Binding of neomycin to phosphatidylinositol 4,5-biphosphate (PIP 2)

Abstract

Schacht (Schacht, J. (1976) J. Neurochem. 27, 1119–1124) demonstrated that neomycin, an aminoglycoside antibiotic, binds with high affinity to phosphatidylinositol 4,5-bisphosphate (PIP 2). We investigated the binding of neomycin to PIP 2 by making electrophoretic mobility measurements with multilamellar bilayer vesicles and surface potential measurements with monolayers. The bilayers and monolayers were formed from mixtures of PIP 2 and egg phosphatidylcholine (PC) in 0.1 M KCl at pH 7. Neomycin does not bind to PC; 10 −3 M neomycin affects neither the zeta potential of PC vesicles nor the surface potential of PC monolayers. In contrast, 10 −6 M neomycin reduces the magnitude of the zeta potential of PC/PIP 2 vesicles (5, 9, and 17 mol% PIP 2) and the surface potential of monolayers (17 mol% PIP 2) to < 50% of their initial values. The electrophoretic mobility results indicate that neomycin forms an electroneutral complex with PIP 2; high concentrations (> 10 −4 M) of neomycin reduce the zeta potential of the PC/PIP 2 vesicles to zero. We could describe our data with the Gouy-Chapman-Stern theory assuming the intrinsic association constant of the 1:1 neomycin-PIP 2 complex is 10 5 M −1. Neomycin is widely used in cell biology to interfere with the generation second messengers, we discuss the relevance of our results to these studies. Specifically, 10 −6 M neomycin binds > 50% of the PIP 2 in a bilayer or monolayer but 10 −5−10 −3 M neomycin is required to affect the turnover of PIP 2 in permeabilized platelets, mast cells, and sea urchin eggs. This result is consistent with a hypothesis that most of the PIP 2 in the inner leaflet of these plasma membranes is not accessible to neomycin because it is associated with proteins.