Hanatoxin inserts into phospholipid membranes without pore formation.

Hanatoxin (HaTx), a 35-residue polypeptide from spider venom, functions as an inhibitor of Kv2.1 channels by interacting with phospholipids prior to affecting the voltage-sensor. However, how this water-soluble peptide modifies the gating remains poorly understood, as the voltage-sensor is deeply embedded within the bilayer. To determine how HaTx interacts with phospholipid bilayers, in this study, we examined the toxin-induced partitioning of liposomal membranes. HPLC-results from high-speed spin-down vesicles with HaTx demonstrated direct binding. Dynamic light scattering (DLS) and leakage assay results further indicated that neither membrane pores nor membrane fragmentations were observed in the presence of HaTx. To clarify the binding details, Langmuir trough experiments were performed with phospholipid monolayers by mimicking the external leaflet of membrane bilayers, indicating the involvement of acyl chains in such interactions between HaTx and phospholipids. Our current study thus describes the interaction pattern of HaTx with vesicle membranes, defining a membrane-partitioning mechanism for peptide insertion involving the membrane hydrocarbon core without pore formation.