Aquaporin-0 (AQP0) is the main water channel in the mammalian lens and is involved in accommodation and maintaining lens transparency. AQP0 binds the Ca2+-sensing protein calmodulin (CaM) and this interaction is believed to gate its water permeability by closing the water-conducting pore. Here, we express recombinant and functional human AQP0 in Pichia pastoris and investigate how phosphorylation affects the interaction with CaM in vitro as well as the CaM-dependent water permeability of AQP0 in proteoliposomes. Using microscale thermophoresis and surface plasmon resonance technology we show that the introduction of the single phospho-mimicking mutations S229D and S235D in AQP0 reduces CaM binding. In contrast, CaM interacts with S231D with similar affinity as wild type, but in a different manner. Permeability studies of wild-type AQP0 showed that the water conductance was significantly reduced by CaM in a Ca2+-dependent manner, whereas AQP0 S229D, S231D and S235D were all locked in an open state, insensitive to CaM. We propose a model in which phosphorylation of AQP0 control CaM-mediated gating in two different ways (1) phosphorylation of S229 or S235 abolishes binding (the pore remains open) and (2) phosphorylation of S231 results in CaM binding without causing pore closure, the functional role of which remains to be elucidated. Our results suggest that site-dependent phosphorylation of AQP0 dynamically controls its CaM-mediated gating. Since the level of phosphorylation increases towards the lens inner cortex, AQP0 may become insensitive to CaM-dependent gating along this axis.

中文翻译：

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磷酸化在钙调蛋白介导的人 AQP0 门控中的作用

水通道蛋白-0 (AQP0) 是哺乳动物晶状体中的主要水通道，参与调节和维持晶状体透明度。AQP0 与 Ca2+ 感应蛋白钙调蛋白 (CaM) 结合，这种相互作用被认为可以通过关闭导水孔来控制其水渗透性。在这里，我们在毕赤酵母中表达重组和功能性人 AQP0，并研究磷酸化如何影响体外与 CaM 的相互作用以及蛋白脂质体中 AQP0 的 CaM 依赖性水渗透性。使用微尺度热泳和表面等离子共振技术，我们证明在 AQP0 中引入单一磷酸模拟突变 S229D 和 S235D 会减少 CaM 结合。相反，CaM 与 S231D 相互作用，其亲和力与野生型相似，但方式不同。野生型AQP0的渗透性研究表明，CaM以Ca2+依赖性方式显着降低水电导率，而AQP0 S229D、S231D和S235D均锁定在开放状态，对CaM不敏感。我们提出了一个模型，其中 AQP0 的磷酸化以两种不同的方式控制 CaM 介导的门控（1）S229 或 S235 的磷酸化消除结合（孔保持开放）和（2）S231 的磷酸化导致 CaM 结合而不导致孔闭合，其功能作用仍有待阐明。我们的结果表明，AQP0 的位点依赖性磷酸化动态控制其 CaM 介导的门控。由于磷酸化水平向晶状体内皮层方向增加，AQP0 可能对沿着该轴的 CaM 依赖性门控变得不敏感。