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KCNQ5 Potassium Channel Activation Underlies Vasodilation by Tea

Background/Aims: Tea, produced from the evergreen Camellia sinensis, has reported therapeutic properties against multiple pathologies, including hypertension. Although some studies validate the health benefits of tea, few have investigated the molecular mechanisms of action. The KCNQ5 voltage-gated potassium channel contributes to vascular smooth muscle tone and neuronal M-current regulation. Methods: We applied electrophysiology, myography, mass spectrometry and in silico docking to determine effects and their underlying molecular mechanisms of tea and its components on KCNQ channels and arterial tone. Results: A 1% green tea extract (GTE) hyperpolarized cells by augmenting KCNQ5 activity >20-fold at resting potential; similar effects of black tea were inhibited by milk. In contrast, GTE had lesser effects on KCNQ2/Q3 and inhibited KCNQ1/E1. Tea polyphenols epicatechin gallate (ECG) and epigallocatechin-3-gallate (EGCG), but not epicatechin or epigallocatechin, isoform-selectively hyperpolarized KCNQ5 activation voltage dependence. In silico docking and mutagenesis revealed that activation by ECG requires KCNQ5-R212, at the voltage sensor foot. Strikingly, ECG and EGCG but not epicatechin KCNQ-dependently relaxed rat mesenteric arteries. Conclusion: KCNQ5 activation contributes to vasodilation by tea; ECG and EGCG are candidates for future anti-hypertensive drug development.


Introduction
Since its initial use in China over 4000 years ago, tea has become one of the most commonly consumed beverages worldwide, second only to water [1]. The leaves of the evergreen species Camellia sinensis are used to make the most prevalent caffeinated teas (Fig. 1A).
Since they are produced from the same plant, the differences between tea varieties (green, oolong, and black) are due to leaf fermentation levels (unfermented, partially fermented, and fully fermented, respectively), which impart the characteristic properties and flavors of the teas [1].
Though there is evidence of health benefits conferred by all tea produced by Camellia sinensis [2], green tea is the most studied as it has the highest antioxidant properties [3]. In vivo and in vitro studies show green tea extract inhibits carcinogenesis, as well as reduces hypertension and risk of heart disease [4][5][6][7]. However, the mechanism behind these therapeutic properties remains incompletely resolved. Medicinal plants contain bioactive compounds that either act independently or synergize to produce therapeutic results. The leaves of Camellia sinensis contain polyphenols belonging to the catechin family -specifically epicatechin (EC), epigallocatechin (EGC), epicatechin gallate (ECG), and epigallocatechin-3-gallate (EGCG) [1]. These catechins are known for their antioxidant properties and are thought to convey the therapeutic benefits of tea, with some studies showing anticancer and cardiovascular health benefits [8]. The fermentation process of the leaves that results in the different tea varieties causes the oxidization of catechins, resulting in green tea containing a higher concentration of catechins [3].
vasoconstriction when these channels are blocked [20]. Due to their roles in a variety of physiological processes and different expression patterns, KCNQ channels are both linked to various inherited disease states and a potential drug target for a variety of diseases [12,21].
Here, we hypothesized that KCNQ5 activation might underlie purported hypotensive properties of tea. We report that green and black tea extracts strongly and isoform-selectively activate KCNQ5. Screening the common catechins in tea, we found that ECG and EGCG selectively activate KCNQ5, making them potential candidates for future medicinal chemistry optimization and drug development for hypertension and even KCNQ5 loss-of function encephalopathy.

