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The Contribution of TWIK-Related Acid-Sensitive K⁺-Containing Channels to the Function of Dorsal Lateral Geniculate Thalamocortical Relay Neurons

Institut für Experimentelle Epilepsieforschung, Westfälische Wilhelms-Universität Münster, Münster, Germany (T.B., H.-C.P.); Abteilung für Klinische Neurobiologie, Universität Heidelberg, Heidelberg, Germany (M.I.A., W.W.); Institut für Physiologie, Otto-von-Guericke-Universität, Magdeburg, Germany (T.M.); Institut für Physiologie I, Westfälische Wilhelms-Universität Münster, Münster, Germany (T.Se., H.-C.P.); Klinik für Neurologie, Julius-Maximilians-Universität Würzburg, Würzburg, Germany (S.G.M., T.Sc., C.K., H.W.); and Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen, Scotland, United Kingdom (W.W.)

Received November 8, 2005; accepted January 18, 2006

	Abstract

Top Abstract Methods and Materials Results Discussion References

 
A genetic knockout was used to determine the specific contribution of TWIK-related acid-sensitive K⁺ (TASK)-1 channels to the function of dorsal lateral geniculate nucleus (DLG) thalamocortical relay (TC) neurons. Disruption of TASK-1 function produced an 19% decrease in amplitude of the standing outward current (I_SO) and a 3 ± 1-mV depolarizing shift in resting membrane potential (V_rest) of DLG neurons. We estimated that current through TASK-1 homodimers or TASK-1/TASK-3 heterodimers contribute(s) approximately one third of the current sensitive to TASK channel modulators in DLG TC neurons. The effects of the TASK channel blocker bupivacaine (20 µM), of muscarine (50 µM), and of H⁺ on I_SO were reduced to approximately 60%, 59%, and shifted to more acidic pH values, respectively. The blocking effect of anandamide on I_SO [30 µM; 23 ± 3% current decrease in wild type (WT)] was absent in TASK-1 knockout (TASK-1^-/-) mice (9 ± 6% current increase). Comparable results were obtained with the more stable anand-amide derivative methanandamide (20 µM; 20 ± 2% decrease in WT; 4 ± 6% increase in TASK-1^-/-). Current-clamp recordings revealed a muscarine-induced shift in TC neuron activity from burst to tonic firing in both mouse genotypes. Electrocorticograms and sleep/wake times were unchanged in TASK-1^-/- mice. In conclusion, our findings demonstrate a significant contribution of TASK-1 channels to I_SO in DLG TC neurons, although the genetic knockout of TASK-1 did not produce severe deficits in the thalamocortical system.

The current through TASK-1 and -3 subunits reveals out-ward rectification of whole-cell currents under physiological K⁺ concentrations and is inhibited by acidic pH; in recombinant systems and in vivo, channel subunits can assemble heteromerically if the cell type expresses both genes at suitable levels (Czirjak and Enyedi, 2002; Berg et al., 2004; Kang et al., 2004; Aller et al., 2005); alternatively, they can function as homomers if either TASK-1 or TASK-3 dominates the expression (Czirjak and Enyedi, 2003; Clarke et al., 2004). Because of 1) overlapping gene expression patterns, 2) similarities of whole-cell currents through different channel subtypes, and 3) the formation of heterodimeric channels resulting in intermediate properties, it is hard to specify the molecular origins of native leak currents. Thus, knockout mice are valuable to assess the in vivo contributions and functional roles of specific K2P channel subtypes.

For example, previous work with TASK-1 knockout mice (TASK-1^-/-) dissected K2P channel diversity in adult cerebellar granule cells (Aller et al., 2005). Granule cells strongly express four K2P genes: TASK-1, TASK-3, the TWIK-related K⁺ channel gene-2c splice variant, and tandem pore domain halothane-inhibited K⁺ channel-2. In TASK-1 knockout mice, although the standing outward K⁺ current (I_KSO) remained unchanged, and granule cells were not more depolarized, the I_KSO became Zn²⁺-sensitive, suggesting that normally most TASK channels in granule cells are TASK-1/TASK-3 heteromers, and that in the TASK-1^-/- granule cells, this heteromer was replaced by homomeric TASK-3 channels. The failure to see a reduction in I_KSO magnitude in the TASK-1^-/- granule cells could be because of contributions from the remaining K2P genes.

