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We investigated the role of tandem pore domain potassium channel (K2P) TRESK in neurobehavioral function and volatile anesthetic sensitivity in genetically modified mice.
Exon III of the mouse TRESK gene locus was deleted by homologous recombination using a targeting vector. The genotype of bred mice (wildtype, knockout or heterozygote) was determined using the polymerase chain reaction. Morphologic and behavioral evaluations of TRESK knockout mice were compared to wildtype littermates. Sensitivity of bred mice to isoflurane, halothane, sevoflurane and desflurane were studied by determining the minimum alveolar concentration preventing movement to tail clamping in 50% of each genotype.
TRESK knockout mice had normal development and behavior except for decreased number of inactive periods and increased thermal pain sensitivity (20% decrease in latency with hot plate test). TRESK knockout mice showed a statistically significant 8% increase in isoflurane minimum alveolar concentration compared to wildtype littermates; sensitivity to other volatile anesthetics was not significantly different. Spontaneous mortality of TRESK knockout mice following initial anesthesia testing was nearly threefold higher than that of wildtype littermates.
TRESK alone is not critical for baseline central nervous system function but may contribute to the action of volatile anesthetics. The inhomogenous change in anesthetic sensitivity corroborates findings in other K2P knockout mice and supports the idea that the mechanism of volatile anesthetic action involves multiple targets. Although it was not shown in this study, a compensatory effect by other K2P channels may also contribute to these observations.
Tandem pore potassium ion channels (K2P) are important contributors to background potassium (K+) currents in excitable cells. These channels are composed of two subunits, each with four transmembrane domains and two pore-forming domains arranged in tandem – a unique structure distinguishing them from other K+ channel subunits.1 Background currents conducted by K2P channels help maintain the resting membrane potential of cells and regulate the action potential of excitable cells.2–4 K2P channels are modulated by biophysical and biochemical factors such as pH, temperature, stretch, molecular oxygen, phospholipids, cyclic nucleotides, neuroprotective agents, protein kinases, G-protein-coupled receptors and neurotransmitters.5–14 K2P channels participate in regulating adrenal aldosterone secretion,15,16 renal proximal tubule cell volume,17,18 cardiac action potential and rhythms,19,20 and neuronal apoptosis and neuroprotection.21,22 K2P channels may also be involved with significant pathologic states such as tumorigenesis and depression.23,24
Many in vitro studies have suggested a role of K2P channels in the mechanism of action of volatile anesthetics. Currents passed by TWIK (Tandem pore Weak Inward rectifying K+ channel)-related Acid-Sensitive K+ channel (TASK)-2 expressed in Xenopus oocytes are potentiated by halothane, isoflurane, enflurane, desflurane and chloroform.25 Human TWIK-RElated K+ channel (TREK)-1 and TREK-2 currents expressed heterologously in COS-7 cells are strongly activated by chloroform, halothane and isoflurane.26,27 TASK-1 currents found in rat motorneurons and locus ceruleus cells are also strongly potentiated by halothane and sevoflurane.28
These cellular findings have been corroborated in whole animal studies. TREK-1 knockout mice are resistant to the anesthetizing action of several volatile anesthetics (chloroform, halothane, isoflurane, sevoflurane, desflurane) requiring higher concentrations to achieve inhibition of righting reflex and nociception.29 However, inactivation of other K2P channel genes produce knockout mice with less definitive changes in anesthetic sensitivity. Both TASK-1 and TASK-3 knockout mice show small changes in sensitivity to some volatile anesthetics30,31 Furthermore, TASK-2 and KCNK7 knockout mice do not have altered sensitivity to volatile anesthetics.32,33 Thus, the involvement of K2P channels in the mechanisms of volatile anesthetics appears variable and must be determined individually for each K2P family member.