Preparation of extracts and compounds
A methanolic extraction (80% methanol/20% water) was performed on 5 bags of Organic Green Tea sourced from Trader Joe's (Irvine, CA, US) and on the contents of 5 PG Tips black teabags sourced from Amazon.com. Each methanol mixture (100 ml) was occasionally inverted over a 48-hour incubation at room temperature (20-25°C). After filtering with Whatman filter paper #1 (Whatman, Maidstone, UK), the methanol was evaporated off over 24 hours at room temperature (20-25°C) in a fume hood. The remaining extract was centrifuged for 10 minutes at 23°C, 4000 RCF to remove any remaining particulates and stored at −20°C. For electrophysiological recordings, the extract was thawed and diluted to 1:100 in bath solution (see below) immediately before use. EC and ECG were solubilized in bath solution to a concentration of 1 mM (more concentrated solutions would require a different solvent) and diluted to appropriate concentrations before use. Likewise, a 1 mM solution of EGCG was prepared in bath solution and used fresh, with the remaining stock solution heated at 35°C for approximately 3 hours and stored at 4°C for four weeks before use. tea extract directly in bath solution (in mM, 96 NaCl, 4 KCl, 1 MgCl 2 , 1 CaCl 2 , 10 HEPES; pH 7.6). We introduced tea extract/compounds via gravity perfusion (1ml/minute for 3 minutes) before recording with pipettes of 1-3 MΩ resistance when filled with 3 M KCl. For intracellular ECG injection, we recorded a control voltage family, then immediately injected 50 nl of 2 mM ECG into each oocyte, giving an approximate intracellular ECG concentration of 100 μM assuming 1 μl oocyte volume. The post-ECG voltage family was recorded 20 minutes after ECG injection.
Recordings were performed with voltage pulses between −80 mV and +40 mV at 20 mV increments for the prepulse from a holding potential of −80 mV and then holding at −30 mV for the tail pulse before repolarizing to −80 mV (see figures for protocols). Clampfit (Molecular Devices) and GraphPad Prism software (GraphPad, San Diego, CA, USA) were used to analyze data and produce figures. Raw and normalized tail currents were fitted with a single Boltzman function: where g is the normalized tail conductance, A 1 is the initial value at −∞, A 2 is the final value at +∞, V 1/2 is the half-maximal voltage of activation and V s the slope factor. We fitted activation and deactivation kinetics with single exponential functions.

Chemical structures and silico docking
We plotted and viewed chemical structures and electrostatic surface potential using Jmol, an open-source Java viewer for chemical structures in 3D: http://jmol.org/. For in silico ligand docking predictions of binding to KCNQ5 we performed unguided docking of ECG using SwissDock with CHARMM forcefields [22,23] and a chimeric human neuronal KCNQ/ Xenopus KCNQ1 cryo-electron microscopy model as previously described [24].

Ultra-Performance Liquid chromatography-mass spectrometry (UPLC-MSe)
A 30-minute UPLC-MSe protocol was performed using a Waters Synapt G2 with a flow rate of 0.1 mL/min. Starting with 98% solution A (0.1% formic acid in water) and 2% solution B (100% acetonitrile), the protocol linearly ramps to 70% solution A and 30% solution B for the first 20 minutes. From minutes 20-25, another linear ramp begins until reaching 3% solution A and 97% solution B, which is held for 2.5 additional minutes; after which, a 1.5 minute linear ramp brings solution A back to 98% and solution B to 2%, which is held until the end of the run. MSe continuum data was obtained in positive mode with the analyzer mode set to resolution, with a mass rage of 105 Da to 2000 Da, scanning every 250ms. The low energy was 6V, high energy ramped from 20-45V, with the cone voltage being 30V. The temperature was kept at 110°C. The resulting chromatograms were analyzed using MassLynx. The spectra for the prominent peaks was analyzed and searched using the Human Metabolomics Database (HMDB), protonated under positive mode with a 0.05 Da molecular weight tolerance. The 459.110 Da peak was identified as HMDB0003153 (EGCG).

Mesenteric artery myography
Male wistar rats, 12 weeks old (Janvier Labs, France) were euthanized by cervical dislocation and used in accordance with Directive 2010/63EU on the protection of animals used for scientific purposes, approved by the national ethics committee, Denmark. Rats were group-housed with regular 12-hour light/dark cycles, in clear plastic containers with ad libitum access to food and water and underwent at least one week of habituation.
The intestines were removed, and third-order mesenteric arteries were dissected in ice cold physiological saline solution containing (in mM): 121 NaCl, 2.

