Methadone is a local anaesthetic-like inhibitor of neuronal Na+ channels and blocks excitability of mouse peripheral nerves  C. Stoetzer, K. Kistner, T. Stüber, M. Wirths, V. Schulze, T. Doll, N. Foadi, F. Wegner, J. Ahrens, A. Leffler  British Journal of Anaesthesia  Volume 114, Issue 1, Pages 110-120 (January 2015) DOI: 10.1093/bja/aeu206 Copyright © 2015 The Author(s) Terms and Conditions

Fig 1 Inhibition of neuronal Na+ channels by methadone. (a) Representative families of current traces generated by Na+ channels in ND7/23 cells in the presence of control solution or 100 µM methadone. Cells were held at −120 mV and currents were elicited by 20 ms long test-pulses ranging from −90 to 40 mV in steps of 10 mV. (b) Concentration-dependent tonic block of resting (filled symbols) and inactivated (empty symbols) Na+ channels by methadone. Resting channels were examined from a holding potential of −120 mV, inactivated channels were induced by a 10 s long pre-pulse at −70 mV followed by a 100 ms long pulse at −120 mV allowing recovery from fast inactivation. Peak amplitudes of Na+ currents at different drug concentrations were normalized with respect to the peak amplitude in control solution and plotted against the concentration of methadone. Data were fitted with the Hill equation represented by the solid line. (c) Development of use-dependent block of neuronal Na+ channels by 10 and 100 μM methadone. Peak currents were normalized to the amplitude of the first pulse and plotted against the pulse number. (d) Normalized current–voltage curves of currents obtained in (a). Currents were normalized to the peak currents amplitude and plotted against the corresponding membrane potential. The lines are drawn to guide the eye. (e) Voltage-dependency of fast inactivation of Na+ channels in ND7/23 cells in control solution or in 10 µM and 100 µM methadone. Fast inactivation was induced by 50 ms long pre-pulses ranging from −120 to −30 mV in steps of 5 mV, and the remaining fraction of available channels was examined with a 20 ms long pre-pulse to 0 mV. The solid lines represent fits calculated with the Boltzmann equation. (f) Voltage-dependency of slow inactivation of Na+ channels in ND7/23 cells in control solution and in the presence of 3 µM methadone. Slow inactivation was induced by 10 s long pre-pulses ranging from −120 to −10 mV in steps of 10 mV followed by a 100 ms long inter-pulse at −120 mV allowing recovery from fast inactivation. The solid lines represent fits calculated with the Boltzmann equation. (g) Concentration-dependent tonic block of resting Na+ channels by bupivacaine examined and calculated as described above for methadone. (h) Fractional inhibition of resting Na+ channels by 50 µM bupivacaine with and without methadone at 10 and 100 µM. Peak current amplitudes were normalized with respect to the recording in control solution and each cell was subsequently treated with each test solution. (i) Use-dependent block of neuronal Na+ channels by 10 µM bupivacaine with and without 10 µM methadone. Peak currents were normalized to the amplitude of the first pulse and plotted against the pulse number. All data are presented as mean (sd). British Journal of Anaesthesia 2015 114, 110-120DOI: (10.1093/bja/aeu206) Copyright © 2015 The Author(s) Terms and Conditions

Fig 2 Non-selective inhibition of neuronal α-subunits of Na+ channels by methadone. (a) Representative currents traces of Nav1.8 expressed in ND7/23 cells. Cells were held at −120 mV and test pulses to 0 mV were applied at 0.1 Hz. Methadone at increasing concentrations were applied until a steady-state block by each concentration was established. (b) Concentration-dependent tonic block of resting Nav1.2, Nav1.3, Nav1.7 channels expressed in HEK 293t cells and Nav1.8 channels expressed in ND7/23 cells. Peak amplitudes of Na+ currents at different drug concentrations were normalized with respect to the peak amplitude in control solution and plotted against the concentration of methadone. Data were fitted with the Hill equation represented by the solid line. (c) Typical original trace from Nav1.7 expressed in HEK 293 cells. For use-dependent block, cells were held at −120 mV and currents were activated at 10 Hz by test pulses to 0 mV. (d) Development of use-dependent block of Nav1.2, Nav1.3, Nav1.7, and Nav1.8 by 100 μM methadone. Peak currents were normalized to the amplitude of the first pulse and plotted against the pulse number. British Journal of Anaesthesia 2015 114, 110-120DOI: (10.1093/bja/aeu206) Copyright © 2015 The Author(s) Terms and Conditions

