The plant alkaloid harmaline blocks the human voltage-gated sodium channel Na v 1.7 expressed in CHO cells: a study using automated patch clamp
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1 Ernst Moritz Arndt University of Greifswald Institute of Pathophysiology The plant alkaloid harmaline blocks the human voltage-gated sodium channel Na v 1.7 expressed in CHO cells: a study using automated patch clamp Heinrich Brinkmeier Vancouver, August 9, 2017
2 The voltage-gated sodium channel Na v 1.7, -subunit is expressed in the neuroendocrine system and peripheral nociceptive neurons is absolutely essential for nociception is highly sensitive to TTX and can be blocked by local anaesthetics, such as ranilozine and lidocaine From: Hoeijmakers et al. (2015) Neurosci Lett 596:51 59
3 Harmaline one of several alkaloids produced by the syrian rue, Peganum harmala (insecticide and antimicrobial substance) intake can cause neurological symptoms in humans, such as visual hallucination, agitation, tremor is used against pain syndromes in North African traditional medicine is a reversible monoamine oxidase inhibitor MW: Da blocks voltage-gated Ca 2+ channels and several potassium channels (> 100 µm)
4 Main questions of this study Are Na v 1.7 currents inhibited by harmaline? and if How does the plant alkaloid harmaline affect the human voltage-gated sodium channel Na v 1.7? pilot study with the Cytopach, an automated system for patch clamping
5 Verification of expression of the hna v subunit in CHO cells First row: CHO K1 (original cell line) Second row: CHO hnav1.7 #4438/S4 (transfected) blue: DAPI, red Na v 1.7 IF bars: 20 µm
6 Use of an automated patch clamp system to record Na v 1.7 currents from single CHO cells - whole cell recording (2-4) - temperature control - fast perfusion system CytoPatch Pipette - mechanical stimulation - current clamp mode Cytocentering Channel CytoBioSience, San Antonio, USA Cell Cytocentering opening G seal CyoPatch TM opening Pressure Low pressure Recording
7 relative peak current (%) Effect of ranolazine on Na v 1.7 currents recorded from CHO cells Late currents 10 ms 200 pa IC 50 = 12.1 µm ranolazine, 10 µm square voltage pulses -90 mv to -10 mv, 40 ms Ranolazine (µm)
8 Inhibitory effect of harmaline on sodium current transients recorded from a CHO cell
9 Time course of Na + current amplitude after whole cell formation: inhibition by harmaline and TTX
10 Concentration-response relation of the harmaline effect IC 50 = 35,5 µm Applied concentrations (µm): 3, 10, 30, 100, 300 and 1 mm
11 Influence of harmaline on the current/voltagecurve (I/V-curve) Recordings after reaching stable current amplitudes in external solution (black) and after application of 30 µm harmaline (red), normalized, means ± STD Peak current: at about + 5mV
12 Effect of harmaline on voltage-dependence of activation of Na v 1.7 currents inflection points control: - 8,9 mv harmaline: -10,2 mv
13 Influence on the steady-state-inactivation curve Recordings after reaching stable current amplitudes in external solution (black) and after application of 30 µm harmaline (red), means ± STD inflection points: control: mv harmaline: mv
14 Effect of harmaline on late Na v 1.7 currents - evaluation of the last 5 ms of the 40 ms traces - peak late
15 Conclusions Harmaline is an unspecific, but effective blocker of the human sodium channel Na v 1.7. The harmaline effect occurs slowly (3-5 min), the concentration of half maximum inhibition was calculated to 35 µm. Specific characteristics of the Na v 1.7 currents, such as kinetics, voltage-dependence of activation and inactivation are not affected by the drug. The harmaline block of Na v 1.7 is voltage-independent. The results support the view that harmaline or P. harmala extracts may be beneficial for certain pain syndromes. The Cytopatch gives reliable results at well defined conditions.
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17 Hansestadt Greifswald Germany Stephan Wierschke Thomas Knott, CyotBioScience Marina Schumacher, Jörg Eisfeld Mirjam Krautwald European Union German Duchenne Foundation German Ministry of Economy
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