DOI: 10.5061/dryad.98sf7m0td
title: Quercetin metabolite 4-methylcatechol does not directly augment the activity of Kv7.4 or Kv7.5

description: Vascular smooth muscle voltage-gated potassium (Kv) channels, particularly
 Kv7.4 and Kv7.5 homomers and/or heteromers, are increasingly being
 recognized to play a role in regulating vascular smooth muscle cell
 excitability. Thus, augmenting Kv7.4 and Kv7.5 activity to induce
 vasorelaxation is being investigated as a mechanism for antihypertensive
 drug development and the underlying molecular mechanism for the
 antihypertensive effects of dietary components and traditional botanical
 medicines. Recently, Dias and colleagues wrote that “Dietary polyphenols
 have been associated with many beneficial cardiovascular effects. However,
 these effects are rather attributed to small phenolic molecules
 formed by the gut microbiota…4-Methylcatechol (4-MC) is one such
 metabolite.” Dias and colleagues demonstrate that 4-MC (15 µM)
 augments vasorelaxation induced by sodium nitroprusside or forskolin in
 rat aortic rings. The vasorelaxation was inhibited by pan
 voltage-gated potassium channel modulator 4-aminopyridine and to a lesser
 extent by Kv7 inhibitor linopirdine, but not by soluble
 guanylyl cyclase inhibitor ODQ. The authors concluded that “in
 silico reverse docking confirmed that 4-MC binds to Kv7.4 through hydrogen
 bonding and hydrophilic interactions” and “our findings
 suggested that 4-MC exerts vasorelaxation by opening Kv channels
 with the involvement of Kv7.4”. Here, we report that 4-MC has no direct
 functional effect on Kv7.4 and Kv7.5 and may be weakly inhibitory to
 Kv7.4/Kv7.5 heteromers at depolarized potentials. At 100 µM 4-MC has mild
 augmenting effects at hyperpolarized potentials on the activity of Kv1.2,
 Kv1.5, and Kv2.1, but not Kv1.1. In conclusion, it is critical that in
 silico docking predictions be experimentally validated in order to
 accurately draw conclusions about the identity of specific proteins as
 pharmacological targets.
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.  4-methylcatechol 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).  4-methylcatechol 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>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: # Quercetin metabolite 4-methylcatechol does not directly augment the
 activity of Kv7.4 or Kv7.5 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 4-methylcatechol. *Xenopus laevis*
 oocytes were injected with cRNA encoding for each of the aforementioned
 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 4-methylcatechol. The
 parameters that we measured to characterize the effect of 4-methylcatechol
 on Kv7.4, Kv7.5, Kv7.4/7.5, Kv1.1, Kv1.2, Kv1.5, and Kv2.1 channels are as
 follows: #### **Current-voltage (IV) curve** 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 laevis* 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). #### **Gmax** 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 4-methylcatechol. Raw values are in microamps (uA). #### **Resting
 membrane potential** 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 laevis* 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). ####
 **Statistics** All statistical analysis were conducted as either paired
 t-test. #### **Additional Information** Excel files with cells with
 '***n.a.***' means not applicable. No data was obtained for this
 cell.