Vasorelaxant effects of 3-methoxycatechol are not via direct activation of voltage-gated potassium channels

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.

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.  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 MgCl2, 1 CaCl2, 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.

Mesenteric artery myography

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 ad libitum 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 CaCl2, 25 NaHCO3, 1.2 KH2HPO4, 1.2 MgSO4, 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% O2/5% CO2. 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.

Statistics and Reproducibility

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