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CELLULAR AND MOLECULAR
Department of Life Science (J.-S.K., J.-Y.P., H.-W.K., E.-J.L., J.-H.L.) and Interdisciplinary Program of Integrated Biotechnology (J.-H.L.), Sogang University, Seoul, Korea; and Department of Physiology (H.B.), Chung-Ang University, Seoul, Korea
Received February 2, 2005; accepted April 20, 2005.
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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; Kim, 2003; Talley et al., 2003).
Molecular studies have revealed that two-pore K+ channels fall into six subgroups: 1) TWIK channels, consisting of TWIK-1 and TWIK-2; 2) TASK channels consisting of TASK-1, TASK-3, and TASK-5; 3) TALK channels, consisting of TALK-1, TALK-2, and TASK-2; 4) TRAAK/TREK channels, consisting of TREK-1, TREK-2, and TRAAK; 5) THIK channels, consisting of THIK-1 and THIK-2; and 6) TRESK channels, consisting of TRESK-1 and TRESK-2. Reconstitution of these channels in expression systems has allowed their individual biophysical and pharmacological properties to be characterized (Kim, 2003; Talley et al., 2003; Franks and Honore, 2004; Kang et al., 2004).
TREK channels generate outwardly rectifying currents that are increased by unsaturated fatty acids such as arachidonic acid and mechanical stimuli such as cell swelling, stretch, and positive and negative pressure (Fink et al., 1996; Maingret et al., 1999; Bang et al., 2000). In contrast to voltage-activated K+ channels, TREK and other two-pore channels are insensitive to traditional K+ channel blockers such as tetraethylammonium and barium (Ba2+) (Kim, 2003). Anesthetics and neuroprotective agents are reported to modulate some two-pore channels, which could therefore be targets for anesthesia and neuroprotection. However, the physiological role of two-pore channels needs to be further investigated, because their reported drug responses are different on distinct K+ channels. For example, the neuroprotective agents riluzole and sipatrigine affect TREK channels in opposite ways: riluzole activates TREK-1 and TRAAK, whereas sipatrigine inhibits them. In addition, TREK-1 and TREK-2 are activated by volatile anesthetics such as halothane and isoflurane, whereas TRAAK is not (Patel et al., 1999; Lesage et al., 2000). Thus the effects of these drugs are not well enough understood to permit definitive characterization of the physiological functions of the TREK channels (Patel et al., 1998; Maingret et al., 1999; Kim et al., 2001), and specific modulators of the various TREKs are needed.
In the present study, we compared the effects of several metal ions on human TREK-2 channels (Gu et al., 2002). It turned out that TREK-2 currents were differentially affected by metallic ions; there was potent inhibition by Pb2+, dose-dependent stimulation by Zn2+, and little effect of the other metal ions. We investigated the structural elements in TREK-2 responsible for Zn2+ enhancement using a series of chimeras between Zn2+-activated TREK-2 and Zn2+-inhibited TASK-3. These studies suggested that structures including the first pore and its adjacent extracellular loop contribute to the Zn2+-mediated enhancement of TREK-2.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Cloning of Human TREK-2 and TASK-3 cDNAs. The first-strand cDNA was synthesized from 0.5 µg of human brain Total RNA (Invitrogen, Carlsbad, CA) with avian myeloblastosis virus reverse transcriptase (Roche Diagnostics, Indianapolis, IN) by incubating at 42°C for 50 min. The reaction was terminated by heating at 95°C for 5 min. PCR primers were designed from human TREK-2 and TASK-3 sequences (GenBank accession numbers AF385400
[GenBank]
and AF212829
[GenBank]
). The forward and reverse primers for human TREK-2 cDNA were 5'-TCTGGGCAACGAAGCA-3' and 5'-AAGCTTGCATTGTCAGCATCAA-3' and for human TASK-3 cDNA were 5'-TGGCGGCCATGAAGAGGCA-3' and 5'-TTTAAACGGACTTCCGGCGTT-3'. The PCR reaction protocol was denaturation at 95°C for 1 min, followed by 30 cycles of 95°C for 30 s, 54°C for 30 s, and 72°C for 2 min 30 s. PCR products were purified and ligated into TOPO TA vector (Invitrogen). They were identical in sequence to the open reading frames of human TREK-2 and TASK-3 (GenBank accession numbers AF385400
[GenBank]
and AF212829
[GenBank]
), and were subcloned in pGEM-HEA to improve expression in Xenopus oocytes (Lee et al., 1999b).