TWIK-RElated Spinal K+ channel (TRESK) was the last K2P family member isolated and initially found expressed only in human spinal cord.34 Subsequently, TRESK expression was also detected in human brain35 and non-neuronal (liver, testis, spleen, heart and lung) tissues.36,37 Like other K2P channels, TRESK channels conduct background K+ currents to help set the membrane potential near the K+ equilibrium potential.34 In dorsal root ganglion (DRG) neurons TRESK’s primary role is to help shape the action potential.38
As with other K2P channels, biochemical and biophysical factors regulate TRESK activity, including unsaturated free fatty acids (arachidonic, docosahexaenoic and linoleic acids), changes in intracellular and extracellular pH,34,37 intracellular Ca2+ levels39 and volatile anesthetics.35 Human TRESK K+ currents heterologously expressed in mammalian and amphibian cells are strongly potentiated (up to 3-fold) by halothane, isoflurane, sevoflurane, and desflurane at clnically relevant concentrations.35
We generated a mouse with inactivated TRESK gene to study its sensitivity to various volatile anesthetics. We also studied the development, growth and behavior of these mice and compared them to wildtype littermates. The recovery and survival of these mice during and immediately after anesthetic treatments were also monitored and recorded. This report describes the first characterization of a TRESK knockout mouse.
All animal experiments done at University of California San Francisco were carried out according to protocols approved by the Institutional Animal Care and Use Committee of the University of California San Francisco. Mice were housed under standard conditions with a 12-hour light/dark cycles in a pathogen barrier facility at University of California San Francisco and given ad libitum access to food and water. TRESK knockout mice were produced under contract with Ozgene LTD Perth, Australia. A targeting vector was constructed to delete the entire sequence of exon III of mouse TRESK gene by homologous recombination. Exon III is the largest of three TRESK exons and encodes the majority of the TRESK channel subunit. The 5′ and 3′ regions of the C57/BL6 strain chromosomal locus (~5 kilobases each) flanking TRESK exon III were cloned from mouse genomic DNA and used as homologous arms to construct the targeting vector with a neomycin cassette (Fig. 1A). The targeting construct was transfected into C57/BL6 embryonic stem cells and the recombinant embryonic stem cell lines were selected. The isolated TRESK knockout embryonic stem cells were microinjected into C57/BL6 mouse blastocysts to generate chimeric mice. The neo cassette was removed by interbreeding with mice constituitively expressing Cre recombinase. Heterozygous offspring with neo cassette deleted were used to breed TRESK knockout mice. In order to increase the number of animals with the desired TRESK genotypes we also used some homozygous knockout offspring for breeding.
At three weeks of age, newborn mice were weaned, separated and identified by ear tags. Genomic DNA was isolated from tail snip samples (<1 cm length) with DNAeasy Blood and Tissue kit (Qiagen, Valencia, CA). Polymerase chain reaction (PCR) was performed with primers flanking TRESK gene exon III: forward primer (P1) 5′-ACCAACACCAAGCTGTCTTGTTTCTC-3′ and reverse primer (P2) 5′-AGACAGATGGACGGACAGACATAGATG-3′ (figure 1A). The PCR reaction mixture contained 1 μl DNA, 2 μl PCR reaction buffer (10× concentration), 0.4 μl dNTP (10 mM), 0.4 μl of 10 mM forward and reverse primers, 0.2 μl Platinum Taq DNA polymerase (5 U/ml), and 16 μl water (PCR Core Kit, Boehringer Mannheim, Germany). The PCR reaction was carried out with a programmable thermal cycler (DNA Engine PTC-200, MJ Research, Waltham, MA) programmed with initial heating to 95°C for 2 min, then 30 cycles of 92°C for 1 min, 58°C for 30 s and 72°C for 2 min, and a final extension at 72°C for 5 min. PCR products were analyzed by electrophoresis on 1% agarose gels. The expected sizes of bands were: wildtype, 2.6 kilobase (kb); knockout/+neo, 2.0 kb; knockout/−neo, 0.15 kb (representative gel shown in Fig. 1B).
