Free radical signalling underlies inhibition of Ca_V3.2 T‐type calcium channels by nitrous oxide in the pain pathway

Abstract

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.

GABAB receptors inhibit low-voltage activated and high-voltage activated Ca2+ channels in sensory neurons via distinct mechanisms

“…Indeed, Kv7 channels are highly sensitive to oxidation by ROS due to the specific cysteine pocket in the intracellular S2-S3 linker of the channel [27,28]. Interestingly, T-type Ca 2+ channels are also sensitive to redox modulation, albeit in contrast to Kv7 channels, oxidizing compounds and ROS do not activate but inhibit T-type channel activity [21,29,30]. Since ROS generation by NK1 receptor triggering was sensitive to PTX [26] we hypothesized that unconventional signaling cascade causing inhibition of LVA current in DRG neurons by baclofen may also be mediated by a ROSdependent mechanism.…”

“…Acting as intracellular second messengers, such ROS augmented the activity of inhibitory Kv7 channels providing a mechanism for substance P mediated peripheral endogenous analgesia [26]. Because i) T-type channels are inhibited by oxidizing agents and ROS [21,29,30];…”

The abolishment of anesthesia-induced cognitive impairment by timely protection of mitochondria in the developing rat brain: The importance of free oxygen radicals and mitochondrial integrity

“…For example, N 2 O, which is one of the anesthetics used in our regimen can generate free oxygen (most likely hydroxy) radicals in the presence of trace metals such as iron and copper (Orestes et al, 2011), both commonly found in biological fluids and neurons. Upregulation of ROS promotes metal-catalyzed protein oxidation, which in turn permanently alters the function of various cellular proteins (Stadtman and Berlett, 1991; Stadtman, 1993).…”

“…Upregulation of ROS promotes metal-catalyzed protein oxidation, which in turn permanently alters the function of various cellular proteins (Stadtman and Berlett, 1991; Stadtman, 1993). For example, N 2 O causes inhibition of low-voltage-activated calcium channels by oxidizing critical amino acids in the functional pore of the channel (Orestes et al, 2011). However, having considered that possibility we believe that it is unlikely that N 2 O production of ROS and potential protein oxidation is the main culprit for developmental neurotoxicity we present in this study.…”

TTA-P2 Is a Potent and Selective Blocker of T-Type Calcium Channels in Rat Sensory Neurons and a Novel Antinociceptive Agent

“…Behavioral tests for inflammatory pain after formalin injection were done with mice in a clear Plexiglas chamber prefilled with air at a flow of 6 l/min, as we reported recently (Orestes et al, 2011). Each mouse was first placed in the prefilled chamber to accommodate for 30 min and then removed from the chamber, injected in the plantar side of the right paw with 20 l of 5% formalin or vehicle (15% cyclodextrin), and returned to the test chamber.…”