Carpaneto Armando, Boccaccio Anna, Lagostena Laura, Di Zanni Eleonora, Scholz-Starke Joachim
Institute of Biophysics, Consiglio Nazionale delle Ricerche, Genova, Italy.
Institute of Biophysics, Consiglio Nazionale delle Ricerche, Genova, Italy
EMBO Rep. 2017 Jul;18(7):1100-1107. doi: 10.15252/embr.201643814. Epub 2017 May 23.
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.
磷脂酰肌醇-3,5-二磷酸（PI(3,5)P）是一种低丰度信号脂质，与真核细胞中的内溶酶体和液泡膜相关。最近关于PI(3,5)P的研究表明其在脱落酸诱导气孔关闭过程中对液泡酸化和形态具有关键作用，但植物细胞中的分子靶点仍不清楚。通过对液泡进行膜片钳记录，我们在此表明PI(3,5)P不影响液泡H⁺-焦磷酸酶或液泡H⁺-ATP酶的活性。相反，低纳摩尔浓度的PI(3,5)P抑制了一种内向整流电导，该电导在长时间H⁺泵浦活动引起液泡酸化时出现。我们提供证据表明这种新的电导由氯离子通道a（CLC-a）介导，CLC-a是阴离子/H⁺交换器家族的成员，以前涉及气孔运动中的H⁺依赖电流，在CLC-a敲除液泡中不存在该电流，并且低浓度的PI(3,5)P抑制了经典的CLC-a依赖的硝酸盐/H⁺反向转运。最后，使用pH指示剂探针BCECF，我们表明CLC-a抑制有助于液泡酸化。这些数据为PI(3,5)P的重要作用提供了机制解释，并推进了我们对液泡离子运输调控的认识。