Real-time quantitative RT-PCR was performed to analyze the K2P gene expression in bred mice. Poly (A)+ RNA from brain, spinal cord and DRG were extracted using a Micro-FastTrack messenger RNA (mRNA) isolation kit (Invitrogen, Carlsbad, CA) with extraction volumes adjusting to 1 ml per 100 mg wet tissue weight. The final concentrations and the purity of the mRNA preparation were determined by measuring the absorbance at 260 nm (A260) and the ratio of A260/A280. The yields of mRNA from the same tissues of different mice were adjusted by dilution with RNAse-free water to reach the same concentration per mg of tissue. Reverse transcription was performed with 0.01–0.1μg of mRNA using a QuantiTect RT kit (Qiagen). Quantitative PCR was carried out on the MX3000P Real-time PCR system (Stratagene, Cedar Creek, TX) with the following thermal cycles: initial heating to 95°C for 10 min, then 30 cycles of 95°C for 15 s, 56°C for 30 s, and 72°C for 30 s. Total 50 μl of PCR reaction mixture included 25 μl of QuantiTect SYBR Green PCR Master Mix (2x concentrated, from Qiagen kit), 0.5 mM of each primer and 2 μl of reverse-transcribed complementary DNA. Quantitative PCR primers for the four K2P channels were: TRESK forward primer 5′-GACAGTGAGGTGTGGGTCTG-3′, reverse primer 5′-CCAGAGCTGTTGCATAGGAA-3′; T R E K-1 forward primer 5′-TGTGGTTATCACTCTGACG-3′, reverse primer 5′-CAGCCCAACGAGGATCCAG-3′; TASK-1 forward primer 5′-CTGCTCATTCACTCGTCCAT-3′, reverse primer 5′-AAGAACTGCCCAGGTGACTT-3′; T A S K-3 forward primer 5′-GACGCTGGTTATGTTCCAGA-3′, reverse primer 5′-CGGTCACCATGTTCTCCATA-3′. Each sample was set-up in triplicate and the results of three mice of each genotype group were pooled.
To establish standard curves, fragments of TRESK, TREK-1, TASK-1 and TASK-3 were amplified by PCR from C57BL6 mouse mRNA, gel purified and diluted serially prior to the quantitative PCR experiments. Five serial dilutions with a factor of 10 for each K2P standard were included in parallel with the quantitation of the K2P channel expression in every PCR run. A linear standard curve of threshold concentration (Ct) versus concentration of standard was established for each gene. The expression level of each gene was quantified as a relative expression against the standard.
Perfusion and fixation of mice were performed according to our recently described methods.40 Mice were deeply anesthetized with sodium pentobarbital (50 mg/kg, intraperitoneal injection) and injected with heparin sodium (1000 unit/kg, American Pharmacological Partners, Schaumburg, IL) into the left ventricle to prevent blood clotting. About 30 ml of 0.1 M phosphate-buffered saline pre-warmed to 37°C was first perfused into the circulation system through the left ventricle and drained out of the right atrium, followed by a perfusion of approximately 40 ml of fixative containing 4% formaldehyde (v/v) in 0.2 M phosphate buffer pre-cooled to 4°C. After perfusion brain, spinal cord and lumbar DRG were dissected, fixed for another 3-4 hours at 4°C in the same fixative, followed by incubation in 30% sucrose in phosphate-buffered saline overnight.
