磷酸化在钙调素介导的人AQP0门控中的作用。

IF 4.4 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Biochemical Journal Pub Date : 2024-01-10 DOI:10.1042/BCJ20230158
Stefan Kreida, Jennifer Virginia Roche, Julie Winkel Missel, Tamim Al-Jubair, Carl Johan Hagströmer, Veronika Wittenbecher, Sara Linse, Pontus Gourdon, Susanna Törnroth-Horsefield
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引用次数: 0

摘要

水通道蛋白-0 (AQP0)是哺乳动物晶状体中的主要通道,参与调节和维持晶状体的透明度。AQP0结合Ca2+感应蛋白钙调蛋白(CaM),这种相互作用被认为通过关闭导水孔来控制其水渗透性。本研究在帕斯德酵母中表达重组的功能性人类AQP0,并研究磷酸化如何影响体外与CaM的相互作用以及AQP0在蛋白脂质体中依赖CaM的透水性。利用微尺度热泳(MST)和表面等离子体共振(SPR)技术,我们发现在AQP0中引入单磷酸模拟突变S229D和S235D减少了cam结合。相比之下,CaM与S231D的相互作用与野生型具有相似的亲和力,但方式不同。对野生型AQP0的渗透性研究表明,CaM以Ca2+依赖的方式显著降低了AQP0的水电导,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的磷酸化消除了结合(孔保持开放),(2)S231的磷酸化导致cam结合而不导致孔关闭,其功能作用仍有待阐明。我们的研究结果表明,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可能对沿该轴的cam依赖性门控变得不敏感。
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The role of phosphorylation in calmodulin-mediated gating of human AQP0.

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.

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来源期刊
Biochemical Journal
Biochemical Journal 生物-生化与分子生物学
CiteScore
8.00
自引率
0.00%
发文量
255
审稿时长
1 months
期刊介绍: Exploring the molecular mechanisms that underpin key biological processes, the Biochemical Journal is a leading bioscience journal publishing high-impact scientific research papers and reviews on the latest advances and new mechanistic concepts in the fields of biochemistry, cellular biosciences and molecular biology. The Journal and its Editorial Board are committed to publishing work that provides a significant advance to current understanding or mechanistic insights; studies that go beyond observational work using in vitro and/or in vivo approaches are welcomed. Painless publishing: All papers undergo a rigorous peer review process; however, the Editorial Board is committed to ensuring that, if revisions are recommended, extra experiments not necessary to the paper will not be asked for. Areas covered in the journal include: Cell biology Chemical biology Energy processes Gene expression and regulation Mechanisms of disease Metabolism Molecular structure and function Plant biology Signalling
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