HCN1 Channels Contribute to the Effects of Amnesia and Hypnosis but not Immobility of Volatile Anesthetics.

BACKGROUND: Hyperpolarization-activated, cyclic nucleotide-gated (HCN) subtype 1 (HCN1) channels have been identified as targets of ketamine to produce hypnosis. Volatile anesthetics also inhibit HCN1 channels. However, the effects of HCN1 channels on volatile anesthetics in vivo are still elusive. This study uses global and conditional HCN1 knockout mice to evaluate how HCN1 channels affect the actions of volatile anesthetics. METHODS: Minimum alveolar concentrations (MACs) of isoflurane and sevoflurane that induced immobility (MAC of immobility) and/or hypnosis (MAC of hypnosis) were determined in wild-type mice, global HCN1 knockout (HCN1) mice, HCN1 channel gene with 2 lox-P sites flanking a region of the fourth exon of HCN1 (HCN1) mice, and forebrain-selective HCN1 knockout (HCN1: cre) mice. Immobility of mice was defined as no purposeful reactions to tail-clamping stimulus, and hypnosis was defined as loss of righting reflex. The amnestic effects of isoflurane and sevoflurane were evaluated by fear-potentiated startle in these 4 strains of mice. RESULTS: All MAC values were expressed as mean ± SEM. For MAC of immobility of isoflurane, no significant difference was found among wild-type, HCN1, HCN1, and HCN1: cre mice (all ~1.24%-1.29% isoflurane). For both HCN1 and HCN1: cre mice, the MAC of hypnosis for isoflurane (each ~1.05% isoflurane) was significantly increased over their nonknockout controls: HCN1 versus wild-type (0.86% ± 0.03%, P < 0.001) and HCN1: cre versus HCN1 mice (0.84% ± 0.03%, P < 0.001); no significant difference was found between HCN1 and HCN1: cre mice. For MAC of immobility of sevoflurane, no significant difference was found among wild-type, HCN1, HCN1, and HCN1: cre mice (all ~2.6%-2.7% sevoflurane). For both HCN1 and HCN1: cre mice, the MAC of hypnosis for sevoflurane (each ~1.90% sevoflurane) was significantly increased over their nonknockout controls: HCN1 versus wild-type (1.58% ± 0.05%, P < 0.001) and HCN1: cre versus HCN1 mice (1.56% ± 0.05%, P < 0.001). No significant difference was found between HCN1 and HCN1: cre mice. By fear-potentiated startle experiments, amnestic effects of isoflurane and sevoflurane were significantly attenuated in HCN1 and HCN1: cre mice (both P < 0.002 versus wild-type or HCN1 mice). No significant difference was found between HCN1 and HCN1: cre mice. CONCLUSIONS: Forebrain HCN1 channels contribute to hypnotic and amnestic effects of volatile anesthetics, but HCN1 channels are not involved in the immobilizing actions of volatile anesthetics.

TASK Channel Deletion Reduces Sensitivity to Local Anesthetic-induced Seizures.

BACKGROUND: Local anesthetics (LAs) are typically used for regional anesthesia but can be given systemically to mitigate postoperative pain, supplement general anesthesia, or prevent cardiac arrhythmias. However, systemic application or inadvertent intravenous injection can be associated with substantial toxicity, including seizure induction. The molecular basis for this toxic action remains unclear. METHODS: We characterized inhibition by different LAs of homomeric and heteromeric K channels containing TASK-1 (K2P3.1, KCNK3) and TASK-3 (K2P9.1, KCNK9) subunits in a mammalian expression system. In addition, we used TASK-1/TASK-3 knockout mice to test the possibility that TASK channels contribute to LA-evoked seizures. RESULTS: LAs inhibited homomeric and heteromeric TASK channels in a range relevant for seizure induction; channels containing TASK-1 subunits were most sensitive and IC50 values indicated a rank order potency of bupivacaine > ropivacaine >> lidocaine. LAs induced tonic-clonic seizures in mice with the same rank order potency, but higher LA doses were required to evoke seizures in TASK knockout mice. For bupivacaine, which produced the longest seizure times, seizure duration was significantly shorter in TASK knockout mice; bupivacaine-induced seizures were associated with an increase in electroencephalogram power at frequencies less than 5 Hz in both wild-type and TASK knockout mice. CONCLUSIONS: These data suggest that increased neuronal excitability associated with TASK channel inhibition by LAs contributes to seizure induction. Because all LAs were capable of evoking seizures in TASK channel deleted mice, albeit at higher doses, the results imply that other molecular targets must also be involved in this toxic action.

The neuroprotective impact of the leak potassium channel TASK1 on stroke development in mice.

Oxygen depletion (O(2)) and a decrease in pH are initial pathophysiological events in stroke development, but secondary mechanisms of ischemic cell death are incompletely understood. By patch-clamp recordings of brain slice preparations we show that TASK1 and TASK3 channels are inhibited by pH-reduction (42+/-2%) and O(2) deprivation (36+/-5%) leading to membrane depolarization, increased input resistance and a switch in action potential generation under ischemic conditions. In vivo TASK blockade by anandamide significantly increased infarct volumes at 24 h in mice undergoing 30 min of transient middle cerebral artery occlusion (tMCAO). Moreover, blockade of TASK channels accelerated stroke development. Supporting these findings TASK1(-/-) mice developed significantly larger infarct volumes after tMCAO accompanied by worse outcome in functional neurological tests compared to wild type mice. In conclusion, our data provide evidence for an important role of functional TASK channels in limiting tissue damage during cerebral ischemia.