Sectioning of fixed tissues of brain (section thickness 30 mm), spinal cord (20 mm) and lumbar DRG (10 mm) was performed on a cryostat (CM1900, Leica, Solms, Germany), from three different TRESK wildtype and knockout mice. Immunostaining using specific antisera against TRESK (goat polyclonal IgG, TRESK/V-12, Santa Cruz Biotechnology, Santa Cruz, CA) was carried out according to the avidin-biotin peroxidase method described by Hsu et al.41 with minor modifications.40 Selected representative sections were immunostained in pretreated 12-well tissue culture plates (Becton Dickinson, Franklin Lakes, NJ). Tissue sections in each well were washed three times for 5 min each with washing buffer containing 10 mM phosphate-buffered saline, 1% normal rabbit serum (Vector Laboratories, Burlingame, CA) and 0.3% Triton X-100. After wash, the sections were incubated for 1 hour in blocking buffer (washing buffer containing 3% normal rabbit serum), and then incubated with 1:100 diluted TRESK antisera for 4 hours with a gentle shaking at room temperature, followed by a continuous incubation at 4°C overnight. After incubation with the TRESK antisera, the sections were washed and then incubated with a biotinylated rabbit-anti-goat antibody (the secondary antibody) that reacted to avidin/biotinylated peroxidase complex (Vector Laboratories, Burlingame, CA) for 2 hours with a gentle shaking at room temperature. In parallel three control incubations were done - without primary antisera, without secondary antibody, or with the primary antisera pre-neutralized with a blocking peptide that was used to raise the antisera. Diaminobenzidine substrate kit for peroxidase (Vector Laboratories) was used to localize the peroxidase reaction sites. The immunostained sections were mounted on glass microscopic slides, air dried and covered with a coverslip using Permount histological mounting medium (Fisher Scientific, Fair Lawn, NJ).
Images taken at 10×, 30× and 50× magnification were captured with a microscope (Leica MZFL III, Wetzlar, Germany) connected to a digital camera (Nikon Dxm 1200, Melville, NY), and images at 200× and 400× magnification were captured with different microscope (Nikon Eclipse E400, Yokohama, Japan) connected to a digital camera (Nikon D300, Yokohama, Japan). All images were converted to gray scale and digitalized. The stereotaxic coordinates of coronal sections of the mouse brain were assigned according to Paxinos and Franklin.42 The levels of the spinal cord sections were determined according to Paxinos.43
The following standardized tests were applied to TRESK knockout mice and wildtype littermates to elicit neurobehavioral differences:
mice were placed in an open field box free of distinct odor or objects and allowed to freely explore open field box for 5 minutes. Behavior was observed and scored as follows: active moves=sum of running, rearing or jumping events; inactive periods=sum of sniff and immobile events; grooming; total defecations and urinations.
mice were placed on one-inch square mesh grid and then shaken briefly to ensure full grip. The grid was then inverted to allow the mouse to maintain grasp for a maximum of 60 seconds. The timer was stopped when the mouse fell (all four paws no longer grasping mesh grid). If the mouse climbed up onto the top of the grid, the grid was re-inverted to ensure upside down grasping. Each animal was tested once.
mice were suspended by their tail (using electrical tape) to a wooden bar 60 cm above table. The following parameters were scored: latency to immobility, cumulative duration of immobility over 4-minute period, clasping (curling into fetal position).
mice were habituated to testing room 30 minutes prior to testing. Fixed Rotarod: On the first day, mice were placed on the rotarod apparatus with the rod rotating at the constant speed of 16 rpm (rotations per minute) for three trials. The trial ended when the mouse fell or after 5 minutes. Accelerated Rotarod: On the second day, mice were placed on the rotarod apparatus with the rod rotating at an accelerating speed, from 4 rpm to 40 rpm. Each 30 seconds, the rotation speed was increased by 4 rpm. The trial ended when the mouse fell or after five minutes.
mice were trained for two consecutive days to traverse down a tapered cue stick, with the tip leading to their home cage. On the first day, mice received two assisted trials. Second day of training was resumed with three unassisted trials. On third day mice were tested with a mesh grid (1 cm squares) placed over the cue stick leaving one inch between the grid and the cue surface. Animals were then videotaped for three trials. Latency to finish, the number of falls and the number of missed footsteps were scored for each mouse. The average for all three trials was used for the analysis.
