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

Phosphatidylinositol-3,5-bisphosphate (PI(3,5)P) is a low-abundance signaling lipid associated with endo-lysosomal and vacuolar membranes in eukaryotic cells. Recent studies on indicated a critical role of PI(3,5)P 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 vacuoles, we show here that PI(3,5)P does not affect the activity of vacuolar H-pyrophosphatase or vacuolar H-ATPase. Instead, PI(3,5)P 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 H-dependent currents were absent in knock-out vacuoles, and canonical CLC-a-dependent nitrate/H antiport was inhibited by low concentrations of PI(3,5)P 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)P and advance our knowledge about the regulation of vacuolar ion transport.