DOI: 10.5061/dryad.2z34tmpwp
title: Vasorelaxant effects of 3-methoxycatechol are not via direct activation of voltage-gated potassium channels

description: The Kv7 (KCNQ) family of voltage-gated potassium (Kv) channels comprises
 Kv7.1-7.5, which are expressed throughout the human body where they
 regulate numerous essential physiological processes . As such, there has
 been a lot of interest in developing and identifying small molecules that
 can modulate specific Kv7 channel isoforms. We previously discovered that
 plant metabolites such as aloperine from Sophora flavescens (Ku Shen),
 carnosol and carnosic acid from rosemary (Salvia rosmarinus), and gallic
 acid and tannic acid from Native American bark extracts are efficacious
 Kv7.5 channel activators with prominent vasorelaxant effects. Kv7.1,
 Kv7.4, and Kv7.5 channels are important regulators of smooth muscle
 contractility, regulating the membrane potential of vascular smooth muscle
 and subsequently vascular tone. Pharmacological targeting of these Kv7
 channels with pan-Kv7 inhibitors XE-991 and linopirdine provokes membrane
 depolarization and vasoconstriction, while several structurally disparate
 activators enhance Kv7 channel currents resulting in membrane
 hyperpolarization and vasorelaxation. Recently, it was concluded based
 in ex vivo pharmacology and in silico docking that the
 vasorelaxant effects of 3-methoxycatechol (3-MOC) arise from direct
 binding to and activation of Kv channels in the vascular smooth muscle,
 specifically Kv7.4, but the direct effects on Kv channels were not
 substantiated experimentally. Here, we report that the mechanism
 of vasodilation by 3-MOC is not via direct Kv channel activation
 and strongly advise against pharmacological target
 assumptions using non-experimentally validated in silico docking,
 a technique not appropriate for this purpose.
description: <strong><em>cRNA prepeartion and Two-electrode
 voltage clamp (TEVC) </em></strong> cRNA
 transcripts encoding human Kv7.4, Kv7.5, Kv1.1, Kv1.2, Kv1.5, and Kv2.1
 were generated by <em>in vitro</em> transcription using the
 mMessage mMachine kit (Thermo Fisher Scientific), after vector
 linearization, from cDNA sub-cloned into plasmids incorporating
 <em>Xenopus laevis</em> β-globin 5’ and 3’ UTRs flanking the
 coding region to enhance translation and cRNA stability.  Defolliculated
 stage V and VI <em>Xenopus laevis</em> oocytes (Xenoocyte,
 Dexter, MI, US) were injected with KCNQ cRNAs (0.1-25 ng) and  incubated
 at 16 <sup>o</sup>C in ND96 oocyte storage solution containing
 penicillin and streptomycin, with daily washing, for 1-4 days prior to
 two-electrode voltage-clamp (TEVC) recording.TEVC was performed at room
 temperature using an OC-725C amplifier (Warner Instruments, Hamden, CT)
 and pClamp10 software (Molecular Devices, Sunnyvale, CA) 1-4 days after
 cRNA injection as described in the section above. For recording, oocytes
 were placed in a small-volume oocyte bath (Warner) and viewed with a
 dissection microscope.  3-methoxycatechol was sourced from Sigma and made
 into 250 mM stock solutions in DMSO prior to dilution in recording
 solution (in mM): 96 NaCl, 4 KCl, 1 MgCl<sub>2</sub>, 1
 CaCl<sub>2,</sub> 10 HEPES (pH 7.6).  3-methoxycatechol was
 introduced into the oocyte recording bath by gravity perfusion at a
 constant flow of 1 ml per minute for 3 minutes prior to recording. 
 Pipettes were of 1-2 MΩ resistance when filled with 3 M KCl.  Currents
 were recorded in response to voltage pulses between -80 mV and +40 mV at
 10 mV intervals from a holding potential of -80 mV, to yield
 current-voltage relationships and examine activation kinetics. Data was
 analyzed using Clampfit (Molecular Devices) and Graphpad Prism software
 (GraphPad, San Diego, CA, USA), stating values as mean ± SEM. Raw or
 normalized tail currents were plotted versus prepulse voltage and fitted
 with a single Boltzmann function.
 <strong><em>Mesenteric artery
 myography</em></strong> In accordance with
 the methods of killing animals described in annex IV of the EU Directive
 2010/63EU on the protection of animals used for scientific purposes, male
 wistar rats, 12 weeks old (Janvier Labs, France), were made unconscious by
 a single, percussive blow to the head. Immediately after the onset of
 unconsciousness, cervical dislocation was performed.  Rats were
 group-housed with regular 12-hour light/dark cycles, in clear plastic
 containers with <em>ad libitum</em> access to food and water
 and underwent at least one week of habituation. After euthanasia, the
 intestines were removed, and third-order mesenteric arteries were
 dissected in ice-cold physiological saline solution containing (in mM):
 121 NaCl, 2.8 KCl, 1.6 CaCl<sub>2</sub>, 25
 NaHCO<sub>3</sub>, 1.2
 KH<sub>2</sub>HPO<sub>4</sub>, 1.2
 MgSO<sub>4</sub>, 0.03 EDTA, and 5.5 glucose. Segments, 2 mm
 in length, of mesenteric artery were mounted on 40 µm stainless steel
 wires in a myograph (Danish Myo Technology, Aarhus, Denmark) for isometric
 tension recordings. The chambers of the myograph contained PSS maintained
 at 37°C and aerated with 95% O<sub>2</sub>/5%
 CO<sub>2</sub>. Changes in tension were recorded by PowerLab
 and Chart software (ADInstruments, Oxford, United Kingdom). The arteries
 were equilibrated for 30 minutes and normalized to passive force.  Artery
 segments were precontracted with 10 µM methoxamine (Sigma; Copenhagen,
 Denmark) in the absence or presence of linopirdine (10 µM) and BIBN-4096
 (100 nM)  (Sigma; Copenhagen, Denmark), before application of
 3-methoxycatechol at logarithmic concentrations.