Green tea extract inhibits KCNQ1/E1 and activates an endogenous oocyte Na + current
EGCG is one of the major components of green tea, with the average cup containing approximately 78 mg of EGCG [25]. EGCG inhibits some voltage-gated potassium channels, such as the cardiac potassium channels human ether-a-go-go (HERG) [26] and KCNQ1/E1 (IC 50 of 6 and 30.1 μM, respectively) [27]. Due to the high concentration of EGCG in green tea, we hypothesized that green tea extract (GTE) would inhibit KCNQ1/E1.
Using two-electrode voltage clamp (TEVC) recording of currents expressed in Xenopus laevis oocytes injected with cRNA encoding KCNQ1 and KCNE1 (10 ng and 2 ng), we studied the effects on KCNQ1/E1 of 1% GTE prepared using a methanolic extraction of organic green tea (Fig. 1D, 1E). As expected, we saw inhibition (66.3 ± 8.8 % at +40 mV prepulse) with the addition of 1% GTE, which is also reflected in the tail and prepulse current analysis (Fig. 1F), with no statistically significant change in E M (Fig. 1G).
Interestingly, a 'hook' was clearly present in the KCNQ1/E1 tail current at the more depolarized prepulse sweeps (0-40 mV), only in the presence of GTE (Fig. 1H). In inactivating channels, such as KCNQ1 without accessory proteins (Fig. 1I), a similar hook appears in the tail current as the membrane potential becomes more negative, causing the channel to pass through an open state when going from an inactivated state to a closed state [28]. As the inactivating property of KCNQ1 is eliminated when KCNQ1 is associated with KCNE1 [29], the appearance of a hook in the tail current was highly unusual.
Because it seemed unlikely that GTE was inducing inactivation in a non-inactivating channel [30], we next investigated the impact GTE had on endogenous oocyte currents (Fig. 1J). In the presence of 1% GTE, a negative current was induced by the more depolarizing pulses ( Fig. 1J, K). In addition, GTE shifted the unclamped oocyte E M from approximately −30 mV to +60 mV, which is close to the reversal potential for Na + under the ionic conditions used (Fig. 1L). These data suggest that GTE activates an endogenous voltage-dependent Na + channel, similar to one previously observed [31][32][33][34].
Since this inward current could explain the hook induced by GTE in recordings of KCNQ1/E1 currents, we subtracted the endogenous currents from the KCNQ1/E1 currents -causing the "hook" to disappear (Fig. 1M). We still observed inhibition in the corrected prepulse and tail current (Fig. 1N).

Black tea activates KCNQ5 in the absence of milk
Black tea, typically consumed with milk, is preferred over green tea in many countries, such as United Kingdom. Black tea extract (1%, made from PG Tips brand teabags) was effective at hyperpolarizing the voltage-dependence of KCNQ5 activation with a V 0.5activation shift of −10.7 ± 2.4 mV ( Milk had no effect alone on KCNQ5 activity, but in combination with black tea resulted in KCNQ5 current inhibition ( Fig. 3A-C) and depolarization of KCNQ5-expressing oocytes (Fig. 3D).

Epicatechin gallate activates KCNQ5
While effects on some Kv channels have been documented for EGCG, other compounds in tea are little explored in this respect. As mentioned above, the main catechins found in tea other than EGCG are epicatechin (EC), epigallocatechin (EGC), and epicatechin gallate (ECG) (Fig. 4A). Due to the striking effect green and black teas had on KCNQ5, but the unwanted inhibitory effects on KCNQ1/E1 that recapitulate those previously found for EGCG, we next studied the individual effects of EC, EGC, and ECG on KCNQ5. The addition of 100 μM EC yielded negligible effects on KCNQ5 current magnitude, voltage dependence ( Fig. 4B, C) and unclamped E M (Fig. 4D). EGC (100 μM) slightly increased KCNQ5 activity (19 ± 9 % at +40 mV) (Fig. 4E) but did not alter V 0.5activation or unclamped E M (Fig. 4E-G). In contrast, ECG (100 μM) robustly increased KCNQ5 activation at negative potentials by shifting the V 0.5activation by −17.1 ± 3.1 mV (Fig. 4H, I). Accordingly, GTE hyperpolarized the unclamped E M by −11.0 ± 2.1 mV (Fig. 4J). Unlike whole GTE, these compounds did not induce a hook in the tail currents, suggesting these compounds also do not activate the endogenous oocyte voltage-dependent Na + channel.

