Abstract
Potassium K2P (“leak”) channels conduct current across the entire physiological voltage range and carry leak or “background” currents that are, in part, time- and voltage-independent. The activity of K2P channels affects numerous physiological processes, such as cardiac function, pain perception, depression, neuroprotection, and cancer development. We have recently established that, when expressed in Xenopus laevis oocytes, K2P2.1 (TREK-1) channels are activated by several monoterpenes (MTs). Here, we show that, within a few minutes of exposure, other mechano-gated K2P channels, K2P4.1 (TRAAK) and K2P10.1 (TREK-2), are opened by monoterpenes as well (up to an eightfold increase in current). Furthermor\e, carvacrol and cinnamaldehyde robustly enhance currents of the alkaline-sensitive K2P5.1 (up to a 17-fold increase in current). Other members of the K2P potassium channels, K2P17.1, K2P18.1, but not K2P16.1, were also activated by various MTs. Conversely, the activity of members of the acid-sensitive (TASK) K2P channels (K2P3.1 and K2P9.1) was rapidly decreased by monoterpenes. We found that MT selectively decreased the voltage-dependent portion of the current and that current inhibition was reduced with the elevation of external K+ concentration. These findings suggest that penetration of MTs into the outer leaflet of the membrane results in immediate changes at the selectivity filter of members of the TASK channel family. Thus, we suggest MTs as promising new tools for the study of K2P channels’ activity in vitro as well as in vivo.
Introduction
Potassium channels selectively and rapidly enable the movement of K+ ions across biological membranes down the electrochemical K+ gradient at a rate close to that of diffusion (). Members of the potassium leak channel family are structurally unique among potassium channels since each subunit possesses four transmembrane segments and two pore-forming domains (2P/4TM architecture). As such, these channels are often referred to as two pore-domain K+ or K2P channels (; ). These channels conduct current across the entire physiological voltage range and are essential for neurophysiological function, while their activity modulates excitability. It was shown that K2P channels could also increase excitability by supporting high-frequency firing once an action potential threshold is reached (). It was recently reported that the majority of K2P channels are gated by membrane potential in spite of their lack of a voltage sensor, as the outward current of K+ ions through the selectivity filter was found to open this gate (). Members of this family may react to membrane stretch, as well as to intracellular and extracellular pH changes, phosphorylation, the activity of various G-protein coupled receptors, and more (; ; ; ). K2P channels activity was shown to modulate various important physiological processes such as pain perception () and cardiac activity (; ; ). Human K2P3.1 channels (TASK-1) are expressed mainly in the atria and possess a promising target for atrial fibrillation treatment (; ). A mutation in K2P9.1 (TASK-3) is connected to the Birk–Barel syndrome, mental retardation, and unique dysmorphism syndrome (). Also, the effect of several volatile analgesics is mediated, in part, through their action on K2P2.1 and K2P4.1 (TRAAK) ().
Terpenes are a large group of structurally diverse organic chemicals that are mostly produced in plants. Monoterpenes (MTs) are terpenes that are composed of two five-carbon isoprene units. For centuries, MTs have been known for their beneficial effects as antifungal agents (), antibacterial (), and analgesic () agents. Terpenes have been proposed as remedies for the treatment of pain (; ; ) and cardiovascular diseases (; ; ; ; ), and were shown to possess antitumor, local anesthetic, and anti-ischemic abilities ().
Several MTs were found to affect ion channels, both in excitable cells () and in other tissues (). To name a few, carvacrol and thymol were found to activate and sensitize the murine and human transient receptor potential (TRP) channel TRPV3, and acyclic MTs like citronellol, nerol, and their derivatives were found to modulate the activity of TRPA1 (). MTs were found to act upon other TRP channels (; ), as well as on voltage-gated ion channels and GABA receptors (; ). However, their activity on members of the K2P potassium channels had not yet been studied.
Recently (), we reported the activation of K2P2.1 by various MTs. Here, we report that MTs activate the other two mechano-gated K2P channels (i.e., K2P4.1 and K2P10.1), in addition to members of other groups of K2P channel families (e.g., TALK, TRESK). Moreover, we found that MTs display remarkable selectivity towards the different K2P channels, and we report that they selectively inhibited the voltage-dependent current of TASK family members.
