The signaling lipid phosphatidylinositol-3,5-bisphosphate targets plant CLC-a anion/H⁺ exchange activity

Phosphatidylinositol-3,5-bisphosphate (PI(3,5)P₂) is a low-abundance signaling lipid associated with endo-lysosomal and vacuolar membranes in eukaryotic cells. Recent studies on Arabidopsis indicated a critical role of PI(3,5)P₂ in vacuolar acidification and morphology during ABA-induced stomatal closure, but the molecular targets in plant cells remained unknown. By using patch-clamp recordings on Arabidopsis vacuoles, we show here that PI(3,5)P₂ does not affect the activity of vacuolar H⁺-pyrophosphatase or vacuolar H⁺-ATPase. Instead, PI(3,5)P₂ at low nanomolar concentrations inhibited an inwardly rectifying conductance, which appeared upon vacuolar acidification elicited by prolonged H⁺ pumping activity. We provide evidence that this novel conductance is mediated by chloride channel a (CLC-a), a member of the anion/H⁺ exchanger family formerly implicated in stomatal movements in Arabidopsis H⁺-dependent currents were absent in clc-a knock-out vacuoles, and canonical CLC-a-dependent nitrate/H⁺ antiport was inhibited by low concentrations of PI(3,5)P₂ Finally, using the pH indicator probe BCECF, we show that CLC-a inhibition contributes to vacuolar acidification. These data provide a mechanistic explanation for the essential role of PI(3,5)P₂ and advance our knowledge about the regulation of vacuolar ion transport.