Catechin activation of KCNQ5 is isoform-selective
Neither EC, EGC, nor ECG (all 100 μM) altered KCNQ1/E1 activity (Fig. 5A-I). To further test the specificity of ECG, the most KCNQ5-activating of the three, we quantified its effects on KCNQ2/Q3 and found it to be essentially inactive on this heteromeric neuronal channel (Fig. 6A, B). Thus, ECG in particular is a relatively isoform-specific KCNQ5 activator.
The predicted ECG binding site, and critical residue R212, are located deep into the membrane near the intracellular face (Fig. 7B), albeit potentially accessible from the extracellular face via the retigabine/GABA binding pocket entrance between S5 and the VSD (Fig. 7L). We injected ECG into oocytes (to an intracellular concentration of approximately 100 μM) expressing KCNQ5 and recorded the effects 20 minutes post injection compared to pre-injection currents and to water-injected controls. We did not observe KCNQ5 current augmentation from intracellular ECG or water (Fig. 7M, N); neither did either alter E M of KCNQ5-expressing oocytes (Fig. 7O). We therefore conclude that despite the depth of KCNQ5-R212, it is most likely that ECG accesses it via the extracellular side through the retigabine/GABA binding pocket [9,24].
Heated EGCG inhibits KCNQ1/E1 and activates KCNQ5.-Contrary to a prior report [27], we found that freshly prepared EGCG (100 μM) (solubilized at room temperature) had no effect on KCNQ1/E1 current magnitude (Fig. 8A, B); neither did it alter voltage dependence (Fig. 8C) or E M of KCNQ1/E1-expressing oocytes (Fig. 8D). However, when EGCG was first heated to 35°C, then subsequently stored at 4°C before being returned to room temperature before use, its ability to inhibit KCNQ1/E1 at depolarized voltages was restored (Fig. 8E-G). In addition, its ability to hyperpolarize E M of KCNQ1/E1-expressing oocytes, likely because of small increases in current at negative potential (Fig. 8G), was uncovered (Fig. 8H). Freshly made EGCG was able to increase KCNQ5 current at −80 mV at concentrations above 1 μM (Fig. 8I, J) (maximal effect was 2.7-fold, at 10 μM), and at 100 μM shifted the voltage dependence of activation by −7.6 ± 2.5 mV (Fig. 8K) and the E M of KCNQ5-expressing oocytes by −8.0 ± 1.9 mV (Fig. 8L). Strikingly, prior heating of EGCG to 35°C followed by storage at 4°C then returning to room temperature before recording resulted in a KCNQ5 current increase of 25-fold at −80 mV (100 μM EGCG) (Fig.  8M, N). Preheated EGCG also had greater effects than those of freshly prepared EGCG on KCNQ5 voltage-dependence of activation (Fig. 8O) (a negative shift of −11.7 ± 2.1 mV) and E M of KCNQ5-expressing oocytes (Fig. 8P) (a negative shift of −12.0 ± 2.1 mV).