By contrast, not all neuronal cell types express the high K2P channel diversity found in cerebellar granule cells. For example, the main K2P genes expressed in mouse thalamus are TASK-1, TASK-3, and TWIK-1. The TASK-3 gene dominates; however, there are variations in expression depending on the exact thalamic nucleus (Fig. 1). The DLG in the mouse thalamus contains moderate levels of TASK-3 and TASK-1 transcripts, together with some TWIK-1 and other K2P gene transcripts (Aller et al., 2005); indeed, in postnatal day 19 rat DLG cells in slices, both TASK-1 and TASK-3 channels (as assayed by pharmacology and in situ hybridization) significantly contribute to the unique features of the cells (Meuth et al., 2003), although under certain circumstances (removal of covalently attached Small Ubiquitin-like Modifier protein), TWIK-1 has TASK-like channel properties (Rajan et al., 2005). Furthermore, in addition to the contribution from TASK channels, I_SO in TC neurons is carried by additional channel types (persistent Na⁺ channels, inward rectifying K⁺ channels, noninactivating voltage-dependent K⁺ channels, and pacemaker channels; S. G. Meuth and T. Budde, unpublished observations). Regardless, removing expression of a particular K2P family member from any given thalamic relay nucleus might have stronger impact on function than in cerebellar granule cells, simply because there are fewer other K2P channel types expressed in the thalamus that could compensate for the loss.

View larger version (123K): [in this window] [in a new window]   Fig. 1. In situ hybridization showing TASK-1 (A and B) and TASK-3 (C and D) gene expression in the mouse (wild-type) thalamus from P19, an age matching the electrophysiological recordings. CA1, hippocampal CA1 region; Cx, neocortex; DG, dentate granule cells; Rt, reticular thalamus; VLG, ventral lateral geniculate nucleus; and ZI, zona incerta. X-ray film autoradiographs, exposure time 4 weeks. Scale bar, 1 mm.

The likely function for K2P channels in thalamic relay cells is characterized by the closure of TASK-1 and -3 channels mediated by modulatory transmitters such as acetylcholine acting via G proteins, thereby promoting membrane depolarization and a shift in activity mode from burst to tonic firing of action potentials (McCormick, 1992; Steriade et al., 1997; Meuth et al., 2003). To determine the specific contribution of the two TASK subunit variants to the physiology of DLG TC neurons and to analyze their influences on cell's response to cholinergic modulation, we used TASK-1-deficient mice (TASK-1^-/-) (Aller et al., 2005). We addressed the following questions: 1) What are the pharmacological and functional consequences of a TASK-1 knockout in DLG cells and does it differ in its effects from cerebellar granule cells? and 2) Is there a TASK-1^-/- phenotype related to the thalamocortical system?

	Methods and Materials

Top Abstract Methods and Materials Results Discussion References

 
The TASK-1 Knockout Mouse Line. The generation of TASK-1^-/- mice has recently been described (Aller et al., 2005). For the study described here, and as in Aller et al. (2005), TASK-1^-/- mice were always produced by breeding heterozygote +/- animals; the -/- and +/+ littermates could then be used for direct comparisons. Mice were genotyped by Southern blotting (Aller et al., 2005).

Animals. Mice were individually housed in standard cages without running wheels on a 12-h light/dark cycle at an ambient temperature of 23 ± 1°C. Rodent laboratory chow and drinking water were provided ad libitum. All procedures involving the use of animals were approved by the local Animal Care Committee of the Universities of Heidelberg and Magdeburg in agreement with the German laws for animal protection.

In Situ Hybridization. In situ hybridization, with ³⁵S-labeled TASK-1- and TASK-3-specific oligonucleotide probes was performed as described previously (Aller et al., 2005). Brains were from wild-type P19 animals.

Preparation: Thalamic Slices. Wild-type (WT) and TASK-1^-/- mice postnatal day 14 to 22 were deeply anesthetized using halothane and decapitated as described previously for rats (Meuth et al., 2003). In brief, thalamic slices were prepared as coronal sections on a vibratome (Series 1000 Classic; Vibratome Company, St. Louis, MO) in an ice-chilled solution containing 200 mM sucrose, 20 mM PIPES, 2.5 mM KCl, 1.25 mM NaH₂PO₄, 10 mM MgSO₄, 0.5 mM CaCl₂, and 10 mM dextrose, pH 7.35 adjusted with NaOH. Before the recording procedure, slices were kept submerged in artificial cerebrospinal fluid: 125 mM NaCl, 2.5 mM KCl, 1.25 mM NaH₂PO₄, 24 mM NaHCO₃, 2 mM MgSO₄, 2 mM CaCl₂, and 10 mM dextrose, pH adjusted to 7.35 by bubbling with a mixture of 95% O₂ and 5% CO₂.

Patch-Clamp Recordings of DLG Neurons. DLG neurons were visualized in the slice preparation using a microscope equipped with infrared differential interference contrast optics. Whole-cell recording pipettes were prepared from borosilicate glass (GT150T-10; Clark Electromedical Instruments, Pangbourne, UK) having typical resistances of 2 to 3 M. Pipettes were filled with an intracellular solution containing 95 mM K-gluconate, 20 mM K₃-citrate, 10 mM NaCl, 10 mM HEPES, 1 mM MgCl₂, 0.5 mM CaCl₂, 3 mM BAPTA, 2 mM Mg-ATP, and 0.5 mM Na-GTP. In divalent cation-free external solutions the osmolarity was kept constant at 305 mOsm/kg by adding mannitol. The internal solution was set to pH 7.25 with KOH and an osmolarity of 295 mOsmol/kg. For current-clamp recordings, a pipette solution containing 5 mM EGTA and 0.5 mM CaCl₂ was used. Slices were continuously superfused with a solution containing 120 mM NaCl, 2.5 mM KCl, 1.25 mM NaH₂PO₄, 30 mM HEPES, 2 mM MgSO₄, 2 mM CaCl₂, and 10 mM dextrose and pH values were adjusted using HCl. Whole-cell patch-clamp recordings were measured with an EPC-10 amplifier (HEKA Elektronik, Lamprecht, Germany), digitized, and acquired onto computer using Pulse software (HEKA Elektronik).