time spent in an open field was measured in an automated clear plastic chamber (41 × 41 × 30 cm) with two 16 × 16 photobeam arrays detecting horizontal and vertical movements. Mice were habituated to testing room 30 minutes prior to testing. During testing, mice were placed in one of four identical clear plastic chambers, as mentioned above, for 15 minutes. Open field movements were automatically recorded by photobeams.
mice were habituated to testing room 30 minutes prior to testing. The mouse was placed with its tail protruding within a restraining tube on the platform of the Stimulus Unit. The tail was positioned on a slot of adjustable width to guarantee stability of the tail underneath a thermal stimulus. The latency to respond was measured (maximum latency is 15 seconds).
mice were habituated to testing room 30 minutes prior to testing. Surface of the hot plate was heated to a constant temperature of 52–55°C as measured by a built-in digital thermometer. During testing, mice were placed in a clear, open-ended cylindrical (11cm diameter × 15cm height) enclosure placed on top of the hot plate. The latency to respond with a hindpaw lick, hindpaw flick, or jump (whichever occurred first) was timed. The mouse was immediately removed from the hot plate and returned to its home cage. Each animal was tested once.
Bred mice aged 2 months or older underwent measurement of MAC according to methods established by the lab of Dr Edmond Eger II.32,44 Genotyping of the animals was done after MAC testing so the investigators were completely unaware of the genotypes during testing.
First, individually-tagged animals were randomly selected from their cages, weighed and placed in their own gas-tight plastic cylinders connected to a system containing a fan and oxygen source. Up to ten mice were tested in one experimental setting. Temperature was monitored by rectal probe and kept between 35.5–37.5°C throughout the experiment. Volatile anesthetics were administered by agent-specific precision vaporizers and the concentrations monitored by a Datex Ohmeda 5250 RGM monitor (Louisville, CO). Individual mouse going through multiple MAC assays had at least one week rest period between test sessions.
Four volatile anesthetics, isoflurane, halothane, sevoflurane and desflurane were tested. Anesthetic agents were administered at an inspired oxygen concentration of 1.0. Administration began at low concentration and continued for a period of time to allow them to reach equilibrium appropriate for each agent (20 minutes for desflurane, 30 minunes for isoflurane and sevoflurane, and 40 minutes for halothane). At equilibrium, the exact anesthetic concentration was determined by gas chromatography (Gow-Mac Instrument Corp, Bridgewater, NJ) equipped with a flame ionization detector. A mechanical clamp was applied on the middle of the tail to test the responsiveness of the mouse. If the animal responded by movement of an extremity or jerking of the head, the anesthetic concentration was increased by 0.1% for halothane, isoflurane and sevoflurane, and 0.2% for desflurane. The location of tail clamping was changed for subsequent measurement at the next concentration. Upon reaching the concentration of anesthetic at which the mouse did not move in response to tail clamping, it was removed from the chamber and allowed to recover at warm temperature. The MAC value of a volatile anesthetic for a given mouse was defined as the average of two sequential concentrations at which the responsiveness of the mouse to the tail clamping changed from positive to negative.
Survival/recovery or death during or immediately after MAC assays were recorded. In order to characterize the death as a direct result from anesthetic treatment, we counted only deaths that occurred within 24 hours of MAC determination. Deaths due to other causes, for example, infection or diseases not related to anesthetic exposure, were excluded from this death rate analysis.
The GraphPad Prism 4 graphing, curve fitting and statistical package (San Diego, CA) was used for the analysis. We have assumed that a Gaussian distribution was present in the MAC assays and in the behavioral data sets. The choice of a parametric test (ANOVA) was based on discussion with Edmond Eger II M.D., (Professor, Department of Anesthesia and Perioperative Care, University of California San Francisco, San Francisco, California, December 2009) the originator of MAC testing with more than forty years of experience. He advised that population normality can be assumed with genetically pure groups (wildtype, heterozygotes and homozygotes derived from a small number of breeding pairs). Power analysis for sample size computation was also performed after discussion with Dr. Eger. We compared three groups: homozygous, heterozygous and wild type animals. To have a probability (power) of 0.9 of detecting an effect at the 0.05 significance level with the standard deviation in the MAC measurement being 6% and an expected effect size of 10% change in MAC requires 15 mice per group. Thus, we needed at least 60 bred mice to have enough mice representing each genotype given a normal Mendelian distribution of genotypes.