Fresh and heated EGCG have additive effects on KCNQ5 activation.-Liquid chromatography mass spectrometry (LC-MS) analysis revealed that green and black tea extracts each contained (Fig. 9A) the prominent peak at 459 Da also observed in fresh (non-heated) EGCG (Fig. 9B). The 459 Da peak disappeared upon heating of EGCG (Fig.  9C). As both green and black tea activated KCNQ5 even more effectively than heated EGCG (Fig. 2, 3,8), we hypothesized that rather than the fresh EGCG 459 Da peak impairing KCNQ5 activation, its degradation upon heating produces other compounds (or modified versions of EGCG) more effective at KCNQ5 activation and potentially additive with that of the 459 Da moiety. To test this hypothesis, we studied effects of combinations of compounds each at 10 μM. Fresh EGCG could not induce KCNQ5 activation in combination with ECG ( Fig. 10A-C), while at 10 μM heated EGCG caused a −7.1 ± 1.6 mV shift in KCNQ5 activation voltage dependence (Fig. 10D, E) and hyperpolarized E M by −8.7 ± 2.5 mV (Fig. 10F). Strikingly, the combination of heated and fresh EGCG produced larger shifts, in both KCNQ5 activation voltage dependence (−10.4 ± 1.7 mV) (Fig. 10G, H) and E M (−14.2 ± 2.0 mV) (Fig. 10I). Further addition of ECG produced mixed effects and less of a hyperpolarizing shift (Fig. 10J-L). The results suggest that the fresh EGCG 459 Da moiety is not the most effective EGCG moiety with respect to KCNQ5 activation, but it does not impair KCNQ5 activation and may synergize with other EGCG moieties present in tea extract to enhance KCNQ5 activation. Further, it is possible that one or more of these moieties may compete with ECG for a KCNQ5 binding site.

ECG and EGCG KCNQ-dependently relax rat mesenteric arteries
As KCNQ5 is expressed in vascular smooth muscle cells and plays a role in regulating vascular tone, we studied the effects of tea catechins on ex vivo segments of rat mesenteric arterial tone under isometric tension at 37°C in the absence or presence of the relatively KCNQ-specific inhibitor linopirdine. ECG relaxed arterial segments pre-contracted with methoxamine. This relaxation was partially linopirdine-sensitive (Fig. 11A). EGCG was even more efficacious at relaxing mesenteric artery and its effects were highly linopirdine sensitive (Fig. 11B). In contrast, and consistent with lack of effects on KCNQ5 in oocytes, epicatechin did not relax mesenteric artery segments (Fig. 11C). The EC 50 for ECG relaxation of arteries was 3.1 ± 0.4 μM, which was shifted to 26 ± 0.1 μM by linopirdine (n = 6) (Fig. 11D). Quantification of EC 50 values for EGCG in the presence of linopirdine was not possible because it eliminated the relaxation response to EGCG (illustrating the strong KCNQ-dependence of EGCG effects), but the R max for EGCG relaxation of arterial segments was 64 ± 13 %, shifted to 5 ± 4 % by linopirdine. Consistent with in vitro effects on KCNQ5, the myography data strongly suggested that ECG and EGCG are KCNQ dependent vasodilators.

Discussion
Tea played a role in creating the world as we know it today. As the consumption of tea spread across the globe, it changed the areas it encountered. It was used as a status symbol, led to new trading routes, helped with religious practices, and even played a role in instigating the American Revolution [36]. With more than 2 billion people currently consuming tea daily in one form or another, it is still playing a significant role in society. Not only has tea had significant cultural and historical impacts on the world, there have also been many reported health benefits to drinking tea-dating as far back as the Shang dynasty (1766-1050 BC), during which tea leaves were used as an herbal remedy in conjunction with other forest herbs [36]. For as long as tea has been around, it has been considered to have medicinal properties. Though people have only recently started studying the science behind tea's perceived health benefits, there is a growing pool of evidence that drinking tea, especially green tea, can help with cardiovascular health and can inhibit carcinogenesis [4][5][6][7]. As exciting as these results are, the mechanism behind these health benefits has remained largely elusive.
Other commonly consumed plants that, like green and black tea [37], are known to have hypotensive properties, were also recently shown [9] to activate KCNQ5, a voltage-gated potassium channel expressed in vascular smooth muscle that regulates vascular tone. Here, we show that 1% methanol-extracted black and green teas are capable of strongly activating KCNQ5. At hyperpolarized potentials, GTE increased the tail current by nearly 20-fold and diminished the voltage-dependence of the channel. Unlike KCNQ5, KCNQ1/E1, necessary for the cardiac delayed rectifier K + current, was inhibited by GTE-likely due to the high concentration of EGCG in GTE. Similar to other hypotensive plants studied [9], GTE had relatively minimal effects on KCNQ2/Q3 complexes.
