Figure 2. PI(3,5)P₂ activates a TPC-like current.

(a) In a representative melanosome from an Oa1^−/− melanocyte, bath application of the intracellular organelle-specific PI(3,5)P₂ (2 μM), but not the same concentration of plasma membrane PI(4,5)P₂, activated I_PIP2. Average PIP₂-induced increase in current density was measured at −120 mV and normalized to basal currents recorded before PI(4,5)P₂ or PI(3,5)P₂ treatment (± s.e.m., n = 3 melanosomes, p < 0.01 for PI(4,5)P₂- vs. PI(3,5)P₂-elicited currents). Gluc⁻-based luminal solutions were used to block outwardly rectifying Cl⁻-dependent currents while measuring inward I_PIP2. (b) 2 μM PI(3,5)P₂ but not the TRPML1 agonist ML-SA1 (25 μM) activated I_PIP2 in a representative melanosome. Average fold increase in current density measured at −120 mV (± s.e.m., n = 3 melanosomes, p < 0.001 for PI(3,5)P₂- vs. ML-SA1-elicited currents). (c) In a representative melanosome, I_PIP2 was inhibited by the TPC antagonist verapamil (150 μM). Average fold increase in current density measured at −120 mV (± s.e.m., n = 4 melanosomes, p < 0.0001 for I_PIP2 vs. I_PIP2 in the presence of verapamil). (d) In a representative melanosome, PI(3,5)P₂ activated an inward current in response to negative voltage steps; no significant PI(3,5)P₂-dependent currents were elicited by depolarizing voltages (representative of 3 melanosomes, p < 0.001 for basal currents vs I_PIP2). (e) PI(3,5)P₂ activated-currents were selective for Na⁺, but not K⁺, Ca²⁺, or the impermeant cation NMDG⁺. Currents were recorded from the same melanosome with 140 mM luminal NaGluc while substituting symmetrical concentrations of cytoplasmic K⁺, NMDG⁺, or 100 mM Ca²⁺ in a Gluc-based solution. (f) I_PIP2 permeability ratios based on E_rev measurements (± s.e.m., n = 4 melanosomes, p < 0.0001 for K⁺, NMDG⁺ or Ca²⁺ vs. Na⁺).