M-channels are formed by the voltage-gated potassium channel a-subunits KCNQ1 to KCNQ5. KCNQ2 to KCNQ5 channels are expressed mostly in the central nervous system, whereas KCNQ1 is mainly expressed in the heart and epithelial cells. Assembled with the b-subunit KCNE1, KCNQ1 conducts the slowly activating potassium current I_Ks, which causes the depolarization of myocyte membranes during the cardiac action potential. Many identified KCNQ1 mutations cause the long QT syndrome, a disorder leading to syncope, cardiac arrhythmias, and cardiac sudden death. A known regulator of M-channels is the lipid phosphatidylinositol 4,5-bisphosphate (PIP₂), which increases the macroscopic current amplitude. The goal of this study was to identify the residues responsible for the interaction between KCNQ1 and the lipid. With regard to the assumption of electrostatic forces responsible for the interaction, 12 mutants of positively charged amino acids to cysteines were characterized by the two electrode voltage clamp technique under the influence of diC₈-PIP₂. To clarify the underlying structural mechanism, molecular dynamics simulations of a KCNQ1-tetramer containing two PIP₂-molecules in each subunit at specific sites were performed. Of the 12 examined amino acids we identified a subset participating in the interaction, whereas others do not. The results are in good agreement with the molecular dynamics simulation, which shows a clustering of many positively charged residues around the PIP₂-headgroups, supporting the assumption of electrostatic interactions. The results indicate the existence of two specific PIP₂-binding sites in each KCNQ1 subunit.