Interestingly, in the present study GTE shifted the unclamped, non-injected oocyte E M by approximately 90 mV to +60 mV, close to the reversal potential for Na + under the ionic conditions used (Fig. 1L) and induced a negative current. These data are consistent with GTE activating an endogenous voltage-dependent Na + current, similar to one previously described by several different groups [31][32][33][34]. The previously described current is non inactivating, has a shallow voltage dependence and activates at more depolarized potentials than typical voltage-dependent Na + channels; it is also activated during staurosporine induced apoptosis in Xenopus oocytes [33]. It is blocked by the calcium channel blocker verapamil (which is known to be relatively nonselective) and by lidocaine, and is relatively insensitive to tetrodotoxin [32,33]. The Na + that enters the cell through this channel is thought to be required for staurosporine-induced apoptosis, rather than it being simply activated by staurosporine but unrelated to the apoptotic process [33]. The current has been previously annotated as Na x [34]. There is to date no known molecular correlate, and no known mammalian analog, suggesting the possibility that this channel has a unique molecular architecture, or is confined to non-mammalian chordates and possibly solely amphibians. Future studies could determine whether GTE induces Xenopus oocyte apoptosis and if there is a mammalian Na x channel with unusual sequence or structure.
As we discovered in the present study for ECG (Fig. 7), we previously found that aloperine, the KCNQ5-selective activator from the hypotensive plant Sophora flavescens, binds at or close to R212 [9]. This site is also required for activation of other KCNQ isoforms by botanical therapeutic compounds; E-2-dodecenal from Coriandrum sativum and Eryngium foetidum binds to the equivalent position in KCNQ2 [38], as do mallotoxin and 3-ethyl-2 hydroxy-2-cyclopenten-1-one from Mallotus oppositifolius in KCNQ1, and mallotoxin in KCNQ2/Q3 channels [39]. The R212 site is juxtapositioned between the voltage sensor foot and the S4-S5 linker that connects the voltage-sensing domain to the pore module in KCNQ channels. Despite this location close to the intracellular side, we found that ECG was only effective at shifting the voltage dependence of KCNQ5 activation if applied from the extracellular face, suggesting it accesses R212 from the outside of the cell. The region containing KCNQ5-R212 is highly influential in KCNQ channel gating, and regulation by the endogenous soluble lipid-derived phosphatidylinositol 4,5-bisphosphate (PIP 2 ) that is required for normal KCNQ activation [40]. R212 and its equivalents form part of a binding pocket that accommodates not just plant metabolites, but also endogenous mammalian metabolites and synthetic drugs such as the anticonvulsants retigabine and gabapentin, and the neurotransmitter γ-aminobutyric acid (GABA) [24,[41][42][43][44].
The polyphenols in tea leaves have multiple promising bioactive properties. The main catechins are EC, EGC, ECG, and EGCG. Of these compounds, EGCG has been the most widely studied. It has pro-oxidant properties in cancer cells, which help induce apoptosis. Additionally, EGCG has the highest antioxidant activity out of the catechins and has been shown to chelate metal ions, which can help maintain metal homeostasis in chronic diseases like diabetes, cardiovascular disease, and atherosclerosis. EGCG also was shown to inhibit KCNQ1/E1 channels, which are expressed in cardiac myocytes and epithelia, but not in vascular smooth muscle, which lacks KCNE1, and the human ether-a-go-go (hERG) channel [27]. Inhibition of these channels could be problematic as life-threatening disorders, such as congenital long QT syndrome, are often associated with dysfunction or loss-of-function mutations in these channels [45,46], and hERG blockade by drugs can cause acquired long QT syndrome, especially when combined with loss-of-function mutations in hERG or its cardiac β subunits [47,48]. However, EGCG also inhibits the cardiac voltage-gated sodium channel and cardiac L-type calcium channel, and did not prolong the QT interval when applied directly to isolated guinea pig hearts [27], which express similar I Ks and I Kr currents to those of human hearts (generated by KCNQ1/E1 and hERG/KCNE2). Thus, if the human heart is safely exposed to EGCG in the tens of micromolar range and above, it could be because of the overall balance of inhibitory effects on both excitatory and inhibitory currents, such as occurs, e.g., with the anti-arrhythmic drug, amiodarone [49,50].