One-way analysis of variance (ANOVA) was used to analyze the differences in MAC testing among the three genotypes of TRESK knockout mice. The criteria for determining statistical significance was a p value < 0.05. Data are presented as mean ± standard error of the mean (SEM) with number of animals undergoing each MAC test shown above upper error bar.
For the behavioral testing, although fewer animals were available for testing the same assumption of genetic homogeneity and therefore normal distribution of behaviors was appropriate. For analysis of behavioral tests, two-tailed Student’s t-test was used. To compare differences in death rate after MAC assays between different genotypes, Fisher’s exact test was used.
Figure 1 shows details of the procedure used to generate TRESK knockout mice. Exon III of mouse TRESK gene was targeted for deletion by homologous recombination because it is the largest of three TRESK exons and encodes the majority of the TRESK channel subunit. Panel A shows the genomic structure of the mouse TRESK gene locus and the elements of the targeting vector that was constructed to delete exon III. To eliminate potential off-targeting effects, mice transgenic for the homologously recombined deletion vector were bred with mice constituitively expressing Cre recombinase to produce the final knockout strain in which the neomycin selection cassette had been deleted.
A total of 186 mice were bred for this study (97 males and 89 females). The genotypes of mouse pups were determined at 8–10 days of age using PCR on DNA samples isolated from tail snips. Typical results from PCR genotyping are shown in Figure 1B. The presence of a 2.6 kilobase band indicated a wildtype allele whereas the presence of a 0.15 kilobase band indicated a knockout allele. The distribution of genotypes of the offspring from crossings of heterozygous parents closely followed a normal Mendelian ratio (1:2:1): wildtype (+/+) 36 (25%), wildtype/TRESK knockout heterozygous (+/−) 65 (46%), homozygous TRESK-knockout (−/−) 41 (29%).
To confirm the elimination of TRESK expression in homozygous knockout animals and to assess the effect of knockout on the expression of other K2P channels, mRNA levels of TRESK, TREK-1, TASK-1 and TASK-3 in brain, spinal cord and DRG were measured by quantitative RT-PCR. In TRESK (−/−) mice, expression of TRESK mRNA was undetectable in the whole brain, spinal cord and DRG; mice heterozygous for TRESK knockout (+/−) expressed approximately half the amount of TRESK mRNA compared to wildtype (Figure 2A). Quantitative RT-PCR results also showed that the expression of TREK-1, TASK-1 and TASK-3 mRNA in the whole brain, spinal cord and DRG of TRESK −/− and +/− mice remained at similar levels as that of their wildtype (+/+) littermates (Fig. 2B–D).
As final proof of the absence of TRESK expression immunocytochemical staining of spinal cord and brain sections showed complete elimination of staining in brain, spinal cord and DRG sections from knockout animals (Figure 3).
TRESK knockout (−/−) and heterozygous (+/−) mice had normal growth and showed no structural abnormalities. Mice bred from homozygous TRESK knockout (−/−) mice parents also developed and grew normally. Body size, body weight and the appearance of fur and whiskers were normal as compared to wildtype littermates. Knockout and wildtype littermates were evaluated in more detail for neurobehavioral differences (10 knockout and 9 wildtype) (Figure 4). Basic behavioral screening including analysis of active and inactive events, grooming, defecation, urination, movement in response to tail suspension and ability to suspend from wire grid were not significantly different from wildtype littermates (Figure 4A, C and D). Knockout mice did show a small but significant decrease in inactive events compared to wildtype littermates (Figure 4B). Motor function was evaluated in two ways: ability to traverse a challenge beam and to maintain position on a rotorod (fixed and accelerating); in these tests there were no significant differences in performance between wildtype and knockout mice (Figure 4E and F). However, TRESK knockout mice had increased thermal nociceptive sensitivity, showing 20% decreased latency in hot plate testing (Figure 4G).
