from knockout mice demonstrates that suppression of Kv3 channels causes impaired motor skill, muscle contraction, abnormal EEG patterns and epileptic seizures.4 Inhibition of human Kv3 channels by bupivacaine may contribute to the neuronal excitation and epileptic seizures observed during accidental intravascular application of this drug.


    Methods and results
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 Abstract
 Introduction
 Methods and results
 Comment
 References
 
Voltage-sensitive outward currents in SH-SY5Y cells5 were recorded with the whole-cell patch-clamp technique6 at room temperature (22–25°C) with an EPC-7 amplifier (List Electronic, Darmstadt, Germany) and pClamp software version 5.71 (Axon Instruments, Foster City, CA, USA) as described previously.7 The peak outward potassium current in a trace was determined by fitting the current to a two-exponential time course (activation and inactivation) and determining the maximal amplitude of the fit. Peak currents were converted to conductances using the Nernst potential for potassium and the voltage was corrected using the measured series resistance and compensation level. The conductance–voltage data were fitted to a Boltzmann function. Inhibition (b) was defined as 1 minus the ratio of the maximal potassium conductance (Gmax) in the presence of the drug to the mean of the current before and after application of the drug. Changes in activation midpoint (Vmid) were quantified as the difference between Vmid in the presence of the drug and the mean of Vmid before and after application of the drug. Concentration–response curves were fitted with the Hill equation. Data are given as mean (standard deviation) for n experiments unless stated otherwise. Statistical comparison was performed with the unpaired Student’s t-test.

Bupivacaine reversibly inhibited human Kv3 potassium channels. The potassium current traces in Figure 1 demonstrate the inhibitory effect. Bupivacaine accelerated macroscopic current decline (Fig. 1) by inducing inactivation-like behaviour. In the presence of bupivacaine, the potassium currents declined more than 100–150 times faster at potentials between +40 and +70 mV compared with potassium currents in the absence of the local anaesthetic. The time constants of this inactivation-like behaviour did not differ between test potentials of +40 and +70 mV. The time constants depended on concentration [4.4 (1.5) and 2.9 (1.2) ms at 10 and 100 µM respectively; P<0.05, n=8–10].



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Fig 1 Superimposed traces of voltage-dependent Kv3 channels activated by depolarizing steps from a holding potential of –80 mV to test potentials ranging from –50 to +70 mV. The interpulse duration was 1s. The traces show current during control (left), with bupivacaine (middle) and after the drug had been washed out (right). Below is the concentration–response curve for inhibition of maximal Kv3 whole-cell conductance (Gmax) by bupivacaine. Data are mean and standard deviation (n=8–12) and are expressed as inhibitory fraction (maximum 1.0).

 
The effect of the local anaesthetic on these potassium channels was measured as inhibition of the maximal whole-cell conductance (Gmax; for details, see above). Under control conditions, Gmax was 7.5 (1.8) nS (n=60). Inhibition of Gmax was concentration-dependent and reversible (Fig. 1). It was described mathematically by Hill functions. The half-maximal inhibitory concentration (IC50) for inhibition of Gmax by bupivacaine was 57 µM. The Hill coefficient was 0.7 (n=60). The membrane potential at which half the channels were activated (Vmid) was shifted by bupivacaine in the depolarizing direction. The reversible shift was concentration-dependent and had a maximum value of 26 mV. The IC50 for the shift was 47 µM and the Hill coefficient was 2.4 (n=60).


    Comment
 Top
 Abstract
 Introduction
 Methods and results
 Comment
 References
 
The local anaesthetic agent bupivacaine reversibly inhibited human neuronal Kv3 channels in a concentration-dependent manner. The midpoint of current activation was shifted to more depolarized potentials by the local anaesthetic. Both drug effects would be compatible with a contributory role of Kv channel inhibition in bupivacaine-induced convulsion.8

Human Kv3 channels belong to a superfamily of voltage-dependent potassium channels consisting of six transmembrane domains.3 4 Mutations of several members of this potassium channel superfamily cause inherited forms of human epilepsy by suppression of neuronal potassium channel activity.3 Kv3 knockout mice exhibit abnormal neuronal behaviour ranging from impaired motor skill to abnormal EEG patterns and epileptic seizures.4 Estimates based on in vitro and animal experiments suggest that inhibition of voltage-dependent potassium channels by 25–50% may cause epilepsy.3 As calculated from the Hill equation, the inhibition produced by the free plasma concentrations of bupivacaine that are estimated to cause seizure in man (35 µM)9 and dogs (32–126 µM)10 would suppress the Kv3 channels by at least 40%. The observed inhibition of human Kv3 channels by bupivacaine would thus be sufficient to account for excitatory side-effects.

The depolarizing shift of the activation threshold by 10–20 mV induced by bupivacaine would add to the possible increase in neuronal excitability induced by inhibition of the potassium channel conductance.8 Both a 50% channel block and a 15 mV depolarizing shift would alone induce spontaneous firing of the Hodgkin–Huxley squid axon model.8 In the presence of a 15 mV depolarizing shift in potassium channel activation, spontaneous firing of the axon would persist if 90% of the sodium channels were blocked.8 The combination of potassium channel inhibition and a depolarizing shift in the activation threshold, as observed in our study, would be compatible with neuronal excitation during bupivacaine intoxication. Establishing structural requirements for local anaesthetic action on human neuronal Kv channels may therefore help to elucidate the molecular determinants of the convulsive side-effects of this drug.


    Acknowledgements
 
We are grateful to Z. Dorner for cell culture. This study was supported by a grant from the Bonner Forschungskommission, Bonn, Germany (PF, O-117.0005).


    References
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 Abstract
 Introduction
 Methods and results
 Comment
 References
 
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5 Friederich P, Dilger JP, Pongs O, Urban BW. Kv3.1 expression in human neuroblastoma SH-SY5Y cells. Pflügers Arch 2000; 439: R427

6 Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflügers Arch 1981; 391: 85–100[ISI][Medline]

7 Friederich P, Benzenberg D, Trellakis S, Urban BW. Interaction of volatile anesthetics with human Kv channels in relation to clinical concentrations. Anesthesiology 2001; 95: 954–8[ISI][Medline]

8 Elliott AA, Elliott JR. Voltage-dependent inhibition of RCK1 K+ channels by phenol, p-cresol, and benzyl alcohol. Mol Pharmacol 1997; 51: 475–83[Abstract/Free Full Text]

9 Tucker GT. Pharmacokinetics of local anaesthetics. Br J Anaesth 1986; 58: 717–31[ISI][Medline]

10 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]