The electrical behaviour of outer hair cells (OHC) is mainly determined by the voltage-dependent K⁺ current, I_K,n that is mediated by the voltage-dependent K⁺ channel KCNQ4 (Kv7.4). Highlighting its essential role for OHC function, mutations in KCNQ4 lead to degeneration of OHCs and thereby cause progressive human hereditary deafness, DFNA2. Activity of all KCNQ (Kv7) channels strongly depends on the membrane phospholipide phosphatidylinositol-4,5-bisphosphate (PIP₂ ). Since alterations in phosphoinositide homeostasis have been implicated in OHC degeneration, we investigated a possible PIP₂ dependence of I_K,n. Plasma membrane PIP₂ levels were experimentally changed by intracellular application via a patch pipette of PIP₂-binding polycations in OHCs while current was monitored by voltage clamp. For comparison, similar experiments were performed on CHO cells heterologously expressing KCNQ4 channels. Intracellular application of poly-D-lysine (200 mg/ml), known to chelate phosphoinositides through electrostatic interactions, inhibited I_K,n in OHCs and recombinant KCNQ4 channels. Polycationic aminoglycoside antibiotics, such as neomycin, have also previously been used to functionally deplete PIP₂. We found that neomycin blocked I_K,n (IC50=712 mM) and recombinant KCNQ4 (IC50=225 mM) in a dose-dependent manner. Strong binding of neomycin to PIP₂ at these concentrations was confirmed by observing displacement of the GFP-tagged, genetically encoded PIP₂ binding domain PLCd₁-PH from the membrane upon introduction of the aminoglycoside. We conclude that I_K,n activity requires membrane PIP₂ and that this dependence may play a role in aminoglycoside ototoxicity.