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

Abstract

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 Ca V 3.1 and Ca V 3.2 isoforms of T-channels. We also performed behavioral testing of anesthetic endpoints in mice lacking Ca V 3.2. 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 (Ϫ65 versus Ϫ100 mV) as evidenced by hyperpolarizing shifts in channel availability curves and a 2.5-fold decrease in IC 50 values. Iso slowed recovery from inactivation and enhanced deactivation in both Ca V 3.1 and Ca V 3.2 in a comparable manner but caused a depolarizing shift in activation curves and greater use-dependent block of Ca V 3.2 channels. In behavioral tests, Ca V 3.2 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.The effects of general anesthetics on ion channels have been the subject of intense research since studies describing specific interactions between anesthetics and proteins (Franks and Lieb, 1982, 1994). It is now known that some ligand-gated channels (e.g., GABA A ), voltage-gated channels, and background potassium channels display anesthetic sensitivity in vitro that is within the concentration range achieved during general anesthesia (Franks, 2008). These channels have overlapping physiological roles and pharmacological profiles, making it difficult to assign aspects of the anesthetic state to individual channel types. Thus, it has become clear that further study of anesthetic mechanisms of action on specific ion channels is needed.Low voltage-activated calcium channels activate with small depolarizations and allow calcium influx at resting potentials so that small differences in channel activity can result in large changes in cellular excitability and/or secondmessenger pathways. Recent molecular studies have indicated that at least three isoforms of T-channels exist: Ca V 3.1 (␣1G), Ca V 3.2 (␣1H), and Ca V 3.3 (␣1I) (Perez-Reyes, 2003). These channels are located throughout the spinothalamic pathway, in which nociceptive information passes from peripheral sensory neurons to the cortex. T-channels in smallsized dorsal root ganglia (DRG) neurons are believed to functi...

Mechanisms of inhibition of CaV3.1 T-type calcium current by aliphatic alcohols

“…This slowing of recovery from inactivated states of T-channels in TC cells is in agreement with our recent study examining the effects isoflurane on kinetic properties of recombinant human Ca V 3.1 and Ca V 3.2 channels (Orestes et al, 2009). Finally, Figure 6D shows that similarly to isoflurane (Orestes et al, 2009), octanol slowed recovery form inactivation of recombinant Ca V 3.1 current, as evident by about 2-fold shift to the right in curves representing best fits to the average data points. Thus, it appears that the unique effect of aliphatic alcohols on recovery from inactivation of Ca V 3.1 T-channels is confined to the native TC cells.…”

“…This was also well documented for different voltage-dependent blockers of T-type channels such as isoflurane (Orestes et al, 2009) and arachidonic acid (Talavera et al, 2004), which inhibit recombinant Ca V 3.1 currents. This is important since inhibitory effects of these drugs are consequently more prominent at depolarized membrane potentials that could occur during awake states and less prominent at hyperpolarized membrane potentials such as those which dominate during sleep states thought to be initiated upon activation of nRT neurons that provide inhibitory postsynaptic potentials (IPSPs) to TC cells.…”

“…The clones are expressed in human embryonic kidney 293 (HEK-293) cells as reported previously (Joksovic et al, 2006; Orestes et al, 2009). HEK-293 cells (CRL-1573; American Type Culture Collection, Manassas, VA) were grown in DMEM/F-12 media (Invitrogen) supplemented with 10% fetal bovine serum, penicillin G (100mg/ml), and streptomycin (0.1mg/ml).…”

CaV3.2 calcium channels control NMDA receptor-mediated transmission: a new mechanism for absence epilepsy

“…Organotypic hippocampal cultured slices were prepared from postnatal 6-to 7-d-old (P6-P7) male and female Sprague Dawley rats, wild-type mice, and Ca V 3.2 knockout (Orestes et al 2009) mice bred congenically on the C57BL/6 background, following our previous studies (Qin et al 2005;Lim et al 2014). All procedures for animal surgery and maintenance were performed following protocols approved by the animal care and use committee of the University of Virginia and in accordance with National Institutes of Health guidelines.…”

T-type calcium channel blockers as neuroprotective agents

“…Lomerizine is an antimigraine medication used in Japan and has been shown to have neuroprotective effects [32]. Many anesthetic agents such as isoflurane, propofol, and nitrous oxide can effectively block T-type calcium channels [70,69,50], although they also block many other ion channels as well.…”