All cells had a resting membrane potential more negative than -65 mV, the access resistance was in the range of 5 to 15 M, and series resistance compensation of more than 40% was routinely used. A liquid junction potential of 8 ± 2 mV (n = 10) was measured and taken into account.

The pH response curve of the standing outward current at -28 mV was drawn according to the equation I_SO = I_max/{1 + (EC₅₀/A)^nH}, where I_SO represents the current response, I_max is the maximal relative current amplitude, EC₅₀ is the half-maximal effective pH value, A is the pH value, and n_H is the Hill coefficient.

Drugs. The following drugs were used for electrophysiological recordings: bupivacaine and muscarine were obtained from Sigma Chemie (Deisenhofen, Germany), prepared as stock solutions in distilled water, and added to the perfusion medium. ZD7288 was delivered from Tocris Cookson Inc. (Köln, Germany) and prepared as described above. Anandamide, methanandamide, and arachidonic acid were obtained from Calbiochem (Schwalbach/Ts., Germany) and dissolved in ethanol. The solvent concentration in the final recording solution did not exceed 1%. Application of the solvent alone (1%) had no effect on the recorded current.

Surgery and Electrode Implantation. Experiments were performed on male mice (between 8 and 12 weeks old; n = 5 for TASK-1^-/-- and wild-type littermates, respectively) and were approved by the Landesverwaltungsamt Halle (AZ 2-663Uni Magdeburg). After anesthesia with pentobarbital (50 mg/kg i.p.; Sigma Chemical Co., St. Louis, MO), the mouse was positioned in a stereotaxic instrument (David Kopf Instruments, Tujunga, CA) with bregma and lambda in a horizontal plane. For bilateral epidural electrocorticogram (ECoG) recordings, silver electrodes were positioned over the central region (AP, -1.0 mm; L, 2 mm from bregma) of both hemispheres and fixed on the skull with dental acrylic cement (Paladur; Heraeus Kulzer GmbH, Wehrheim/Ts, Germany). In addition, reference and ground electrodes were implanted over the nasal and cerebellar region, respectively. Xylocaine cream (2%; Astra GmbH, Germany) was applied to all pressure points and wound edges. Four days after surgery, ECoG was recorded for 4 to 6 h to get a wide spectrum of different behavioral states (i.e., wake and sleep) using a swivel commutator.

ECoG recordings were performed using the spike2-software package (Cambridge Electronic Design, Cambridge, UK). Recorded electrical activities were fed through a differential amplifier (EXT-20F or DPA 2F; npi electronic GmbH, Tamm, Germany) collected at a sampling rate of 1 kHz with a filter cut-off of 100 Hz, transformed by an A/D interface (CED 1401plus; Cambridge Electronic Design) and stored on-line on a personal computer. In addition, data were stored on a magnetic tape recorder via a neuro-corder (DR-890; NeuroData Instr. Corp., Delaware Water Gap, PA) for off-line analysis.

Behavioral Observations. The activity of WT (n = 5; 27.58 ± 3 g) and TASK-1^-/- mice (n = 8; 28.39 ± 1.4 g) was continuously recorded in their home cages by infrared detectors for 24 h. These recordings were analyzed by two experienced observers blinded for the genetic background of the animals. Forms of behavior (sleep, resting, leaning, rearing, grooming, nutrition, and moving) were calculated as percentage of 24 h.

Statistics. All results are presented as mean ± S.E. and differences were considered statistically significant if p < 0.05. Substance effects were tested for statistical significance using a modified Student's t test for small samples (Dixon and Massey, 1969).

	Results

Top Abstract Methods and Materials Results Discussion References

 
Expression of the TASK-1 and TASK-3 Genes in the Mouse DLG. We first examined the DLG by in situ hybridization and focused on the expression of the TASK-1 and -3 genes in our target thalamic nucleus. Mouse brains were selected from an age that matched the majority of electrophysiological recordings, postnatal day 19. Figure 1 shows coronal brain sections through the thalamus at the DLG level. We reported previously for the adult mouse brain (Aller et al., 2005), that the TASK-1 gene's highest expression is in cerebellar granule cells, motor neurons, raphe, and locus ceruleus, whereas TASK-3 gene expression is at higher levels throughout the brain (e.g., CA1 pyramidal cells, dentate granule cells, neocortex, and many hypothalamic nuclei). This is reflected in the coronal images shown in Fig. 1 at the level of the thalamus for P19. Nevertheless, specific thalamic nuclei have quite different expression profiles of the TASK-1 and TASK-3 genes. For example, the ventral lateral geniculate nucleus (VLG) has more TASK-3 than TASK-1 gene expression, whereas the midline thalamic nuclei have more TASK-1 transcripts (Fig. 1, A and C). More caudally (Fig. 1, B and D), TASK-3 expression dominates in the thalamus and zona incerta (Fig. 1D). TASK-1 expression dominates in the reticular thalamus (Fig. 1A). Despite the overall predominance of TASK-3 gene expression in the fore- and midbrain, both TASK-1 and TASK-3 mRNAs can be detected in the DLG at approximately roughly equal amounts (Fig. 1, A and C).

