Quercetin metabolite 4-methylcatechol does not directly augment the activity of Kv7.4 or Kv7.5

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.

Methods
cRNA prepeartion and Two-electrode voltage clamp (TEVC) 

cRNA transcripts encoding human Kv7.4, Kv7.5, Kv1.1, Kv1.2, Kv1.5, and Kv2.1 were generated by in vitro transcription using the mMessage mMachine kit (Thermo Fisher Scientific), after vector linearization, from cDNA sub-cloned into plasmids incorporating Xenopus laevis β-globin 5’ and 3’ UTRs flanking the coding region to enhance translation and cRNA stability.  Defolliculated stage V and VI Xenopus laevis oocytes (Xenoocyte, Dexter, MI, US) were injected with KCNQ cRNAs (0.1-25 ng) and  incubated at 16 oC 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 MgCl2, 1 CaCl2, 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.

Statistics and Reproducibility

All values are expressed as mean ± SEM.  One-way ANOVA was applied for all tests; all p values were two-sided.