Anionic phospholipid phosphatidic acid (PA) behaves as an important second messenger involved in many cellular processes, such as development, cytoskeletal dynamics, vesicle trafficking, and stress response. Recently, it was reported that PA can directly bind with the rice Shaker K+ channel OsAKT2 to inhibit its channel activity. Two adjacent arginine residues (R644 and R645) in ANK domain were identified as a PA-binding site essential to the PA-mediated inhibition of OsAKT2. However, there may be still other PA-binding sites unidentified in OsAKT2. Here, using a PA biosensor (PAleon), we found that the exogenous PA treatment significantly increased the PA level at the plasma membrane of Xenopus oocytes which were used to express OsAKT2 for electrophysiological assays. As reported previously, exogenous PA markedly inhibited OsAKT2 K+ currents. Replacement of two adjacent basic residues (R190 and K191) in the S4 voltage sensor by glycine completely abolished the time-dependent K+ currents of OsAKT2, but this variant was insensitive to PA treatment. In addition, we also identified other two adjacent arginines (R755 and R756) located in the cytosolic domain as a PA-binding site, which were also essential to the PA-mediated inhibition of OsAKT2. These results provide a more comprehensive understanding of the PA-K+ channel interaction mechanism. Combining the findings here with the previous study, we propose that multiple basic residues (R190/K191, R644/R645, and R755/R756) in different domains of OsAKT2 contribute to PA-mediated regulation of OsAKT2.
Keywords: AKT2; OSAKT2; PA; Phospholipid signaling; phosphatidic acid; rice; shaker K+ channel.