Mechanisms and Functional Significance of Inhibition of Neuronal T-Type Calcium Channels by Isoflurane

Previous data have indicated that T-type calcium channels (low-voltage activated T-channels) are potently inhibited by volatile anesthetics. Although the interactions of T-channels with a number of anesthetics have been described, the mechanisms by which these agents modulate channel activity, and the functional consequences of such interactions, are not well studied. Here, we used patch-clamp recordings to explore the actions of a prototypical volatile anesthetic, isoflurane (Iso), on recombinant human CaV3.1 and CaV3.2 isoforms of T-channels. We also performed behavioral testing of anesthetic endpoints in mice lacking CaV3.2. Iso applied at resting channel states blocked current through both isoforms in a similar manner at clinically relevant concentrations (1 minimum alveolar concentration, MAC). Inhibition was more prominent at depolarized membrane potentials (-65 versus -100 mV) as evidenced by hyperpolarizing shifts in channel availability curves and a 2.5-fold decrease in IC50 values. Iso slowed recovery from inactivation and enhanced deactivation in both CaV3.1 and CaV3.2 in a comparable manner but caused a depolarizing shift in activation curves and greater use-dependent block of CaV3.2 channels. In behavioral tests, CaV3.2 knockout (KO) mice showed significantly decreased MAC in comparison with wild-type (WT) litter mates. KO and WT mice did not differ in loss of righting reflex, but mutant mice displayed a delayed onset of anesthetic induction. We conclude that state-dependent inhibition of T-channel isoforms in the central and peripheral nervous systems may contribute to isoflurane's important clinical effects

Novel neuroactive steroid with hypnotic and T-type calcium channel blocking properties exerts effective analgesia in a rodent model of post-surgical pain

Background and Purpose: Neuroactive steroid (3β,5β,17β)-3-hydroxyandrostane-17-carbonitrile (3β-OH) is a novel hypnotic and voltage-dependent blocker of T-type calcium channels. Here, we examine its potential analgesic effects and adjuvant anaesthetic properties using a post-surgical pain model in rodents. Experimental Approach: Analgesic properties of 3β-OH were investigated in thermal and mechanical nociceptive tests in sham or surgically incised rats and mice, with drug injected either systemically (intraperitoneal) or locally via intrathecal or intraplantar routes. Hypnotic properties of 3β-OH and its use as an adjuvant anaesthetic in combination with isoflurane were investigated using behavioural experiments and in vivo EEG recordings in adolescent rats. Key Results: A combination of 1% isoflurane with 3β-OH (60 mg·kg−1, i.p.) induced suppression of cortical EEG and stronger thermal and mechanical anti-hyperalgesia during 3 days post-surgery, when compared to isoflurane alone and isoflurane with morphine. 3β-OH exerted prominent enantioselective thermal and mechanical antinociception in healthy rats and reduced T-channel-dependent excitability of primary sensory neurons. Intrathecal injection of 3β-OH alleviated mechanical hyperalgesia, while repeated intraplantar application alleviated both thermal and mechanical hyperalgesia in the rats after incision. Using mouse genetics, we found that CaV3.2 T-calcium channels are important for anti-hyperalgesic effect of 3β-OH and are contributing to its hypnotic effect. Conclusion and Implications: Our study identifies 3β-OH as a novel analgesic for surgical procedures. 3β-OH can be used to reduce T-channel-dependent excitability of peripheral sensory neurons as an adjuvant for induction and maintenance of general anaesthesia while improving analgesia and lowering the amount of volatile anaesthetic needed for surgery

Are neuronal voltage-gated calcium channels valid cellular targets for general anesthetics?

The effects of anesthetics and analgesics on ion channels have been the subject of intense research since recent reports of direct actions of anesthetic molecules on ion channel proteins. It is now known that ligand-gated channels, particularly γ-amino-butyric acid (GABAA) and N-methyl-D-aspartate (NMDA) receptors, play a key role in mediating anesthetic actions, but these channels are unable to account for all aspects of clinical anesthesia such as loss of consciousness, immobility, analgesia, amnesia and muscle relaxation. Furthermore, an assortment of voltage-gated and background channels also display anesthetic sensitivity and a key question arises: What role do these other channels play in clinical anesthesia? These channels have overlapping physiological roles and pharmacological profiles, making it difficult to assign aspects of the anesthetic state to individual channel types. Here, we will focus on the function of neuronal voltage-gated calcium channels in mediating the effects of general anesthetics

Mechanisms of Inhibition of T-Type Calcium Current in the Reticular Thalamic Neurons by 1-Octanol: Implication of the Protein Kinase C Pathway

Recent studies indicate that T-type calcium channels (T-channels) in the thalamus are cellular targets for general anesthetics. Here, we recorded T-currents and underlying low-threshold calcium spikes from neurons of nucleus reticularis thalami (nRT) in brain slices from young rats and investigated the mechanisms of their modulation by an anesthetic alcohol, 1-octanol. We found that 1-octanol inhibited native T-currents at subanesthetic concentrations with an IC50 of approximately 4 μM. In contrast, 1-octanol was up to 30-fold less potent in inhibiting recombinant CaV3.3 T-channels heterologously expressed in human embryonic kidney cells. Inhibition of both native and recombinant T-currents was accompanied by a hyperpolarizing shift in steady-state inactivation, indicating that 1-octanol stabilized inactive states of the channel. To explore the mechanisms underlying higher 1-octanol potency in inhibiting native nRT T-currents, we tested the effect of the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) and PKC inhibitors. We found that PMA caused a modest increase of T-current, whereas the inactive PMA analog 4α-PMA failed to affect T-current in nRT neurons. In contrast, 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole (Go 6976), an inhibitor of calcium-dependent PKC, decreased baseline T-current amplitude in nRT cells and abolished the effects of subsequently applied 1-octanol. The effects of 1-octanol were also abolished by chelation of intracellular calcium ions with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid. Taken together, these results suggest that inhibition of calcium-dependent PKC signaling is a possible molecular substrate for modulation of T-channels in nRT neurons by 1-octanol

TRPV1-lineage neurons are required for thermal sensation

The TRPV1 ion channel is expressed in sensory neurons and is involved in sensing noxious heat. By using a molecular genetic approach to generate mice that lack TRPV1-expressing neurons, the findings show that the TrpV1-lineage neurons are needed for sensing hot and cold temperatures, but not for normal touch and mechanical pain sensation