The role of phosphorylation in calmodulin-mediated gating of human AQP0
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The role of phosphorylation in calmodulin-mediated gating of human AQP0. / Kreida, Stefan; Roche, Jennifer Virginia; Missel, Julie Winkel; Al-Jubair, Tamim; Hagströmer, Carl Johan; Wittenbecher, Veronika; Linse, Sara; Gourdon, Pontus; Törnroth-Horsefield, Susanna.
I: The Biochemical journal, Bind 481, Nr. 1, 2024, s. 17-32.Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › fagfællebedømt
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TY - JOUR
T1 - The role of phosphorylation in calmodulin-mediated gating of human AQP0
AU - Kreida, Stefan
AU - Roche, Jennifer Virginia
AU - Missel, Julie Winkel
AU - Al-Jubair, Tamim
AU - Hagströmer, Carl Johan
AU - Wittenbecher, Veronika
AU - Linse, Sara
AU - Gourdon, Pontus
AU - Törnroth-Horsefield, Susanna
N1 - © 2024 The Author(s).
PY - 2024
Y1 - 2024
N2 - 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.
AB - 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.
KW - Animals
KW - Humans
KW - Aquaporins/genetics
KW - Calcium/metabolism
KW - Calmodulin/genetics
KW - Eye Proteins/genetics
KW - Lens, Crystalline/metabolism
KW - Mammals/metabolism
KW - Phosphorylation
KW - Water/metabolism
U2 - 10.1042/BCJ20230158
DO - 10.1042/BCJ20230158
M3 - Journal article
C2 - 38032258
VL - 481
SP - 17
EP - 32
JO - Biochemical Journal
JF - Biochemical Journal
SN - 0264-6021
IS - 1
ER -
ID: 389362534