Construction of Chimeric Channel Proteins and TREK-2 Mutants. Five chimeras were created by introducing the corresponding regions of TASK-3 into TREK-2 as follows (where the subscripts refer to the regions of TREK-2 substituted): TREKM1M2, TREKM3M4, TREKP1, TREKP1a, and TREKP1b. The structure of each chimera is shown schematically in Fig. 3. All chimeras were made using a standard PCR overlap extension method (Horton et al., 1989). For TREKM1M2, amino acids 1 to 221 of TREK-2 were replaced by residues 1 to 143 of TASK-3. For TREKM3M4, residues 222 to 506 of TREK-2 were replaced by residues 144 to 374 of TASK-3. For TREKP1, residues 95 to 171 of TREK-2 were replaced by residues 31 to 97 of TASK-3. For TREKP1a, residues 95 to 153 of TREK-2 were replaced by residues 31 to 79 of TASK-3, and for TREKP1b, residues 154 to 171 of TREK-2 were replaced by residues 80 to 97 of TASK-3. TREK-2 mutants (H121A, H135A, H156A, D158A, N177A, and N177H) with single-point mutations were made by the PCR overlap extension method (Horton et al., 1989), and the mutated regions were confirmed by sequencing.
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). The oocytes were injected with 0.5 to 1 ng of cRNA of TREK-2 or TREK-2-like chimeric channels showing activation to zinc, and with 2 ng of cRNA of TASK-3 or TASK-3-like chimeric channels showing inhibition to zinc, using a Drummond Nanoject injector (Drummond Scientific Company, Parkway, PA). For TREK-2 mutants with single-point mutations, 1 ng of cRNA was injected into oocytes to compare their relative expression levels. There was no significant difference between current amplitudes of the mutant channels.
Electrophysiological Recordings and Data Analysis. K+ currents were measured in SOS solution (2 mM KCl, 100 mM NaCl, 1.8 mM CaCl2, 1 mM MgCl2, 5 mM HEPES, 2.5 mM pyruvic acid, and 50 µg/ml gentamicin, pH 7.4) using a voltage-clamp amplifier (OC-725C; Warner Instruments, Hamden, CT). Serial zinc solutions were prepared just before experiments by diluting a stock solution of 10 mM ZnCl2 that was made in slightly acidified SOS, and then diluted zinc solutions were readjusted to pH 7.4 with NaOH. Microelectrodes (Warner Instruments) were filled with 3 M KCl, and their resistances were 0.2 to 1.0 M
. The currents were sampled at 5 kHz and low-pass filtered at 1 kHz using the pClamp system (Digidata 1320A and pClamp 8; Axon Instruments, Foster City, CA). Peak currents were analyzed with Clampfit software (Axon Instruments), and graphical representations of the data were obtained with Prism software (GraphPad, San Diego, CA). Dose-response curves were fitted to the Hill equation B = (1 + EC50/[divalent ion]n)–1, where B is the normalized block, EC50 is the concentration of a divalent ion giving half-maximal enhancement, and n is the Hill coefficient. The concentration of a divalent ion giving half-maximal inhibition is represented as IC50. Data are presented as means ± S.E.M. and tested for significance using Student's unpaired t test.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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fast) of TREK-2 activation was not significantly changed by any of the divalent ions examined; however, the slow time constant (slow) was increased by barium but not by the other metal ions (Fig. 1, I and J).