Four volatile anesthetics, isoflurane, halothane, sevoflurane and desflurane were tested. Table 1 shows the number and gender of the animals tested with each agent. The sensitivity of TRESK knockout and heterozygous mice to isoflurane was significantly reduced as compared to that of wildtype mice: MAC values (mean + SEM) of isoflurane for knockout, 1.54 ± 0.016; heterozygote, 1.52 ± 0.017; and wildtype, 1.42 ± 0.016 (p=0.001 between knockout and wildtype) (Figure 5A). However, the sensitivities of TRESK knockout or heterozygous mice to desflurane, halothane and sevoflurane were not different from wildtype. MAC values of desflurane were knockout, 7.59 ± 0.09; heterozygote, 7.58 ± 0.07; and wildtype, 7.47 ± 0.10 (Figure 5B). MAC values of halothane were knockout, 1.16 ± 0.02; heterozygote, 1.15 ± 0.03; and wildtype, 1.16 ± 0.02 (Figure 5C). MAC values of sevoflurane were knockout, 2.86 ± 0.03; heterozygote, 2.89 ± 0.03; and wildtype, 2.87 ± 0.05 (Figure 5D).
The recovery and survival of wildtype mice after MAC assays were consistent with previous studies using this mouse strain (death rate for C57BL6 ~12–15% during MAC testing, verbal communication James Sonner M.D., Ph.D., Professor, Department of Anesthesia and Perioperative Care, University of California San Francisco, San Francisco, California, December 2009). However, the recovery and survival of the TRESK knockout mice after the MAC assays were significantly impaired – knockout mice recovered slowly and incompletely after the MAC assay as compared with wildtype littermates. Some knockout mice appeared to have recovered but then were found dead in their cages the next day. Mortality of knockout mice during and within the first 24 hours after MAC assay was significantly higher than that of wildtype littermates (Table 2). The difference in mortality between genotypes was significantly different for animals after a first MAC study (31.6% for knockout vs. 11.5% for wildtype, p<0.05 by Fisher’s exact test). Overall death rates after multiple MAC assays (mice undergoing two or more MAC assays) were also increased (73.7% for knockout vs. 53.8% for wildtype) but the difference was not statistically significant.
In the present study, we evaluated the phenotype of mice with deletion of the K2P channel TRESK gene. TRESK homozygous knockout mice appeared no different than wildtype littermates in growth, overall appearance and reproduction, suggesting that TRESK is not an essential gene for these aspects of mouse development. Knockout of the TRESK gene produced no noticeable impairment in the gross behaviors and motor ability of the mice but on closer testing knockout mice were found to have mildly increased thermal sensitivity and decreased inactive behavior compared to wildtype mice. Although we found no compensatory changes in expression of other major K2P channels when TRESK expression is lost, the lack of behavioral phenotype in these mice implies there is redundancy behind the functional contribution that TRESK makes to the function of the central nervous system.
We also investigated the role of TRESK in the sensitivity to volatile anesthetics and found that mice with inactivated TRESK were less sensitive to isoflurane, but had unchanged sensitivity to desflurane, halothane and sevoflurane. The difference in MAC for isoflurane was small but statistically significant. This isolated finding is similar to previously reported changes in anesthetic sensitivity in other K2P knockout mice. TASK-1 knockout mice have partially reduced sensitivity to isoflurane or halothane depending on the assay30 while TASK-3 knockout mice show decreased sensitivity to halothane but not isoflurane in standard MAC assay.31 Furthermore, knockout mice with deletion of other proteins implicated in anesthesia mechanisms such as the β3 subunit of the γ-aminobutyric acid type A receptor45 or the stomatin gene show variable changes in volatile anesthetic sensitivity.46 Only TREK-1 knockout mice manifest a consistent reduction in volatile anesthetic sensitivity, yet even in those mice the changes in MAC varied widely, from as low as 7% for desflurane to as high as 48% for halothane.29 We believe that our finding of decreased anesthetic sensitivity for only one drug in TRESK knockout mice adds to a growing consensus that volatile anesthetics are acting on multiple targets, possibly including K2P channels, whose sum total effect achieves the pharmcodynamic action of volatile anesthetics.
