Assembled with the beta-subunit KCNE1, Kv7.1 conducts the slowly activating potassium current IKs, which is one of the major currents underlying the repolarization of the cardiac action potential. A known regulator of Kv7 channels is the lipid phosphatidylinositol 4,5- bisphosphate (PIP2). PIP2 increases the macroscopic current amplitude by stabilization of the open conformation of Kv7.1/KCNE1 channels whereas the exact nature of interaction is incomplete. To identify amino acid residues responsible for the interaction between Kv7.1 and PIP2 13 charge neutralizing point mutations at the intracellular membrane border were generated. The influence of DiC8-PIP2 on the Kv7.1/KCNE1 channel complex was analysed. Nine residues participating in the functional interaction of PIP2 with Kv7.1/KCNE1 were found. To clarify the underlying structural mechanism, molecular modelling and molecular-dynamics simulations of Kv7.1/KCNE1 complexes containing two PIP2 molecules in each subunit at specific sites were performed. These residues may form at least two binding pockets per subunit, leading to the stabilization of channel conformations upon PIP2 binding.