The application of Zn2+ increased the TREK-2 currents in a dose-dependent manner (Fig. 2, A and B). The effect was immediate and usually saturated in 
1.5 min (Fig. 2B). A similar effect of Zn2+ was observed on TREK-2 currents evoked by a ramp protocol (Fig. 2C). Curve fitting gave an EC50 of 87.1 ± 3.1 µM and a Hill coefficient n of 1.1 ± 0.2 (Fig. 2D). EC50 values at different potentials were similar. Consistent with this, the values for percentage of stimulation of TREK-2 currents by 100 µM zinc were not significantly changed at different test potentials (Fig. 2I), suggesting that Zn2+ activation is voltage-independent. The activation time constants fast and slow of the TREK-2 currents tended to be diminished by Zn2+, but the differences were not significant (Fig. 2, G and H; P > 0.05, Student's t tests).
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) cloned from human brain RNA by RT-PCR (see Materials and Methods). Expression of pure TASK-3 channels generated strong outward currents in response to depolarizing potentials, and Zn2+ inhibited these currents (IC50 = 25.4 ± 1.2 µM, Hill coefficient = 1.0 ± 0.1, Fig. 2, E and F). The activation time constants (fast and slow) of TASK-3 were elevated by 100 and 1000 µMZn2+ (Fig. 2, G and H; P < 0.01, 0.05, Student's t test). The percentage of inhibition of the TASK-3 currents by 100 µM Zn2+ was similar at different potentials, suggesting that Zn2+ inhibition of TASK-3 is voltage-independent (Fig. 2I). To help identify the site(s) modulated by Zn2+, chimeras were first constructed by substituting either the N-terminal or C-terminal halves of TREK-2 with the corresponding regions of TASK-3. Expression of TREKM1M2 (first half of TREK-2 replaced with that of TASK-3), gave strong outward currents inhibited by Zn2+ (IC50 = 27.7 ± 2.0 µM; Fig. 3B). The TREKM1M2 currents were activated instantaneously in response to step test potentials; consequently, the activation kinetics were not properly fitted by exponential equations. Apparently, the replacement of the first half of TREK-2 with that of TASK-3 creates a chimera that is activated much faster than wild-type TREK-2 or TASK-3.
In contrast, expression of TREKM3M4 (second half of TREK-2 replaced with that of TASK-3) did not generate significant outward currents so that we could not evaluate the effect of Zn2+ ions (Fig. 3C). However, the fact that the Zn2+ sensitivity of the first-half chimera (TREKM1M2) resembled that of TASK-3 suggests that the N-terminal half of TREK-2 plays an important role in controlling the enhancement by Zn2+.
We further dissected the first half of TREK-2 to localize the element(s) influencing the Zn2+ enhancement effect. Our hypothesis was that the first pore and/or its neighboring extracellular loop would be more important for the Zn2+ effect than the transmembrane or cytoplasmic loops. Accordingly, we constructed TREKP1 (the first-pore loop and preceding extracellular loop of TREK-2 replaced by those of TASK-3) and examined its response to Zn2+. Superfusion of Zn2+ solution inhibited channel activity (Fig. 3D) with an IC50 of 12.6 ± 2.1 µM, about 2-fold lower than that for TASK-3. These data support the idea that the first pore and the preceding extracellular loop are responsible for the opposing effects of Zn2+ on TREK-2 and TASK-3. The reason why substitution of the TASK-3 regions actually renders TREKP1 more sensitive to Zn2+ than TASK-3 itself remains to be investigated.