Methods
Animals
All experiments using animals were performed following the guidelines of the Institutional Animal Care and Use Committee. The project approval number is IL-61-09-2015.
Cloning
Channels were cloned into plasmid pRAT that included a T7 RNA polymerase promoter to enable cRNA synthesis, as well as the 3′-UTR and 5′-UTR sequences of the Xenopus laevis β-actin gene to ensure efficient expression in Xenopus oocytes. Competent Escherichia coli DH5α cells were transformed by heat shock. Plasmid DNA was purified with a Wizard Plus SV Miniprep kit (Promega). Restriction enzyme digestions were performed according to the manufacturer’s instructions (Fermentas or NEB). Point mutations were generated according to the Quickchange site-directed mutagenesis technique (Stratagene) and confirmed by sequencing. cRNA was transcribed in vitro by T7 polymerase using an AmpliCap High Yield Message Maker (Epicentre) kit.
Electrophysiology
Xenopus laevis oocytes were isolated and injected with 20–40 nl of solutions containing 0.3–40 ng cRNA using a 3.5″ Drammond#3-000-203-G/X glass capillary, pulled in a Sutter P97 capillary puller and a Drummond manual oocyte microinjection pipette (3-000-510-X). Whole-cell currents were measured 1–3 days after injection by the two-electrode voltage-clamp technique (GeneClamp 500B, Axon Instruments). Data were filtered at 2 kHz and sampled at 5 kHz with Clampex 9.0 software (Axon Instruments). For two-electrode voltage-clamp experiments, the pipette contained 3M KCl and the bath solution contained (in mM) unless otherwise noted: 4 KCl, 96 NaCl, 1 MgCl2, 0.3 CaCl2, 5 HEPES, pH 7.4 with NaOH (standard solution). All measurements of K2P5.1 and K2P17.1 channels were performed at pH = 9.0. When needed, bath solution sodium ions were isotonically replaced by potassium ions and vice versa. When testing MT activity, the standard bath solution was supplemented with the same concentration of the solvent (ethanol) as of the tested chemical.
Injection of cRNA into oocytes was done in OR-2 solution (in mM: 5 HEPES, 1 MgCl2, 2.5 KCl, 82.5 NaCl, pH = 7.4). Post-injection oocytes were maintained in ND-91 solution (in mM: 5 HEPES, 1 MgCl2, 1.8 CaCl2, 2 KCl, 91 NaCl, pH = 7.4). Specific recording protocols are mentioned in the relevant figure legends. To determine the voltage-dependent fraction of the current, the initial, voltage-independent (instantaneous) current was estimated by fitting the current to an exponential decay slope as the initial currents are masked by the capacitive transient current, as was previously described ().
Chemicals
Carvacrol (cat#282197), thymol (cat#T0501), p-cymene (cat#C121452), 4-isopropylphenol (cat# 175404), eugenol (cat#E51791, cinnamaldehyde (cat#W228613), menthol (cat#M2772), beta-citronelol (cat# C83201), geraniol (cat#16333, 4-methylcatechole (cat# M34200), and arachidonic acid (cat#A3611) were all purchased from Sigma-Aldrich.
Preparation of Compounds
Compounds delivered as powders were dissolved into stock solutions (4–6 M) in 100% ethanol. Compounds delivered as liquid oils (6.5–7.5 M) were diluted 1:1 with ethanol to form stock solutions and were kept at −20°C for up to two weeks. Just before testing, stock solutions were diluted in the bath solution to the desired concentration, and diluted compounds were vigorously vortexed until completely dissolved. All solutions were supplemented with ethanol to a final concentration of 0.1% (v/v) (confirmed not to harm the oocytes). The pH was corrected to 7.4 ± 0.05 using NaOH or HCl.
Statistical Analysis
Data were expressed as the mean ± standard error of the mean (SEM) and analyzed and presented using Microsoft Excel 2016. Groups of two paired data sets were analyzed using a Wilcoxon Signed Ranks test and groups of two unpaired data sets were analyzed using Mann–Whitney U test with IBM SPSS Statistics ver. 20 software. Values were considered to be significantly different when the z-value was ≤0.05 (*), ≤0.01 (**), or ≤0.001 (***). All experiments were repeated with at least five oocytes.
