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The role of phosphorylation in calmodulin-mediated gating of human AQP0

Kreida, Stefan LU ; Roche, Jennifer Virginia LU ; Missel, Julie Winkel ; Al-Jubair, Tamim LU ; Hagströmer, Carl Johan LU orcid ; Wittenbecher, Veronika ; Linse, Sara LU ; Gourdon, Pontus LU and Törnroth-Horsefield, Susanna LU (2024) In The Biochemical journal 481(1). p.17-32
Abstract

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... (More)

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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author
; ; ; ; ; ; ; and
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
aquaporin, gating, membrane protein calmodulin, phosphorylation, protein–protein interaction, water permeability
in
The Biochemical journal
volume
481
issue
1
pages
16 pages
publisher
Portland Press
external identifiers
  • pmid:38032258
  • scopus:85181765893
ISSN
0264-6021
DOI
10.1042/BCJ20230158
language
English
LU publication?
yes
id
615f0f1e-edc5-4916-8d37-8ff68fdd8d5b
date added to LUP
2024-02-12 10:19:24
date last changed
2024-04-14 01:48:48
@article{615f0f1e-edc5-4916-8d37-8ff68fdd8d5b,
  abstract     = {{<p>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.</p>}},
  author       = {{Kreida, Stefan and Roche, Jennifer Virginia and Missel, Julie Winkel and Al-Jubair, Tamim and Hagströmer, Carl Johan and Wittenbecher, Veronika and Linse, Sara and Gourdon, Pontus and Törnroth-Horsefield, Susanna}},
  issn         = {{0264-6021}},
  keywords     = {{aquaporin; gating; membrane protein calmodulin; phosphorylation; protein–protein interaction; water permeability}},
  language     = {{eng}},
  month        = {{01}},
  number       = {{1}},
  pages        = {{17--32}},
  publisher    = {{Portland Press}},
  series       = {{The Biochemical journal}},
  title        = {{The role of phosphorylation in calmodulin-mediated gating of human AQP0}},
  url          = {{http://dx.doi.org/10.1042/BCJ20230158}},
  doi          = {{10.1042/BCJ20230158}},
  volume       = {{481}},
  year         = {{2024}},
}