The behavioral evaluation of mice with inactivated TRESK gene are also congruent with previous studies of other K2P family knockout mice.47 TREK-1, TWIK-1, TASK-2 and KCNK7 knockout mice all are healthy, fertile and have normal morphology and behaviors.29,32,33,48 TASK-1 knockout mice act normally in several behavioral tests, with only minor changes in paw withdrawal response and motor function.30,49 TASK-3 knockout mice show no gross abnormality apart from a minor increased locomotor activity during the dark phase and slower swimming ability.31 Even the TASK-1/TASK-3 double-knockout mouse shows no obvious neurological abnormalities or health problems.50 These observations reinforce the idea that the expression of individual members of the K2P gene family is not essential for normal growth, development and reproduction.
We did document a greater mortality rate in TRESK knockout mice after undergoing MAC testing compared to their wildtype littermates and speculate that this impaired survival ability may result from reduced tolerance to the stress of MAC testing. The MAC assay itself, consisting of a prolonged anesthetic with varying agent levels and exposure to noxious stimuli under at times a light plane of anesthesia represents a significantly stressful event. We do not believe that the higher mortality of knockout versus wildtype mice occurred due to the difference in isoflurane MAC because knockout and wildtype animals received very similar concentrations of isoflurane during testing. In addition, greater mortality also occurred in animals exposed only to the other volatile anesthetics, for which the knockout mice did not display any change in MAC. The exact mechanism underlying reduced survival ability in these mice remains to be further investigated and could include alterations in respiratory dynamics, abnormal response to changes in PaCO2 or interference with other cardiorespiratory functions.
As with all global knockout studies it is possible that a compensatory effect by other K2P channels or other genes responsible for anesthetic action may occur in response to TRESK gene deletion. Quantitative PCR showed that knockout of TRESK gene did not significantly alter mRNA expression of TREK-1, TASK-1 and TASK-3 in the whole brain, spinal cord and DRG. However, these results do not rule out changes in expression of these K2P family members at a regional or cellular level or of compensation by other ion channel expression. Changes in gene expression of other anesthetic sensitive ion channels could compensate for loss of TRESK, as has been described with μ opioid receptor knockout.51
Another physiologic effect that could be masking the role of TRESK and other K2P channels in knockout mice is the fact that the anesthetics studied in the MAC assays were delivered in 100% oxygen. Given that several K2P channels are activated by molecular oxygen it is possible that the anesthetic response of knockout animals may have blunted by this factor.
In conclusion, inactivation of TRESK gene expression in mice does not cause a significant effect on development, growth, reproduction and gross behavior, but does cause a slightly decrease in the sensitivity of the mice to isoflurane and increased thermal nociception. Decreased sensitivity to isoflurane but not to halothane, sevoflurane and desflurane indicates that TRESK may partially mediate the action of this volatile anesthetic. A significantly higher death rate in TRESK knockout mice after the MAC assays could indicate a role for TRESK in an endogenous survival mechanism in response to stress.
We thank Yun Weng Ph.D, Postdoctoral Fellow and Irene Oh B.S, Staff Research Associate Department of Anesthesia and Perioperative Care, University of California San Francisco for their technical assistance.
Summary statement: This study is the first characterization of genetically modified mice with a knockout of the tandem pore potassium channel TRESK, focusing on their sensitivity to volatile anesthetics.
Disclosure of funding: Supported by United States National Institute of Health (Bethesda, Maryland) grant GM58149 to Dr. Yost.