pH Sensitivity of Leak Potassium Channels in WT and TASK-1^-/- DLG Neurons: Consequences for the Resting Membrane Potential. TASK-1 and -3 channels contribute to the I_SO (Fig. 3A) in several cell types, such as cerebellar granule cells, motor neurons, and DLG TC cells (Millar et al., 2000; Brickley et al., 2001; Bayliss et al., 2003; Meuth et al., 2003). To assess the contribution of the TASK-1 subunit to the total TASK-1 and -3 current in TC neurons of the DLG, we probed a number of TASK-1 and -3 channel features (pH sensitivity, block by bupivacaine, and modulation by ACh) in whole-cell patch-clamp experiments. Both TASK-1 and TASK-3 channels are inhibited by acidification, although over different pH ranges (pK values for channel activity, 7.5 and 6.7 for TASK-1 and TASK-3, respectively) (Duprat et al., 1997; Kim et al., 2000; Rajan et al., 2000). Tandem-linked heterodimeric TASK channel constructs displayed pH sensitivity (pK 7.3) closer to that of TASK-1 than TASK-3 (Berg et al., 2004). In a first experimental step, we compared the pH dependence of I_SO in WT and TASK-1^-/- mice. At a holding potential of -28 mV I_SO averaged 368 ± 17 pA in WT mice (n = 38) and 299 ± 16 pA in TASK-1^-/- mice (n = 37; data not shown; p = 0.005) under experimental control conditions (pH 7.25). Varying the external pH (pH_O) from 8.0 to 5.0 stepwise resulted in a reduction of I_SO, which was different in both genotypes. Whereas I_SO averaged 438 ± 47 pA (n = 4) and 415 ± 32 pA (n = 3) at pH 7.5 in WT and TASK-1^-/-, respectively, current amplitudes were strongly reduced to 39 ± 22 pA (n = 4; WT) and 35 ± 29 pA (n = 3; TASK-1^-/-) at pH 5.0. From populations of cells (a total of 24 cells were tested for each genotype) recorded at different pH values, the EC₅₀ value of I_SO inhibition could be determined at 6.6 and 5.9 for WT and TASK-1^-/-, respectively, according to the Hill equation (Fig. 2A).

View larger version (24K): [in this window] [in a new window]   Fig. 3. Effect of bupivacaine on TASK current in WT and TASK-1-/- DLG neurons. A and B, currents evoked by ramping the membrane potential from -28 to -138 mV over 800 ms (see inset) under control conditions (black trace) and during application of bupivacaine (gray trace) in WT (A) and TASK-1-/- mice (B). C, mean I-V relationship of current components sensitive to bupivacaine obtained by graphical subtraction of currents during drug action from control currents (i.e., control - bupivacaine). Both components show a clear outward rectification and a reversal at the expected EK. D, mean bar graph representation of ISO reduction at -28 mV by bupivacaine in WT (black column) and TASK-1-/- (gray column).

View larger version (10K): [in this window] [in a new window]   Fig. 2. pH sensitivity of the standing outward current in WT and TASK-1-/- DLG neurons. A, dependence of ISO at -28 mV on extracellular acidification (mean ± S.E.) for groups of three to five relay neurons in a range from pH 5.0 to 8.0 is shown. The curve was drawn according to the Hill equation through the data points for WT (black line) and TASK-1-/- (gray line), respectively. The EC50, Imax, and Hill values were WT, 6.6, 106.4, and 0.53; and TASK-1-/-, 5.9, 101.4, and 0.55. B, mean value of the resting membrane potential under different recording conditions (squares, control conditions; circles, presence of ZD7288; triangles, presence of ZD7288 at pH 5.0; WT, closed symbols; TASK-1-/-, open symbols).

