Novel Kv7.1-Phosphatidylinositol 4,5-Bisphosphate Interaction Sites Uncovered by Charge Neutralization Scanning

K(v)7.1 to K(v)7.5 alpha-subunits belong to the family of voltage-gated potassium channels (K-v). Assembled with the beta-subunit KCNE1, K(v)7.1 conducts the slowly activating potassium current I-Ks, which is one of the major currents underlying repolarization of the cardiac action potential. A known regulator of K(v)7 channels is the lipid phosphatidylinositol 4,5-bisphosphate (PIP2). PIP2 increases the macroscopic current amplitude by stabilizing the open conformation of 7.1/KCNE1 channels. However, knowledge about the exact nature of the interaction is incomplete. The aim of this study was the identification of the amino acids responsible for the interaction between K(v)7.1 and PIP2. We generated 13 charge neutralizing point mutations at the intracellular membrane border and characterized them electro-physiologically in complex with KCNE1 under the influence of diC(8)-PIP2. Electrophysiological analysis of corresponding long QT syndrome mutants suggested impaired PIP2 regulation as the cause for channel dysfunction. To clarify the underlying structural mechanism of PIP2 binding, molecular dynamics simulations of K(v)7.1/KCNE1 complexes containing two PIP2 molecules in each subunit at specific sites were performed. Here, we identified a subset of nine residues participating in the interaction of PIP2 and K(v)7.1/KCNE1. These residues may form at least two binding pockets per subunit, leading to the stabilization of channel conformations upon PIP2 binding.