To further localize the key controlling structures, we constructed TREKP1a and TREKP1b (the extracellular loop and first pore of TREK-2, respectively, replaced by the corresponding regions of TASK-3) (Fig. 3, E and F). Zn2+ inhibited the activity of TREKP1a with an IC50 of 32.6 ± 2.6 µM, whereas 58% of TREKP1a activity was maintained at 1000 µM zinc (Fig. 3E). These results indicate that introduction of the extracellular loop of TASK-3 causes TREKP1a to be partially inhibited by Zn2+ rather than strongly enhanced by it. It also implies that the extracellular loop of TREK-2 contributes to the Zn2+ modulation effect. The outcome with the other chimera TREKP1b was similar, with an IC50 of 29.6 ± 2.2 µM (Fig. 3F). Incomplete inhibition (34%) at more than 1000 µM Zn2+ was also found with TREKP1b. The levels of residual inhibition in TREKP1a and TREKP1b suggest that the first pore is more important than the intracellular loop for the enhancement of TREK-2 channel activity by Zn2+. The activation time constants (fast and slow) of the chimeric channels are shown as a function of Zn2+ concentration in Fig. 3G. No significant difference was found between the fast activation time constants of the chimeras in either the presence or absence of Zn2+. The slow activation constants of the three mutant channels in the absence of Zn2+ were similar and significantly lower than that of wild-type TREK-2 (P < 0.05, Student's t test; Figs. 2H and 3G). The slow constants of TREKP1 and TREKP1b were increased by Zn2+, whereas that of TREKP1a was unaffected (Fig. 3G).
Zn2+ can interact with histidine and cysteine residues and the acidic amino acids aspartic acid and glutamic acid. These types of residue are present in the extracellular loop; we examined the roles of the three histidine residues (His121, His135, and His156) by replacing them individually with alanine. TREKH121A activity was increased by 10 µM Zn2+ and somewhat further by 100 µM Zn2+. Thereafter, there was partial inhibition by 1000 µM Zn2+, with activity dropping to about the level produced by 10 µM zinc. Similarly, TREKH156A activity was enhanced by 10 µM Zn2+ but was not further increased by 100 µM Zn2+ and was partially inhibited by 1000 µM Zn2+ (Fig. 4B); the level of the inhibited activity tended to be slightly lower than the control activity in the absence of Zn2+. These biphasic profiles suggest that His121 and His156 in the extrafascial loop contribute to activation by Zn2+. Unlike TREKH121A and TREKH156A, however, TREKH135A was activated by Zn2+, and its activation profile was similar to that of wild-type TREK-2. Therefore, of the three histidine residues, His121 and His156 seem to be critical for Zn2+ activation.
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In the first-pore region, we selected Asp158 and Asn177 (asparagine177) and individually mutated them to alanine, because Asp158, an acidic amino acid, was recently reported to play a role in Cu2+ activation of TREK-1 (Gruss et al., 2004), and Asn177 corresponds to His98 of TASK-3, which is essential for Zn2+ inhibition of TASK-3 (Clarke et al., 2004). TREKD158A activity was stimulated by Zn2+ up to 100 µM, and its Zn2+ activation profile was similar to that of wild-type TREK-2 over that range. However, there was no further activation but rather slight inhibition by 1000 µM Zn2+. The -fold activations of TREKD158A by 100 and 1000 µMZn2+ were greater than those of TREKH121A or TREKH156A. Nevertheless, the partial inhibition of TREKD158A by 1000 µMZn2+ suggests that Asp158 may participate in Zn2+ activation of TREK-2, although it may be less important than His121 and His156. Similarly, the activities of TREKN177A and TREKN177H were stimulated by low concentrations of Zn2+ up to 100 µM and then partially inhibited by 1000 µM Zn2+, and the -fold stimulations by 1000 µM Zn2+ were less than that of wild-type TREK-2 (P < 0.05, Student's t test; Fig. 4B), implying that Asn177 also participates in Zn2+ stimulation.