Actions of the Local Anesthetic Bupivacaine on I_SO in DLG Neurons. Next, the local anesthetic bupivacaine, known to block TASK-1 and -3 channels (Leonoudakis et al., 1998; Kindler et al., 1999; Meadows and Randall, 2001; Meuth et al., 2003) was tested. Bath application of 20 µM bupivacaine resulted in a marked reduction in I_SO amplitude, which was significantly (p = 0.027) stronger in WT (55 ± 8%; n = 8) compared with TASK-1^-/- (33 ± 4%; n = 8; Fig. 3, A, B, and D). To determine the current-voltage (I/V) relationship of the bupivacaine-sensitive current, the membrane potential was ramped from -28 to -138 mV over 800 ms (Fig. 3, A and B, inset). The resulting current revealed a complex waveform indicative for outwardly and inwardly rectifying components (Fig. 3, A and B). By subtracting currents in the presence of bupivacaine from control currents, and assigning the appropriate membrane potential to each time point, the I-V relationship of the bupivacaine-sensitive current was constructed (Fig. 3C). The bupivacaine-sensitive currents in DLG neurons revealed clear outward rectification and a reversal potential of -103 ± 5 (n = 8) and of -103 ± 3 (n = 8) for WT and TASK-1^-/-, respectively [i.e., close to the expected K⁺ equilibrium potential (E_K) of -104 mV]. In summary, these data indicate a reduced bupivacaine-sensitive current component in DLG neurons lacking TASK-1 gene expression.

Muscarine Inhibits I_SO in WT and TASK-1^-/- DLG Cells: Functional Implications. Next, we investigated the functional modulation of TASK-1 and -3 channels in DLG neurons by activation of muscarinic ACh receptors (mAChRs). First, the action of muscarine (50 µM) was analyzed under voltage-clamp conditions. After application of muscarine via the extracellular solution, I_SO amplitude was significantly (p = 0.025) reduced by 41 ± 2% (n = 4) and 24 ± 4% (n = 8) in WT and TASK-1^-/-, respectively (Fig. 4, A, B, and D). The curvature of the muscarine-sensitive I-V relationship was characterized by inward and outward rectification in both genotypes and reversed close to E_K (WT, -105 ± 5 mV, n = 4; TASK-1^-/-, -107 ± 2 mV, n = 8; Fig. 4C). Second, the effect of mAChR activation was recorded under current-clamp conditions. To allow high-frequency burst firing, cells were held at a potential of -72 ± 1 mV (n = 8) via DC current injection.

View larger version (33K): [in this window] [in a new window]   Fig. 4. Muscarine effect on ISO in WT and TASK-1-/- DLG neurons. A and B, membrane responses to ramp protocols (from -28 to -138 mV over 800 ms; see inset) under control conditions (black traces) and during bath application of muscarine (gray traces) in WT (A) and TASK-1-/- DLG cells (B). C, mean I-V relationship of the muscarine-sensitive current was calculated by graphical subtraction of currents during drug action from those under control conditions (i.e., control - muscarine). D, mean bar graph representation of ISO reduction at -28 mV by muscarine in WT (black column) and TASK-1-/- (gray column). E and F, functional consequences of muscarine administration were tested under whole-cell current-clamp conditions in WT (E) and TASK-1-/- (F). DLG TC neurons of both mouse genotypes were held at a potential of 72 ± 1mV(n = 8) by DC current injection. This holding current was not changed during the course of the experiment. Cells were challenged using 100- to 200-pA depolarizing current pulses (800-ms duration). Under control conditions, depolarizing current pulses starting from hyperpolarized membrane potentials evoked typical burst firing (black traces). Addition of muscarine led to a marked depolarization of the membrane potential with a consecutive shift of the activity mode from burst to tonic firing (gray traces).

The Effects of Cannabinoid Ligands on I_SO in DLG Neurons and the Specific Involvement of TASK-1. The cannabinoid agonists anandamide (arachidonylethanolamide; 30 µM) and R-(+)-WIN 55,212-2 have been suggested as TASK-1-specific blockers, having no effect on TASK-3 channels (Maingret et al., 2001). In contrast, other reports have claimed that on recombinant channels anandamide does not distinguish between TASK-1 and TASK-3 (Berg et al., 2004; Aller et al., 2005). In the present study, anandamide induced a reduction in I_SO amplitude at -28 mV by 23 ± 3% (n = 5; Fig. 5B, black column) in WT neurons. In TASK-1^-/- DLG neurons, the blocking effect of anandamide was abolished, and drug application was accompanied by a 9 ± 6% (n = 6; Fig. 5B, gray column) increase in I_SO amplitude. The I-V relationship of the anandamide-sensitive current in WT neurons was characterized by outward rectification and reversed at -103 ± 3 mV (n = 5; Fig. 5A, black trace). In contrast, the anandamide-sensitive current in TASK-1^-/- DLG TC cells was linear and see-sawed around zero (n = 6; Fig. 5A, black circles). To confirm these data, we used an anandamide analog (methanandamide; 10 µM) with higher metabolic stability. Whereas application of methanandamide onto WT neurons reduced I_SO amplitude by 20 ± 2% (n = 6; Fig. 5B, stripped black column), the effect was absent on TASK-1^-/- DLG cells (4 ± 6% increase in I_SO amplitude; n = 6; Fig. 5B, gray stripped column). The I-V relationship of the methanandamide-sensitive current was characterized by outward rectification and a reversal potential at -105 ± 2 mV (n = 6; Fig. 5A, gray trace). In TASK-1^-/- DLG cells, the methanandamide-sensitive current was linear with near zero amplitude (n = 6; Fig. 5A, gray circles). Because anandamide can be rapidly transformed to arachidonic acid (AA), we next investigated the effect of this eicosanoid on I_SO in DLG TC neurons. In both TASK-1^-/- and WT DLG cells, AA induced a significant increase in I_SO amplitude and was thus very different from the effect of the two anandamide derivatives (WT, 25 ± 6% increase, n = 5; TASK-1^-/-, 32 ± 9%, n = 7; data not shown).

