Free radical signalling underlies inhibition of CaV3.2 T-type calcium channels by nitrous oxide in the pain pathway.
Nitrous oxide (N2O, laughing gas) has been used as an anaesthetic and analgesic for almost two centuries, but its cellular targets remain unclear. Here, we present a molecular mechanism of nitrous oxide's selective inhibition of CaV3.2 low-voltage-activated (T-type) calcium channels in pain pathways. Using site-directed mutagenesis and metal chelators such as diethylenetriamine pentaacetic acid and deferoxamine, we reveal that a unique histidine at position 191 of CaV3.2 participates in a critical metal binding site, which may in turn interact with N2O to produce reactive oxygen species (ROS). These free radicals are then likely to oxidize H191 of CaV3.2 in a localized metal-catalysed oxidation reaction. Evidence of hydrogen peroxide and free radical intermediates is given in that N2O inhibition of CaV3.2 channels is attenuated when H2O2 is neutralized by catalase. We also use the adrenochrome test as an indicator of ROS in vitro in the presence of N2O and iron. Ensuing in vivo studies indicate that mice lacking CaV3.2 channels display decreased analgesia to N2O in response to formalin-induced inflammatory pain. Furthermore, a superoxide dismutase and catalase mimetic, EUK-134, diminished pain responses to formalin in wild-type mice, but EUK-134 and N2O analgesia were not additive. This suggests that reduced ROS levels led to decreased inflammation, but without the presence of ROS, N2O was not able to provide additional analgesia. These findings reveal a novel mechanism of interaction between N2O and ion channels, furthering our understanding of this widely used analgesic in pain processing.
Department of Anaesthesiology, University of Virginia Health System, Mail Box 800710, Charlottesville, VA 22908-0710, USA.
Peihan
P
Orestes
Damir
D
Bojadzic
Jeonghan
J
Lee
Emily
E
Leach
Reza
R
Salajegheh
Michael R
MR
Digruccio
Michael T
MT
Nelson
Slobodan M
SM
Todorovic
D000323:Adrenochrome / Q000378:metabolism; D018712:Analgesics, Non-Narcotic / Q000494:pharmacology*; D000818:Animals; D002121:Calcium Channel Blockers / Q000494:pharmacology*; D020747:Calcium Channels, T-Type / Q000187:drug effects* / Q000378:metabolism; D002374:Catalase / Q000378:metabolism; D002614:Chelating Agents / Q000494:pharmacology; D003676:Deferoxamine / Q000494:pharmacology; D004195:Disease Models, Animal; D005260:Female; D005727:Ganglia, Spinal / Q000187:drug effects* / Q000378:metabolism; D057809:HEK293 Cells; D006639:Histidine; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D008297:Male; D008564:Membrane Potentials; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D016297:Mutagenesis, Site-Directed; D009609:Nitrous Oxide / Q000494:pharmacology*; D009942:Organometallic Compounds / Q000494:pharmacology; D010084:Oxidation-Reduction; D010146:Pain / Q000378:metabolism / Q000517:prevention & control*; D004369:Pentetic Acid / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000378:metabolism*; D012459:Salicylates / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects*; D013997:Time Factors; D014162:Transfection
D018712:Analgesics, Non-Narcotic; C479952:CACNA1H protein, human; C504883:Cacna1h protein, mouse; C509958:Cacna1h protein, rat; D002121:Calcium Channel Blockers; D020747:Calcium Channels, T-Type; D002614:Chelating Agents; C433928:EUK-134; D009942:Organometallic Compounds; D017382:Reactive Oxygen Species; D012459:Salicylates; D006639:Histidine; D000323:Adrenochrome; D004369:Pentetic Acid; D006861:Hydrogen Peroxide; D002374:Catalase; D003676:Deferoxamine; D009609:Nitrous Oxide