These mutagenesis experiments suggest that multiple residues spreading across the extracellular loop and first pore contribute to Zn2+ activation of TREK-2. Although His121 and His156 in the extracellular loop and Asp158 and Asn177 in the first pore contribute to Zn2+ activation, we cannot rule out the possibility that residues in other regions contribute to the activation.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Using the information obtained concerning the role of these residues, we attempted to abolish Zn2+ activation of TREK-2 or convert it to Zn2+ inhibition by introducing additional single-point mutation(s). Mutation of Asn177 in the pore to histidine, the corresponding residue in TASK-3, gave a biphasic modulation effect similar to that with N177A. In addition, even when H156A and N177A were combined, the Zn2+ modulation profile resembled that of H156A and N177A. Furthermore, His121 and His156 are identical to the residues in the TREK-1 channel (Fig. 4A), which is reported to be inhibited by Zn2+ (Gruss et al., 2004). These findings suggest that the zinc activation effect is not due to one or two residues, but that multiple residues including the four residues identified here and unidentified residues are likely to be jointly responsible. For example, negatively charged residues (Asp and Glu) in the extracellular loop could be also targets for the effect of Zn2+. Therefore, it will be of interest to establish how a number of different residues interact to bring about Zn2+ activation.
Structure-function studies of TREK and other two-pore channels have revealed the critical regions required for sensitivity to free fatty acids and pH by analyzing deletion mutants and chimeric channels (Kim, 2003; Franks and Honore, 2004). The initial 30-amino acid regions of the carboxyl termini of TREK-1 and TREK-2 were identified as crucial for activation by free fatty acids and protons. A glutamate residue in the region was shown to play a role as a proton sensor that also affects the sensitivity to mechanic stimuli and free fatty acids. The same C-terminal region of TREK-1 was shown to participate in its activation by volatile anesthetics such as halothane. In contrast to the carboxyl termini involved in sensitivity to pH and fatty acids, the regions affected by divalent ions were localized to the first-pore segment and the preceding extrafascial loop. Glu70 in the extrafascial loop of TASK-1 was shown to be critical for inhibition by ruthenium red (Czirjak and Enyedz, 2003). In the case of TASK-3, Glu70 in the extrafascial loop and His98 in the first pore were critical for zinc inhibition (Gruss et al., 2004). These results, in combination with our findings, suggest that the extracellular loop and the first pore contain key motif(s) participating in the modulation of two-pore K+ channel activity by endogenous trace ions such as Zn2+ and Cu2+. Therefore, these regions are potential targets for drugs regulating two-pore channel activity.
Like TREK-2, acid-sensing ion channels and ATP-sensitive K+ channels show Zn2+-mediated activation (Baron et al., 2001; Prost et al., 2004). Zn2+ potentiates acid-sensing channels activated by protons, and the Zn2+-interacting sites were identified as His163 and His339 in the extracellular loop located just after the first trans-membrane domain. Zn2+ activates ATP-sensitive K+ channels by interacting with extracellular His326 and His332 of the sulfonylurea receptor subunit (Bancila et al., 2005). Although issues such as Zn2+-interacting sites have been solved, it still remains to be determined how Zn2+ activates channels. For example, it may modulate either the number of channels in the membrane, their single-channel conductance, or their probability of opening. Single-channel recordings in the outside-out configuration might distinguish between these possibilities. In the case of Shaker- and Shaw-type K+ channels, Zn2+ activates channel activity by increasing subunit tetramerization (Jahng et al., 2002; Strang et al., 2003). Therefore, the question of whether the Zn2+ activation effect on TREK-2 is correlated with dimerization of its subunits should be tested by biochemical approaches.
Cu2+ has been shown to activate TREK-1 (Gruss et al., 2004), and Asp128 plays a critical role in this effect. Therefore, we have tested whether Cu2+ stimulates TREK-2 activity. Application of Cu2+ led to activation of TREK-2 with an activation profile similar to that of Zn2+ (J. S. Kim, H. Bang, and J.-H. Lee, unpublished data).