View larger version (25K): [in this window] [in a new window]   Fig. 5. Specific pharmacological strategies of blocking TASK-1 and TASK-3 channels in DLG TC neurons. A, I-V relationship of anandamide- (black label) and methanandamide-sensitive (gray label) currents in WT (straight lines) and TASK-1-/- (circles). B, mean bar graph representation of ISO changes induced by anandamide (closed columns) and methanandamide (stripped columns) in WT (black label) and TASK-1-/- (gray label). C, I-V relationship of divalent cation-sensitive currents in WT (black line) and TASK-1-/- (gray line). D, mean bar graph representation of ISO induced by removal of divalent cations from the extracellular solution at -28 mV in WT (black column) and TASK-1-/- (gray column).

Electroencephalographic Recordings. To assess possible deficits of TASK-1^-/- mice with respect to whole brain function, we recorded ECoG of freely moving TASK-1^-/- and WT littermates. TASK-1^-/- as well as WT mice expressed very regular field potential patterns on the ECoG without any abnormalities such as polyspike complexes or high-voltage spike activities and showed similar patterns during expression of different behavioral states (e.g., wake and sleep; data not shown). During wake behavioral state, both genotypes revealed predominant frequencies at theta frequency (5-12 Hz). During sleep the ECoG of TASK-1^-/- and WT animals was characterized by higher amplitudes compared with the wake state and low-frequency activity at approximately 2 to 4 Hz (data not shown).

Behavioral Observations Indicate a Comparable Sleep-Wake Cycle of TASK-1^-/- Mice and Controls. Because the dorsal thalamus plays an important role in the mammalian sleep-wake cycle, we next performed behavioral observations on WT and TASK-1^-/- mice. Consistent with the EEG recordings, the behavioral observations offered similar results for both mice genotypes. With respect to sleeping and waking behavior, WT animals were active (addition of moving, rearing, leaning, grooming, and nutrition) and inactive (sleeping and resting) for 44 ± 2 and 56 ± 2% of the day (n = 5, repeated measurements; Fig. 6A). The behavior of TASK-1^-/- mice was indistinguishable with 43 ± 1% active and 57 ± 1% inactive behavior per 24 h (n = 5, repeated measurements; Fig. 6A). Furthermore, the analysis of specific behaviors such as moving, rearing, leaning, grooming, or nutrition showed no statistically significant differences between knockouts and wild-type littermates (Fig. 6B). Like-wise, further differentiation of inactive behavior (separation of resting behavior and sleep) displayed no significant differences between the two groups.

View larger version (16K): [in this window] [in a new window]   Fig. 6. Behavioral analysis of WT and TASK-1-/-. A, mean bar graph representation of active (black columns) and inactive (gray columns) periods in WT and TASK-1-/- (as indicated) in percentage of 24 h. B, pie diagram representation of distinct active (moving, rearing, leaning, grooming, and nutrition) and inactive (resting and sleep) behavior components.

	Discussion

Top Abstract Methods and Materials Results Discussion References

 
This study describes novel aspects of TASK-1 channel function in DLG TC neurons and can be summarized as follows: 1) I_SO amplitude was reduced and the resting membrane potential was more depolarized in TASK-1^-/- cells in comparison with WT; 2) I_SO decreased in amplitude with increasing proton concentration. The pK value of current inhibition was shifted to lower pH values in TASK-1^-/- neurons compared with WT; 3) The TASK-1 and -3 channel blocker bupivacaine reduced the outwardly rectifying TASK current in TASK-1^-/- more effectively than in WT; 4) Muscarine reduced I_SO by inhibiting TASK-1 and -3 channels and inwardly rectifying channels. Although this effect was less pronounced in knockouts, neurons switched from burst to tonic firing in both TASK-1^-/- and WT. 5) Anandamide as well as the more stable methanandamide blocked an outward rectifying TASK current component in WT but was ineffective in TASK-1^-/-. On the other hand, removal of divalent cations from the extracellular medium, a maneuver to specifically unblock TASK-3 channels, was equally effective in the two genotypes. 6) Consistent with the mild electrophysiological effects of TASK-1 gene knockout in the dorsal thalamus, no noticeable differences between WT and TASK-1^-/- mice were found in EEG recordings and in the overall sleep/waking behavior. We conclude 1) that in DLG neurons TASK-1 makes a significant contribution to I_SO and 2) that anandamide blocks TASK-1 or TASK-1 and -3 channels in WT cells but not the presumably homomeric TASK-3 channels remaining in the TASK-1^-/- DLG TC cells.

