Loss-of-function mutations in Kv7.1 often lead to long QT syndrome (LQTS), a cardiac repolarization disorder associated with arrhythmia and subsequent sudden cardiac death. The discovery of agonistic IKs modulators may offer a new potential strategy in pharmacological treatment of this disorder. The benzodiazepine derivative (R)-L3 potently activates Kv7.1 channels and shortens action potential duration, thus may represent a starting point for drug development. However, the molecular mechanisms underlying modulation by (R)-L3 are still unknown. By combining alanine scanning mutagenesis, non-canonical amino acid incorporation, voltage-clamp electrophysiology and fluorometry, and in silico protein modelling, we show that (R)-L3 not only stimulates currents by allosteric modulation of the pore domain but also alters the kinetics independently from the pore domain effects. We identify novel (R)-L3-interacting key residues in the lower S4-segment of Kv7.1 and observed an uncoupling of the outer S4 segment with the inner S5, S6 and selectivity filter segments.
Loss-of-function mutations in Kv7.1 often lead to long QT syndrome (LQTS), a cardiac repolarization disorder associated with increased risk of arrhythmia and subsequent sudden cardiac death. The discovery of agonistic IKs modulators may offer a new potential strategy in pharmacological treatment of this disorder. The benzodiazepine (R)-L3 potently activates Kv7.1 channels and shortens action potential duration, thus may represent a starting point for drug development. However, the molecular mechanisms underlying modulation by (R)-L3 are still unknown. By combining alanine scanning mutagenesis, non-canonical amino acid incorporation, voltage-clamp electrophysiology and fluorometry, and in silico protein modelling, we showed that (R)-L3 not only stimulates currents by allosteric modulation of the pore domain but also alters the kinetics independently from the pore domain effects. We identified novel (R)-L3-interacting key residues in the lower S4-segment of Kv7.1 and observed an uncoupling of the outer S4 segment with the inner S5, S6 and selectivity filter segments with similarity to phosphatidylinositol-4,5-bisphosphat (PI(4,5)P2). Summarizing, we provide structural and functional evidence for two independent Kv7.1 activating mechanisms by a single modulator mimicking PI(4,5)P2
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