Results
Activation of Mechano-Gated Channels by Monoterpenes
As we have recently reported (), the activity of K2P2.1 is modulated by various MTs. K2P2.1 is a member of the mechano-gated K2P channel clade that includes K2P4.1 (TRAAK) and K2P10.1 (TREK-2). We, therefore, investigated whether MTs modulate all mechano-gated K2P channels. An external application of MTs resulted in the increase in currents of K2P10.1 channels by seven compounds and in the increase in currents of K2P4.1 channels by eight of the tested compounds, although to lower levels (Figure 1A). As was found for K2P2.1 (), the phenol-containing compounds (carvacrol and thymol) were more potent in opening both channels, while linear compounds and compounds containing no hydroxyl group were less effective (Figure 1A). Under standard testing conditions, currents of most K2P channels are composed of two components: an instantaneous “leak” current (voltage-independent, VI) and a voltage-dependent (VD) current (), as demonstrated in Figure 1B. We, thus, looked at whether MTs affect the voltage sensitivity of the channels by looking at the change in the proportion of the two current components. For K2P4.1 and K2P10.1 channels, no change in voltage dependency was detected (Figure 1C). However, for K2P2.1 channels, voltage dependency was reduced during carvacrol application, as was previously reported (). As expected (), arachidonic acid had a similar effect to carvacrol (Figure 1C).
Figure 1
Activation of TALK and TRESK Channels
The activity of members of the TALK clade of potassium channels (K2P5.1, K2P16.1, and K2P17.1; TASK-2, TALK-1, and TALK-2, respectively) is sensitive to external pH, as these channels are activated at an alkaline pH (; ). K2P5.1 (TASK-2) is expressed mostly at the tubular epithelial and is involved in pathological conditions such as Balkan endemic nephropathy (BEN) (; ). While most tested compounds had almost no effect on this channel, carvacrol and cinnamaldehyde (Figure 2) activated it by up to 17-fold (15.7 ± 3.0, n = 7 and 4.7 ± 0.5, n = 5, respectively, 0.3 mM for both compounds), although at different rates (Figure 2B). While activation by carvacrol was reversible, activation by cinnamaldehyde was not, even after a 5-min wash. It should be noted that irreversible activation of TRPA1 channels by cinnamaldehyde was previously reported (). The minimal concentration that showed substantial activation of the channel was 12 µM for carvacrol (albeit not statistically significant) (1.9 ± 0.1-fold, n = 5, Figure 2C) and 30 µM for cinnamaldehyde (2.8 ± 0.3-fold, n = 5; statistically significant).
Figure 2
K2P16.1 and K2P17.1 are expressed predominantly in the pancreas and may be involved in the exocrine secretion of bicarbonate. A gain of function mutation in K2P17.1 was associated with progressive cardiac conduction disorder (). While K2P16.1 was not affected by any of the tested MTs (not shown), K2P17.1 was moderately activated by menthol, thymol, β-citronellol, 4MC, and carvacrol (Figure 3A). K2P18.1 (TRESK, KCNK18) is unique among other K2P channels by having an extra-long cytoplasmatic domain that is located between the two pore-forming domains (). This channel is expressed in the dorsal root ganglion, trigeminal ganglion neurons, and spinal cord (; ; ). Mutation in this channel was linked to familial migraine with aurora (). K2P18.1 was opened rapidly and robustly by carvacrol and to a lesser degree by thymol and 4-isopropylphenol (4IPP) (Figure 3B). Other compounds displayed mild to no effect on this channel. Since with mechano-gated K2P channels, we observed a reduction in the proportion of the voltage-dependent current as a result of activation by carvacrol (Figure 1C), we tested this feature in these channels as well. In K2P5.1 channels, the share of the voltage-dependent current indeed decreased (Figure 3C). In contrast, for K2P17.1 channels, the share of the voltage-dependent current did not change. In K2P18.1 channels, where the basal share of the voltage-dependent current was low, currents displayed more sensitivity to voltage after incubation with carvacrol (Figure 3C).