TASK Channels in DLG TC Neurons. The TASK channel genes are differentially expressed in the rodent central nervous system (Talley et al., 2001). In rat DLG TC neurons, the current carried by TASK channels is characterized by outward rectification, reversal at E_K, inhibition by extracellular acidification, block by bupivacaine, and down-regulation by activation of mAChR (Meuth et al., 2003). From the data presented here, we estimate that current through TASK-1 homodimers or TASK-1/TASK-3 heterodimers (Czirjak and Enyedi, 2002) contributes approximately one third of the current sensitive to TASK channel modulators in DLG TC neurons. This conclusion is based on the following evidence: 1) Compared with WT, the bupivacaine-sensitive current in TASK-1^-/- DLG TC neurons is approximately 35% smaller in amplitude. 2) The difference between bupivacaine-sensitive currents in WT and TASK-1^-/- DLG cells at -28 mV is approximately 100 pA. 3) Similar to the effect of bupivacaine, the muscarine-sensitive component is approximately 28% smaller in TASK-1^-/- DLG cells. These findings further indicate that bupivacaine (Leonoudakis et al., 1998; Brown, 2000; Buckler et al., 2000) and mechanisms acting downstream of mAChR (Patel and Lazdunski, 2004) influence TASK-1 and TASK-3 channels similarly. Nevertheless, knockout of the TASK-1 gene significantly reduced I_SO, resulting in a depolarized resting membrane potential, thereby confirming the persistent activity of native TASK-1 channels and their contribution to the neuronal membrane potential (Duprat et al., 1997). However, in adult cerebellar granule cells, deletion of the TASK-1 protein leaves the magnitude of I_KSO unaffected (Aller et al., 2005), whereas deletion of TASK-1 from DLG TC cells produces a decrease in leak conductance. One explanation is that granule cells express more K2P subunit genes at higher levels than DLG TC cells, allowing greater possibilities of compensation (Brickley et al., 2001). Similar considerations may apply to other brain regions because the TASK-1^-/- phenotype shows few neurological deficits.

Both TASK1 and TASK3 channels are inhibited by acidification, although over different pH ranges (pK 7.5 and 6.7 for TASK-1 and TASK-3, respectively) (Duprat et al., 1997; Kim et al., 2000; Rajan et al., 2000). Tandem-linked heterodimeric TASK channel constructs displayed pH sensitivity (pK 7.3) closer to that of TASK-1 than TASK-3 (Berg et al., 2004). Consistent with these findings, I_SO amplitude in TC cells of WT (TASK-3 + TASK-1; EC₅₀ = 6.6) revealed an EC₅₀ value that was shifted to a more alkaline pH value in comparison with TASK1^-/- (TASK-3 only; EC₅₀ = 5.9). Deviation of absolute pK values from those published for heterologously expressed TASK channels and the 90% reduction of I_SO at pH 5.0 probably result from the fact that the steady-state current in TC neurons is carried by a number of different ion channels (persistent Na⁺ channels, inward rectifier K⁺ channels, noninactivating voltage-dependent K⁺ channels, TASK channels, and pacemaker channels; S. G. Meuth and T. Budde, unpublished observations) with unknown or undefined pH sensitivity. Changes of the extracellular pH can influence a variety of different conductances and receptors (Kaila and Ransom, 1998), so that additional contributors to the pH effects can be expected.

Endogenous Cannabinoids and Their Actions on TASK Channels of DLG Cells. Human recombinant TASK-1 but not TASK-3 channels are blocked by anandamide (Maingret et al., 2001). However, the situation seems to be more complicated when taking rodent TASK channels into account, because two other groups claim that anandamide blocks both recombinant (rodent) TASK-1 and TASK-3 channels fairly equally at concentrations >1 µM (Berg et al., 2004; Aller et al., 2005). We were surprised to find that TASK-1 channels convey the anandamide-sensitivity of I_SO in DLG TC neurons, thereby confirming that the endogenous cannabinoid ligand anandamide compounds selectively block TASK-1 channels (Maingret et al., 2001). This conclusion is confirmed indirectly by the effect of specifically activating TASK-3 channels by removing extracellular divalent cations, which produced indistinguishable effects in the two mouse genotypes. Anandamide may be rapidly eliminated by enzymatic hydrolysis, leading to the production of AA. The finding that AA induced an increase in I_SO rather than a reduction further confirms the specific action of anandamide. Although we have no intuitive explanation for this selectivity of anandamide for TASK-1 in native membranes, the following findings should be taken into account: 1) At higher concentrations (10 µM), recombinant rodent TASK-1 channels are more sensitive to anandamide than rodent TASK-3 channels (Berg et al., 2004). 2) The anandamide-sensitive current components in rat and mouse DLG TC neurons are similar: they outwardly rectify, reverse at E_K, and constitute 22 ± 2% (n = 4) and 23 ± 3% (n = 5) of I_SO, respectively. These findings may point to subtle cell type-specific influences. 3) TASK channels are regulated by adapter proteins and phosphatidylinositol-4,5-bisphosphate (Czirjak et al., 2001; Rajan et al., 2002; Chemin et al., 2003; Lopes et al., 2005), thereby indicating complex interplay between multiple factors. 4) TASK-1^-/- mice have changed responses to the cannabinoid ligand WIN55212-2 (Linden et al., 2004); the analgesic, sedative, and hypothermic effects of WIN55212-2 are reduced in TASK-1^-/- mice, implicating channels containing TASK-1 in supraspinal pain pathways, for example, in the thalamus. This behavioral result confirms that endogenous cannabinoid ligands certainly have the potential to influence cell excitability by acting close to TASK-1 and/or TASK-3 channels in vivo.