While we found that addition of cow's milk to black tea eliminated the KCNQ5 augmenting effects of black tea in vitro (Fig. 3), a prior study showed that black tea ingestion resulted in robust increases in plasma levels of total phenols, catechins, quercetin and kaempferol that were unaffected by addition of sem-skimmed cow's milk to the ingested tea [51]. While another study suggested that milk impairs the increased antioxidant capability of whole blood derived from black tea [52], others found that milk did not impair this effect of black tea consumption [53]. We conclude that in our in vitro experiments, milk constituents bind to black tea polyphenols and prevent them from activating KCNQ5, but that when ingested, this binding would be released, e.g., in the stomach, to permit release of polyphenols such as ECG into a free state that could permit KCNQ5 activation. The overall human plasma levels of total tea-derived phenols peak at 400 μM about an hour after a single cup of black tea [51]. Depending on how much of this constitutes ECG, EGCG and KCNQ5-active EGCG derivatives (that form at/after heating to at least 35°C as occurs during preparation or ingestion of tea), this is well within the concentration range where we see robust effects on KCNQ5 activation (e.g., Fig. 10) and vasodilation (Fig. 11). This strongly supports the contention that polyphenol activation of KCNQ5 underlies at least some of the vascular health benefits of tea.
For this study, we also examined EC, EGC and ECG. Though EC and EGC had little to no effect on the KCNQ channels tested, ECG had a striking hyperpolarizing effect on the voltage dependence of KCNQ5 activation, with minimal effects on KCNQ1/E1 and KCNQ2/Q3. Although ECG has been studied for its anticancer properties [54,55], little work has been conducted with ECG and cardiovascular health. Our results suggest that both ECG and EGCG contribute to the hypotensive properties of tea through KCNQ5 activation, making ECG in particular (because it lacks KCNQ1/E1 and hERG inhibition) an interesting candidate for potential drug development, possibly involving medicinal chemistry approaches to optimize its safety and efficacy profiles. Future studies may also target elucidation of the molecular changes that occur in EGCG upon heating to body temperature that enhance its KCNQ5 activation efficacy (after prior heating to 35°C herein) and permit it to relax arteries (studies conducted at 37°C herein). KCNQ5 loss-of-function mutations can also lead to intellectual disability or epileptic encephalopathy [56], therefore the identification of KCNQ5 selective activators could also help drug development for these disorders. In summary, activation of the vascular and neuronal KCNQ5 potassium channel contributes significantly to vasodilation by both green and black tea. The tea polyphenols ECG and EGCG are major contributors to this effect, via hyperpolarization of the voltage dependence of KCNQ5 activation. ECG and EGCG or optimized derivatives of these compounds are candidates for future anti-hypertensive drug development.          ECG and EGCG are KCNQ-dependent vasorelaxants. All error bars indicate SEM. A: Concentration-response for relaxation of methoxamine-precontracted rat mesenteric artery by epicatechin gallate alone (black) or in the presence of linopirdine (red) (n = 6). B: Concentration-response for relaxation of methoxamine-precontracted rat mesenteric artery by epigallocatechin-3-gallate alone (black) or in the presence of linopirdine (red) (n = 7-8). C: Concentration-response for relaxation of methoxamine-precontracted rat mesenteric artery by epicatechin alone (black) or in the presence of linopirdine (red) (n = 4). D: Scatter plot showing epicatechin gallate LogEC 50 values for relaxation of methoxamine precontracted rat mesenteric artery alone or in the presence of linopirdine as indicated (n = 6). *P<0.05 according to an unpaired Student's t test. E: Scatter plot showing epigallocatechin-3-gallate R max values for methoxamine-precontracted rat mesenteric artery alone or in the presence of linopirdine as indicated (n = 7-8). **P<0.001 according to an unpaired Student's t test.