In the brain, Zn2+ is stored in synaptic vesicles and released from presynaptic terminals by depolarization (Howell et al., 1984; Ebadi et al., 1994). The released Zn2+ reaches a concentration exceeding 100 µM upon strong synaptic activation and interacts with structures such as ion channels and ligand-gated receptors to modulate synaptic transmission and neuronal excitability. Hence, TREK-2 may be one of the targets modulated by elevated Zn2+ concentrations. The Zn2+ activation effect could be a mechanism by which Zn2+ modulates neuronal excitability. In addition, it may help, together with other factors such as membrane stretching, unsaturated fatty acids, and hydrogen ions, to protect neurons from damage under pathological conditions by attenuating neuronal excitability. Conversely, Zn2+ deficiency may depolarize the membranes of neuronal cells expressing TREK-2, thus aggravating the damage to them under pathological conditions by increasing their excitability.
Unlike Zn2+, Pb2+ proved to be a strong inhibitor of TREK-2 activity, whereas Co2+ and Ni2+ had no effect (Fig. 1). Pb2+ blocks voltage-gated Ca2+ and K+ channels and hyperpolarization-activated channels but activates SK4, a Ca2+-activated K+ channel, by acting as a Ca2+ surrogate. These findings suggest that Pb2+ acts differently on different ion channels (Cao and Houamed, 1999; Atchison, 2003; Dai et al., 2003; Liang et al., 2004). Therefore, its effects may differ between different types of two-pore K+ channels. Together with the absence of effect of Co2+ and Ni2+, the effect of Pb2+ might be used to distinguish TREK-2 from other two-pore K+ channels.
Recently, Clarke et al. (2004) showed that Zn2+ strongly inhibits human TASK-3 with an IC50 = 19.8 µM but has no effect on human TASK-1 and TASK-2. Gruss et al. (2004) have also shown that Zn2+ inhibits TASK-3, with an IC50 = 12.7 µM, and that the IC50 for TREK-1 was 659 µM. In the light of these reports and our findings, it seems that the effect of Zn2+ could be used as a criterion to distinguish TREK-2 from TREK-1 and other two-pore channels, although the effect of Zn2+ on other two-pore K+ channels remains to be investigated. In addition, the results reported here provide a basis for exploring the pore structure and function of two-pore potassium channels.
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Acknowledgements |
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Footnotes |
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ABBREVIATIONS: TWIK, tandem pore domain weak inwardly rectifying K+ channel; TASK, TWIK-related acid-sensing K+ channel; TALK, TWIK-related alkaline-activated K+ channel; TRAAK, TWIK-related arachidonic acid-activated K+ channel; TREK, TWIK-related K+ channel; THIK, TWIK-related halothane-inhibited K+ channel; TRESK, TWIK-related spinal cord K+ channel; PCR, polymerase chain reaction.
1 These authors contributed equally to this work. 
Address correspondence to: Dr. Jung-Ha Lee, Department of Life Science, Sogang University, Mapo-Gu, Sinsu-Dong 1, Seoul 121-742, Korea. E-mail: jhleem{at}ccs.sogang.ac.kr
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), TREKM3M4 (C), TREKP1 (D, 
), TREKP1a (E, 
), and TREKP1b (F, 
). In the left panels are schematic diagrams of the chimeras. In the middle panels, representative currents of each chimera recorded in the absence (control) and presence of serial concentrations of Zn2+ are overlapped. All the currents were evoked by the same voltage protocol every 10 s. Vertical bars represent 1 µA. In the right panels, all the currents are normalized to the control current recorded in the absence of Zn2+ and plotted against the applied Zn2+ concentration. The smooth curves were fitted using the Hill equation. The estimated IC50 values of TREKM1M2, TREKP1, TREKP1a, and TREKP1b are 27.7 ± 2.0, 12.6 ± 2.1, 32.6 ± 2.0, and 29.6 ± 2.2 µM, and their Hill coefficients are 1.5 ± 0.06, 1.0 ± 0.07, 1.2 ± 0.19, and 1.3 ± 0.1, respectively (n = 5–7). C, no functional expression of TREKM3M4 was detected. G, activation time constants of chimeric channels. Chimeric channel currents were fitted with two exponential curves, and their fast and slow time constants (
), TREKH135A (
), TREKH156A (