Figure 3
Acid-Sensitive K2P3.1 and K2P9.1 (TASK) Channels Are Inhibited by Monoterpenes
K2P3.1 and K2P9.1 (TASK-1 and TASK-3, respectively) channels are expressed in the pancreas and placenta and to a lesser degree in the brain, heart, and kidneys (; ). Unlike all other tested K2P channels, current levels of K2P3.1 and K2P9.1 decreased by all tested MTs (Figure 4A). In contrast to mechano-gated channels, which were affected mostly by cyclic phenolic compounds like thymol and carvacrol, TASK channels were affected mostly by linear MTs such as β-citronellol and geraniol (Figure 4A). Inhibition was rapid (e.g. τ = 3.8 ± 0.2, 6.2 ± 1.2, and 5.6 ± 1.5 s for thymol, β-citronellol, and carvacrol, respectively, n = 7–11) and within the solution change rate in our system (τ = 7.7 ± 1.0 s, n = 5). When using thymol, a monoterpene that affects both channel types, it was obvious that the inhibition rate was remarkably faster than that observed for activation of mechano-gated K2P channels (Figure 4B). To make sure that the differences in rates were not merely a result of a difference in affinities (Kinhibition of K2P3.1 by thymol is 20 ± 5 µM and Kactivation of K2P2.1 by thymol is 290 ± 50 µM, not shown), we measured the current change rates at three different concentrations for each channel (Figure 4C). As expected, no measurable change in the rate of K2P3.1 channels inhibition was observed, while the activation rate for K2P2.1 channels was much slower at all concentrations.
Figure 4
To further examine the unique activity of MTs on TASK channels, we tested the activity of carvone on K2P3.1 channels as a model, as this monoterpene had no activity on K2P2.1 channels (Figure 5A), while readily and rapidly (Figure 5B) decreasing K2P3.1 channel currents with a Kinhibition of 0.90 ± 0.16 mM (Figure 5C). As in most K2P channels, when held at −80 mV, K2P3.1 currents are comprised of two components: an instantaneous, voltage-independent (VI), and a time- and voltage-dependent component (VD) (Figure 5D, control). As was observed with other MTs, the VD component of the current was dramatically reduced due to application of carvone (Figures 5D, E). The inhibition of the VD currents resulted, as anticipated, in the disappearance of K2P3.1 channels tail currents (Figure 5F).
Figure 5
External K+ concentration is known to affect the open probability of the selectivity filter gate of potassium channels () and, in particular, that of K2P channels (). We, thus, tested the effect of external K+ on carvone-induced current inhibition. The concentration of external K+ had a profound effect on K2P3.1 currents. A significant current decrease was observed under low (0 and 4 mM) external K+ concentration (Figure 6A). At all external potassium concentrations, carvone reduced K2P3.1 currents (Figures 6B, C). Currents at 0 mM (no added potassium) external K+ were too low to allow further accurate analysis. When we analyzed the effect of carvone on each current component (VD or VI) at three external K+ concentrations, we found that the VD current was almost completely eliminated at all concentrations, while the VI current was only mildly affected (Figures 6C–G). At 60 mV, inhibition was reduced by high external K+ (Figure 6C).
Figure 6
Discussion
In this study, we used an exogenous expression system to measure the impact of MTs on the activity of various human K2P channels. MTs were found to affect various types of ion channels at high micromolar to millimolar concentrations (; ; ; ; ; ; ; ), comparable to the concentrations that were found here to affect K2P channels. Mechano-gated K2P4.1 and K2P10.1 were activated mainly by the cyclic aromatic phenolic MTs, carvacrol, and thymol, with the latter being the most effective. This is in accordance with our finding that the same MTs are the best activators of the other mechano-gated K2P channel, K2P2.1 (). Unlike in K2P2.1 channels, the voltage dependency of the current did not change as a result of MT activation. Our findings suggest that for best activation of mechano-gated K2P channels, terpenes should be moderately hydrophobic (XLogP3 ~ 3, as is the case for carvacrol and thymol) and to be able to penetrate, yet not become embedded in, the bilayer due to the presence of a small polar area. Also, a phenol moiety was necessary to obtain high channel-stimulating activity. We believe that such molecules are embedded into the outer leaflet of the bilayer and perturbed its structure and/or curvature. Less hydrophobic and more polar molecules (a polar area larger than 20 Å2 and XLogP value lower than 3) will stay in the polar area of the outer leaflet, while more hydrophobic molecules will sink deeper into the bilayer. We showed that for K2P2.1, the cytoplasmic carboxyl-terminal of the channel is needed for the activity of MTs (). It is yet to be determined whether this is the mechanism by which the other two mechano-gated K2P channels are activated by MTs.