TASK-1 Function in the Thalamocortical System. The resting membrane potential of thalamic relay neurons is in the range of -70 mV and has been attributed to currents through leak channels (I_K-leak and I_Na-leak), pacemaker channels (I_h), inwardly rectifying K⁺ currents (I_KIR), and voltage-dependent channels active below threshold (I_A and I_T) (Williams et al., 1997; Zhan et al., 1999). TASK-1 contributes 3 mV hyperpolarization to V_rest and partially counterbalances the depolarizing influence of I_h. Strong extracellular acidification completely inhibited current through TASK-1 and -3 channels and resulted in a marked depolarization of V_rest, indicating some tens of mV hyperpolarizing influence of TASK channels. The value of the prevailing membrane potential in TC neurons is outstandingly important for thalamic function because these cells display burst activity and tonic firing at hyperpolarized and depolarized values of the membrane potential, respectively (Steriade et al., 1997). The burst mode of activity is characterized by two to six action potentials on the ridge of a low threshold calcium spike and occurs during natural sleep and absence epilepsy. Generation of single action potentials can be observed during wakefulness (Steriade et al., 1997). The shift between the two activity modes is mediated by transmitters of the ascending brain stem system (e.g., ACh) acting on I_K-leak (McCormick, 1992). TASK-1 and TASK-3 channels have been shown to constitute the mAChR-sensitive leak current in TC neurons (Meuth et al., 2003). However, removal of TASK-1 is not sufficient to disrupt the mAChR-dependent switch from burst to tonic firing in DLG TC neurons in situ. Although the tonic firing frequency of TASK-1^-/- TC neurons tends toward higher frequencies during ACh application (27 and 32 Hz for WT and TASK-1^-/-, respectively), a comparison with cells from standard C57/BL6 wild-type mice revealed no significant differences (32 ± 2 Hz, n = 7, under identical recording conditions), thereby indicating similar tonic firing properties in different mouse genotypes. In accordance with this finding, no behavioral deficits related to the function of the thalamocortical system (e.g., overall EEG pattern and sleeping/waking behavior) could be observed for TASK-1^-/- mice. However, the idea that sleep deprivation or some other induced stress would reveal functional differences between WT and TASK-1^-/- cannot be excluded. We anticipate that TASK-3 and TASK-1/TASK-3 double or perhaps TASK-1/TASK-3/TWIK-1 triple knockouts will be needed to assess the full impact of K2P channel activity on thalamic function.

	Acknowledgements

 
We thank I. Preugschat-Gumprecht (Heidelberg), R. Ziegler (Magdeburg) and A. Jahn (Magdeburg) for excellent technical assistance. W.W. thanks Prof. Dr. H. Monyer for generous support.

	Footnotes

 
This work was supported by Deutsche Forschungsgemeinschaft grants BU 1019/5-2 (to T.B.) and WI 1951/1-2 (to W.W.), Leibniz-program (to H.-C.P.), the Volkswagen Stiftung I/78 554 (to W.W.), and Fonds der Chemischen Industrie (to W.W.).

ABBREVIATIONS: K2P, two-pore domain potassium channel; TASK, TWIK-related acid sensitive K⁺ channel; DLG, dorsal lateral geniculate nucleus; PIPES, piperazine-N,N'-bis(2-ethanesulfonic acid); BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; ECoG, electrocorticogram; TC, thalamocortical relay neuron; ACh, acetylcholine; pH_O, external pH; E_K, K⁺ equilibrium potential; mAChR, muscarinic acetylcholine receptor; EEG, electroencephalogram; R-(+)-WIN 55,212-2, (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl) pyrrolo-[1,2,3-d,e]-1,4-benzoxazin-6-yl]-1-naphthalenyl-methanone; ZD7288, 4-(N-ethyl-N-phenylamino)-1,2-dimethyl-6-(methylamino) pyridinium chloride.

Address correspondence to: Dr. Thomas Budde, Institut für Experimentelle Epilepsieforschung, Westfälische Wilhelms-Universität Münster, Hüfferstr. 68, D-48149 Münster, Germany. E-mail: tbudde{at}uni-muenster.de

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