Suppression of ih contributes to propofol-induced inhibition of mouse cortical pyramidal neurons.

The contributions of the hyperpolarization-activated current, I(h), to generation of rhythmic activities are well described for various central neurons, particularly in thalamocortical circuits. In the present study, we investigated effects of a general anesthetic, propofol, on native I(h) in neurons of thalamus and cortex and on the corresponding cloned HCN channel subunits. Whole cell voltage-clamp recordings from mouse brain slices identified neuronal I(h) currents with fast activation kinetics in neocortical pyramidal neurons and with slower kinetics in thalamocortical relay cells. Propofol inhibited the fast-activating I(h) in cortical neurons at a clinically relevant concentration (5 microM); inhibition of I(h) involved a hyperpolarizing shift in half-activation voltage (DeltaV1/2 approximately -9 mV) and a decrease in maximal available current (approximately 36% inhibition, measured at -120 mV). With the slower form of I(h) expressed in thalamocortical neurons, propofol had no effect on current activation or amplitude. In heterologous expression systems, 5 muM propofol caused a large shift in V1/2 and decrease in current amplitude in homomeric HCN1 and linked heteromeric HCN1-HCN2 channels, both of which activate with fast kinetics but did not affect V1/2 or current amplitude of slowly activating homomeric HCN2 channels. With GABA(A) and glycine receptor channels blocked, propofol caused membrane hyperpolarization and suppressed action potential discharge in cortical neurons; these effects were occluded by the I(h) blocker, ZD-7288. In summary, these data indicate that propofol selectively inhibits HCN channels containing HCN1 subunits, such as those that mediate I(h) in cortical pyramidal neurons-and they suggest that anesthetic actions of propofol may involve inhibition of cortical neurons and perhaps other HCN1-expressing cells.

HCN subunit-specific and cAMP-modulated effects of anesthetics on neuronal pacemaker currents.

General anesthetics have been a mainstay of surgical practice for more than 150 years, but the mechanisms by which they mediate their important clinical actions remain unclear. Ion channels represent important anesthetic targets, and, although GABA(A) receptors have emerged as major contributors to sedative, immobilizing, and hypnotic effects of intravenous anesthetics, a role for those receptors is less certain in the case of inhalational anesthetics. The neuronal hyperpolarization-activated pacemaker current (Ih) is essential for oscillatory and integrative properties in numerous cell types. Here, we show that clinically relevant concentrations of inhalational anesthetics modulate neuronal Ih and the corresponding HCN channels in a subunit-specific and cAMP-dependent manner. Anesthetic inhibition of Ih involves a hyperpolarizing shift in voltage dependence of activation and a decrease in maximal current amplitude; these effects can be ascribed to HCN1 and HCN2 subunits, respectively, and both actions are recapitulated in heteromeric HCN1-HCN2 channels. Mutagenesis and simulations suggest that apparently distinct actions of anesthetics on V(1/2) and amplitude represent different manifestations of a single underlying mechanism (i.e., stabilization of channel closed state), with the predominant action determined by basal inhibition imposed by individual subunit C-terminal domains and relieved by cAMP. These data reveal a molecular basis for multiple actions of anesthetics on neuronal HCN channels, highlight the importance of proximal C terminus in modulation of HCN channel gating by diverse agents, and advance neuronal pacemaker channels as potentially relevant targets for clinical actions of inhaled anesthetics.

Functional expression of TASK-1/TASK-3 heteromers in cerebellar granule cells.

TASK-1 and TASK-3 are functional members of the tandem-pore K+ (K2P) channel family, and mRNAs for both channels are expressed together in many brain regions. Although TASK-1 and TASK-3 subunits are able to form heteromers when their complementary RNAs are injected into oocytes, whether functional heteromers are present in the native tissue is not known. Using cultured cerebellar granule (CG) neurones that express mRNAs of both TASK-1 and TASK-3, we studied the presence of heteromers by comparing the sensitivities of cloned and native K+ channels to extracellular pH (pHo) and ruthenium red. The single-channel conductance of TASK-1, TASK-3 and a tandem construct (TASK-1/TASK-3) expressed in COS-7 cells were 14.2 +/- 0.4, 37.8 +/- 0.7 and 38.1 +/- 0.7 pS (-60 mV), respectively. TASK-3 and TASK-1/TASK-3 (and TASK-3/TASK-1) displayed nearly identical single-channel kinetics. TASK-3 and TASK-1/TASK-3 expressed in COS-7 cells were inhibited by 26 +/- 4 and 36 +/- 2 %, respectively, when pHo was changed from 8.3 to 7.3. In outside-out patches from CG neurones, the K+ channel with single channel properties similar to those of TASK-3 was inhibited by 31 +/- 7 % by the same reduction in pHo. TASK-3 and TASK-1/TASK-3 expressed in COS-7 cells were inhibited by 78 +/- 7 and 3 +/- 4 %, respectively, when 5 microm ruthenium red was applied to outside-out patches. In outside-out patches from CG neurones containing a 38 pS channel, two types of responses to ruthenium red were observed. Ruthenium red inhibited the channel activity by 77 +/- 5 % in 42 % of patches (range: 72-82 %) and by 5 +/- 4 % (range: 0-9 %) in 58 % of patches. When patches contained more than three 38 pS channels, the average response to ruthenium red was 47 +/- 6 % inhibition (n= 5). These electrophysiological studies show that native 38 pS K+ channels of the TASK family in cultured CG neurones consist of both homomeric TASK-3 and heteromeric TASK-1/TASK-3.