Fig 3 Block of nerve conduction in peripheral C- and A-fibre axons by methadone and bupivacaine. (a and b) Representative traces of the latencies of C-fibre (a) and A-fibre (b) CAPs during application of increasing concentrations of methadone (30–1000 µM). The coloured columns indicate the time-point and duration of each concentration of methadone, and the intervals between these applications represent the time for wash-out and recovery of CAPs. Note that methadone induces an increased latency of CAPs and that this effect is concentration-dependent. (c) Dose–response curves for effects of methadone (circles) and bupivacaine (squares) on the latency of C- (blue) and A-fibre (green) CAPs. The drawn lines represent fits of the data as calculated with the Hill equation. (d and e) Representative traces of the amplitudes of C-fibre (d) and A-fibre (e) CAP during application of increasing concentrations of methadone. Note that methadone induces a concentration-dependent reduction in CAP amplitudes. (f) Dose–response curves for effects of methadone (circles) and bupivacaine (squares) on the amplitudes of C- (blue) and A-fibre (green) CAPs. The drawn lines represent fits of the data as calculated with the Hill equation. British Journal of Anaesthesia 2015 114, 110-120DOI: (10.1093/bja/aeu206) Copyright © 2015 The Author(s) Terms and Conditions

Fig 4 Stereo-selective block of Nav1.7 by methadone. (a) Chemical structures of dextromethadone and levomethadon. (b) Concentration-dependent tonic block of resting Nav1.7 channels by dextromethadone (open squares) and levomethadone (filled squares). Currents were activated at 0.1 Hz in cells held at −120 mV and their peak amplitudes at different drug concentrations were normalized with respect to the peak amplitude in control solution. Data were fitted with the Hill equation represented by the solid line. (c) Development of use-dependent block at 10 Hz of Nav1.7 by 100 μM dextromethadone or levomethadone. Peak currents were normalized to the amplitude of the first pulse and plotted against the pulse number. Error bars represent mean (sd). British Journal of Anaesthesia 2015 114, 110-120DOI: (10.1093/bja/aeu206) Copyright © 2015 The Author(s) Terms and Conditions

Fig 5 Inhibition of HCN2 by methadone. (a). Representative original traces of HCN2 channels expressed in HEK 293t cells held at −40 mV. Currents were induced by 1 s long test pulses to 100 mV applied at 0.1 Hz. Racematic methadone was applied at increasing concentrations until a steady-state block was reached for each concentration. (b) Concentration-dependent block of HCN2 by methadone. Peak current amplitudes at different drug concentrations were normalized with respect to the peak amplitude in control solution. Data were fitted with the Hill equation represented by the solid line. Error bars represent mean (sd). British Journal of Anaesthesia 2015 114, 110-120DOI: (10.1093/bja/aeu206) Copyright © 2015 The Author(s) Terms and Conditions

Fig 6 Cytotoxicity by methadone and bupivacaine in HEK 293t cells. (a and b) PI and Annexin V double staining was explored with flow cytometry on HEK 293t cells after 12 h treatment with methadone (a) and bupivacaine (b) at increasing concentrations. Staining with Annexin V identifies cells in the early phase of apoptosis and staining with both Annexin V and PI identifies necrotic or dead cells. The fraction of cells stained with Annexin V (blue columns) or Annexin V+PI (green shaded columns) are plotted against the applied concentrations of methadone and bupivacaine. Error bars represent mean (sd). British Journal of Anaesthesia 2015 114, 110-120DOI: (10.1093/bja/aeu206) Copyright © 2015 The Author(s) Terms and Conditions