 <strong><em>Statistics and
 Reproducibility</em></strong> All values
 are expressed as mean ± SEM.  One-way ANOVA was applied for all tests; all
 <em>p</em> values were two-sided.
description: Source Data for manuscript: Vasorelaxant effects of 3-methoxycatechol are
 not via direct activation of voltage-gated potassium channels
 ------------------ The datasets included are the original Excel files used
 to generate each panel for figures 1 and 2 in this manuscript. The title
 of each Excel file is labeled to directly correspond to the figure in the
 manuscript: Figure Number & panel &gt; Channel Investigated &gt;
 Condition &gt; Parameter Measured --- The data contained within this
 repository are those obtained from cellular electrophysiology recordings.
 We used two-electrode voltage clamp (TEVC) electrophysiology and the
 Xenopus laevis oocyte expression system to record the electrical activity
 of wild-type Kv7.4, Kv7.5, Kv7.4/7.5, Kv1.1, Kv1.2, Kv1.5, and Kv2.1
 channels in response to 100 uM 3-methoxycatechol. Xenopus laevis oocytes
 were injected with cRNA encoding for each of these channels and were
 incubated at 16 degrees for 1-4 days prior to recording using TEVC. The
 subsequent measurements allow us to characterize the biophysical responses
 of these channels to 3-methoxycatechol. Additionally, this study also used
 mesenteric artery myography to elucidate the molecular mechanism of action
 of 3-methoxycatechol. The technique measures the force of contraction and
 relaxation of ex-vivo mesenteric arteries in response to various stimuli.
 In this study the artery segments were precontracted with methoxamine in
 the absence or presence of ion channel and receptor specific inhibitors,
 before application of 3-methoxycatechol. The parameters that we measured
 to characterize the effect of 3-methoxycatechol on Kv7.4, Kv7.5,
 Kv7.4/7.5, Kv1.1, Kv1.2, Kv1.5, and Kv2.1 channels as well as mesenteric
 artery are as follows: &gt; **Current-voltage (IV) curve** &gt; This is a
 graph representing the relationship between the electrical current (flow
 of ions) and voltage applied across a device (the cell membrane). In
 electrophysiology, I-V curves are used to study the activity of biological
 cells, in this case Xenopus oocytes expressing the aforementioned
 channels. The data contained in these excel files were measured from the
 peak of the prepulse current generated by a voltage protocol that starts
 at a holding potential of -80 mV and increases in +10 mV increments until
 +40 mV. All raw values are in microamps (uA). &gt; **Gmax** &gt; These
 data were used to generate conductance-voltage curves. Graphs were
 generated by taking measurements from the tail current (recorded at -30 or
 -40 mV) immediately following the prepulse current as described above.
 These data enable us to determine the shift in voltage-dependence of
 activation of the channel in response to 3-methoxycatechol. Raw values are
 in microamps (uA). &gt; **Resting membrane potential** &gt; The resting
 membrane potential (RMP) is the electrical potential difference across a
 cell's membrane at rest. The RMP is determined by the concentration
 of ions across the membrane and the membrane permeability to each type of
 ion. Kv channels expressed in Xenopus oocytes whose activity is augmented
 pharmacologically will result in a hyperpolarizing shift in the RMP. Here,
 we measured the RMP (EM) of unclamped Xenopus laevis oocytes expressing
 the aforementioned channels and reported the values in millivolts (mV).
 &gt; **Mesenteric artery myography** &gt; The data collected for this
 analysis was obtained from mesenteric arteries from adult male rats
 pre-contracted with methoxamine (10 µM) in the absence or presence of 10
 µM linopirdine (Kv7-specific antagonist) or 100 nM BIBN-4096 (CGRP
 receptor-specific antagonist), before application of 3-methoxycatechol
 (0.01, 0.03, 0.1, 1, 3, 10, 30, and 100 µM). Raw values are in
 milli-newton (mN). &gt; **Statistics** &gt; All statistical analysis were
 conducted as either paired t-test. &gt; **Additional Information** &gt;
 Excel files with cells with 'n.a.' means not applicable. No data
 was obtained for this cell. &gt; **Version changes** &gt; 10-Feb-2025: IV
 and Gmax graphs for Kv7.4/7.5 were updated to the versions used to
 generate the data in the published manuscript. &gt; &gt; 11-Feb-2025: IV,
 Gmax, and Mesenteric artery myography Excel files have been updated after
 a typo was identified. This has now been resolved.