Functional screen for subtype specificity of voltage sensor–targeted Kv7 potentiators

Background and Purpose Voltage‐gated K v 7 (potassium channel subfamily Q KCNQ) potassium channels are powerful modulators of neuronal excitability. ICA‐069673 is a N‐aryl benzamide drug that targets the voltage‐sensing domain (VSD) of K v 7.2 with strong selectivity over K v 7.3 or K v 7.5, but the molecular basis of this selectivity remains poorly understood. Our objective was to identify novel sequence determinants of subtype specificity of ICA‐069673. Experimental Approach We generated a library of K v 7.2 mutants based on sequence differences between K v 7.2 (ICA‐069673 sensitive) and K v 7.3 or K v 7.5 subtypes (ICA‐069673 insensitive). Xenopus oocytes and/or HEK293 cells were used for electrophysiological characterisation of mutants and identification of residues that influence ICA‐069673 sensitivity. Key Results We identified several substitutions that markedly reduce ICA‐069673 sensitivity in K v 7.2. While the S2–S3 region is particularly important, ICA‐069673 modulation of K v 7.2 is influenced by residues throughout the VSD. K v 7.2I173T had the most prominent effect on ICA‐069673 sensitivity, including accelerated channel closure upon rapid ICA‐069673 wash‐off. Interestingly, different residues contribute to ICA‐069673 resistance in K v 7.3 versus K v 7.5. Conclusion and Implications The sensitivity of K v 7 subtypes to VSD‐targeted potentiators varies markedly, arising from amino acid differences across the VSD. Resistance in K v 7.3 and K v 7.5 appears to arise from distinct sequence differences.