FIGURE 5.

Otoferlin C2 domain interacts with phosphatidylinositols. A, surface plasmon resonance analysis of otoferlin C2F interaction with PIP₂. Purified C2F domain was immobilized on a CM5 sensor chip via an amine coupling reaction (11). 100 nm PIP₂ (analyte) was diluted in HBS binding buffer containing no added Ca²⁺, and the solution was brought to 100 μm Ca²⁺ before injection. For binding assays lacking Ca²⁺, the analyte was adjusted to 1 mm EGTA. Samples were injected at a flow rate of 20 μl/min. Flow cells blocked with ethanolamine served as reference cells. Each sample was analyzed in five replicas, and all experiments were performed at least three times. There was approximately a 40% increase in binding for PIP₂ in the presence of Ca²⁺. B, a 100 nm concentration of each of phosphatidylinositol (PI), PIP, PIP₂, and PIP₃ was analyzed on a C2F-immobilized sensor chip, and the maximum binding response at the end of the association phase (n = 5) was averaged. Error bars denote S.E. Among the different phosphoinositides and phosphatidylinositol, PIP₂ showed the most robust binding to the C2F domain followed by phosphatidylinositol, PIP₃, and PIP. PIP₂ also showed a significantly higher response than the other phosphoinositols in the presence versus absence of 100 μm calcium. Phosphatidylinositol, PIP, and PIP₃ did not show any remarkable increase in C2F binding upon addition of calcium. RU, response units.