The alkaline-sensitive K2P5.1 and K2P17, but not K2P16.1, were also found to be activated by MTs. While K2P17.1 was only mildly activated (up to a fourfold increase in current, Figure 3A), K2P5.1 currents increased by up to 17-fold (0.3 mM, Figure 2A). Even at 60 µM carvacrol, K2P5.1 currents increased by 5.5-fold (Figure 2C). The selectivity of the MTs towards these channels was not the same as for mechano-gated channels. While carvacrol activated, to a degree, all channels, K2P5.1 was uniquely activated by cinnamaldehyde, but not by thymol, and K2P17.1 was uniquely activated by β-citronellol and 4MC (Figure 3A). K2P18.1 channels were activated by the same MTs, up to 5.3-fold (Figure 3B). Our findings indicate a certain degree of selectivity in the sensitivity of different K2P channels to MTS, as some channels are activated by MTs that are inactive against other channels. The origin for this apparent selectivity is unclear since whether membrane-adhered hydrophobic molecules directly bind to channels or if they change membrane properties, causing each channel to react differently to those changes is an ongoing debate (; ; ; ; ; ). This dilemma applies not only to monoterpenes but also to other lipophilic molecules such as general anesthetics and alcohols (), as well as for endocannabinoids () and steroids (), all are allosteric modulators of several structurally different ion channels (; ; ). By changing the physicochemical properties of the surrounding membrane environment (; ; ; ), and energetic requirements for gating-related conformational changes monoterpenes could affect ligand-gated ion channels (LGICs) (; ), and voltage-gated ion channel (VGICs) as reviewed by Oz at al. 2015 (). On the other hand, evidence for direct binding of lipophilic monoterpenes, such as carvacrol and thymol, to specific amino acid residues in the transmembrane domain of Human 5-Hydroxytryptamine Type 3 (5-HT3Rs) were found (). Similarly, the different potency of menthol stereoisomers () on 5-HT3Rs or GABA(A) receptor () suggest also a degree of selectivity in monoterpenes action on ion channels.
For K2P2.1, it was shown that channel opening would result in a reduction in its voltage dependency (; ; ). Schewe et al. displayed that, for all mechano-gated K2P channels, activation causes a gating mode shift within the selectivity filter and that these channels can be converted into a “classical” leak mode when stimulated by arachidonic acid or PIP2 (). This phenomenon was observed in our experiments with K2P2.1, but not with K2P4.1 or K2P10.1 channels. Reduction in the percent of voltage-dependent current was observed with K2P5.1, but not with K2P17.1, while with K2P18.1, an increase in the voltage-dependent current was recorded (Figure 3C). The voltage-dependency of the K2P channels’ current is directly related to their open probability. Since in our experiments, only a relative estimation of the open probability of the channel is measured, we believe that it is plausible that “leak-like” behavior of the channel is achieved only at high open probabilities and that under our experimental conditions, this was not always achieved.
The acid-sensitive TASK channels, K2P3.1 and K2P9.1, were affected differently by MTs: a. most tested MTs caused a decrease in TASK channel currents (Figure 4A); b. the current decrease rate was high (a few seconds), while the activation rate of other channels was at least an order of magnitude slower (Figures 4B, C and not shown); and c. unlike other tested K2P channels, TASK channels were affected mostly by linear MTs (β-citronellol and geraniol, Figure 4A). These three observations suggest that TASK channels are affected by MTs by a different mechanism than the other tested channels. Thus, we further studied the characteristics of the current inhibition of TASK channels by MTs using K2P3.1 and carvone as a model.
