In vitro activity: ML213 (formerly known as CID-3111211) is a potent and selective activator/opener of Kv7.2 (KCNQ2) and Kv7.4 (KCNQ4) channels which enhances Kv7.2 and Kv7.4 channels with EC50 of 230 and 510 nM, respectively. ML213 shows more than 80-fold selectivity for Kv7.2 (KCNQ2) and Kv7.4 (KCNQ4) versus a large battery of related potassium channels such as KV7.1, KV7.3 and KV7.5 in a thallium-based fluorescence assay, it also can afford modest brain levels. ML213, as a potent vasorelaxant in different blood vessels, may have the potential to be developed as therapeutics for various smooth muscle disorders.

Kinase Assay: ML213 (100 nM-30 μM) increases maximal conductance to a peak at 212% ± 27% of control, with an EC50 of 0.8 ± 0.3 μM. ML213 (10 μM) reduces the deactivation rates of Kv7.4 currents by 4.6-fold in the voltage range from –130 mV to –90 mV. ML213 is a potent and effective activator of homomeric Kv7.5 channels overexpressed in A7r5 cells. ML213 increases maximal conductance of Kv7.5 channels with an EC50 of 0.7 ± 0.2 μM. ML213 (10 μM) also reduces deactivation rates of Kv7.5 currents by 5.9-fold on average. ML213 produces similar effects on heteromeric Kv7.4/7.5 channels: 204% ± 11% maximal increase in conductance with an EC50 of 1.1 ± 0.6 μM and a 34.2 ± 3.3 mV maximal negative shift of the activation curve, with an EC50 of 3.8 ± 1.2 μM. ML213 causes a vasorelaxation in different precontracted rat blood vessels. ML213 (10 μM) also hyperpolarizes mesenteric artery smooth muscle cells. ML213 causes a concentration-dependent shift in the V1/2 for KCNQ2 activation with an EC50 340 ± 70 nM and a maximal shift of 37.4 mV.

Cell Assay: A7r5 cells were cultured as described previously. For overexpression studies, subcultured A7r5 cells at 70% confluence were infected with Adv-Kv7.4 or Adv-Kv7.5 or both at a multiplicity of infection of 100 and used for electrophysiologic experiments 2–12 days after infection as previously described. For mutagenesis studies, subcultured A7r5 cells at 70% confluence were transfected with appropriate Kv7 channel mutants inserted into a pIRES2-EGFP (Kv7.5 W235L, Kv7.4 F143A) or pShuttle-IRES-hrGFP-2 (Kv7.4 W242L) vector using Lipofectamine transfection reagent according to the manufacturer’s protocol. Cells expressing the exogenous channels were identified based on the detection of green fluorescence protein (GFP).