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

Abstract

Phosphatidylinositol‐3,5‐bisphosphate ( PI (3,5)P 2 ) 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)P 2 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)P 2 does not affect the activity of vacuolar H + ‐pyrophosphatase or vacuolar H + ‐ ATP ase. Instead, PI (3,5)P 2 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)P 2 . 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 2 and advance our knowledge about the regulation of vacuolar ion transport. image This study identifies inhibition of CLC ‐a dependent anion/H + exchange as a vacuolar target of the signaling lipid phosphatidylinositol‐3,5‐bisphosphate. This provides a clue to the lipid's positive effect on vacuolar acidification in plant cells. PI(3,5)P 2 does not affect major vacuolar H + pump activities. Vacuolar acidification elicited by prolonged H + pumping activity revealed a novel H + ‐dependent chloride conductance mediated by the anion/H + exchanger CLC‐a. CLC‐a anion/H + exchange activity is reversibly inhibited by PI(3,5)P 2 at low nanomolar concentrations. CLC‐a inhibition contributes to vacuolar acidification.