It was clearly evident that carvone almost completely eliminated the voltage-gated portion of K2P3.1 currents (Figures 5D–F). Since voltage-dependent gating was shown to originate from the movement of three to four potassium ions into the high electric field of an inactive selectivity filter () and since the stability of the selectivity filter was shown to be affected by the concentration of external potassium ions in potassium channels in general () and in K2P channels in particular (), we looked at the influence of external potassium concentrations on K2P3.1 activity and sensitivity to carvone. External potassium ion levels had a clear effect on channel gating, as currents decreased dramatically at low external levels (Figure 6A). At all potassium levels, carvone reduced K2P3.1 current, while specifically targeting the voltage-dependent portion (Figures 6C–G). While the voltage-independent portion of the current behaved, as expected, like a potassium GHK-leak, the voltage-dependent portion displayed an outward rectification behavior that was partly dependent on potassium concentration (Figure 6F). We suggest that as external potassium ions stabilize the selectivity filter at its conductive state, they minimize the destabilizing structural changes caused by carvone. For this reason, we suggest that MTs might serve as a useful tool in studying the voltage-dependency of TASK channels as they specifically target the voltage-dependent portion of the current. Recently, it was reported that bupivacaine blocks TASK channels in a voltage-dependent manner by disrupting the K+-flux gating mechanism (), and that it is located laterally in the side fenestrations of K2P3.1 channels and interacts with residues of the pore helix, and the M2, M3, and M4 segments. It is conceivable that both bupivacaine and MTs bind to a similar binding site within the membrane and, thus, affect channels through a similar mechanism.
K2P channels play a role in various physiological processes such as pain signaling () heart function (), and more (; ; ; ). For example, activation of mechano-gated K2P, as well as K2P18.1 channels, is expected to result in reduced pain sensation and neuroprotection. Terpenes have been proposed as analgesic agents (), as remedies for the treatment of pain and cardiovascular diseases (; ; ; ; ; ; ; ) and were shown to possess antitumor, local anesthetic, and anti-ischemic abilities (). Any of these activities of terpenes that stem from their activity on K2P channels remains to be determined. Even though MTs are regularly consumed by people as food additives, due to their low concentration in food, they are unlikely to have any pharmacological effect. However, the extensive use of MTs in traditional medicine might raise the possibility of their beneficial pharmacological use when given in high concentrations.
Funding
This work was supported by a grant from the Israel Science Foundation (1877/15) to NZ.
Statements
Data availability statement
All datasets generated for this study are included in the article/supplementary material.
Ethics statement
The animal study was reviewed and approved by Institutional Animal Care and Use Committee, Ben Gurion University. The project approval number is IL-61-09-2015.
Author contributions
Conception and design of the study: EA and NZ. Acquisition of data: EA and GB. Analysis and interpretation of data: EA. Writing the manuscript: EA and NZ.
Acknowledgments
The authors thank Prof. Dierk Thomas for his generous gift of the K2P4.1, K2P10.1, K2P16.1, and K2P17.1 clones.
Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Abbreviations
MTs, monoterpenes; K2P, two pore-domain potassium channels.
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Summary
Keywords
K2P channel, TREK-1, TRAAK, TASK-1, TALK, monoterpenes, leak channels, voltage-dependent current
Citation
Arazi E, Blecher G and Zilberberg N (2020) Monoterpenes Differently Regulate Acid-Sensitive and Mechano-Gated K2P Channels. Front. Pharmacol. 11:704. doi: 10.3389/fphar.2020.00704
Received
17 September 2019
Accepted
29 April 2020
Published
20 May 2020
Volume
11 - 2020
Edited by
Moran Rubinstein, Tel Aviv University, Israel
Reviewed by
Sharon Weiss, Tel Aviv University, Israel; Yael Stern-Bach, Hebrew University of Jerusalem, Israel
Updates
Copyright
© 2020 Arazi, Blecher and Zilberberg.
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Noam Zilberberg, noamz@bgu.ac.il
This article was submitted to Pharmacology of Ion Channels and Channelopathies, a section of the journal Frontiers in Pharmacology
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