May 2007
Volume 48, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2007
Effects of Calmodulin/AQP0 Interaction on the Calcium Sensitivity of AQP0 Water Permeability
Author Affiliations & Notes
  • K. Kalman
    Physiology and Biophysics, Univ of California-Irvine, Irvine, California
  • K. Németh-Cahalan
    Physiology and Biophysics, Univ of California-Irvine, Irvine, California
  • A. Froger
    Physiology and Biophysics, Univ of California-Irvine, Irvine, California
  • J. E. Hall
    Physiology and Biophysics, Univ of California-Irvine, Irvine, California
  • Footnotes
    Commercial Relationships K. Kalman, None; K. Németh-Cahalan, None; A. Froger, None; J.E. Hall, None.
  • Footnotes
    Support NIH Grant EY5661
Investigative Ophthalmology & Visual Science May 2007, Vol.48, 3994. doi:https://doi.org/
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      K. Kalman, K. Németh-Cahalan, A. Froger, J. E. Hall; Effects of Calmodulin/AQP0 Interaction on the Calcium Sensitivity of AQP0 Water Permeability. Invest. Ophthalmol. Vis. Sci. 2007;48(13):3994. doi: https://doi.org/.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose:: To investigate the role of calmodulin (CaM) and the phosphorylation of the C-terminus on the calcium sensitivity of AQP0 water permeability (Pf).

Methods:: We expressed AQP0 mutants, which mimicked phosphorylation at serines of the CaM-binding site, along with wild-type AQPO in Xenopus oocytes. We measured Pf in response to an osmotic challenge at different calcium concentrations (µM, 2 or 5 mM) in the presence of CaM inhibitors (TFP, CDZ and W7) or crippled CaM (which is unable to bind calcium). Endogenous Xenopus CaM was present in all experiments. We used confocal microscopy to image the co-localization of a fluorescent CaM(Cam488) in the oocyte membrane with either AQP0 or its mutants.

Results:: The low and high Pf state of AQP0 depends on pH, Ca2+ concentration, and the presence of calmodulin. In the presence of CaM inhibitors AQP0 lost its Ca2+ regulation and was locked in a high Pf state. When crippled CaM and AQP0 were co-expressed, 5 mM Ca2+ rather than µM Ca2+ induced a high Pf. Interestingly, when Ser235Asp mutant was co-expressed with crippled CaM, its Pf remained low and lost its Ca2+ regulation. Other CaM-binding-site mutants (Ser229Asp, Ser231Asp, Ser231Asn and Ser229Asn/Ser235Asp) resulted in a high Pf channel. In the oocyte membrane we observed less fluorescence in the CaM488/Ser229Asp co-expression than in the CaM488/AQP0 co-expression.

Conclusions:: CaM inhibitors prevent any binding of CaM to AQP0, and crippled CaM binds to AQP0 but cannot sense the Ca2+ . The opposite effects of CaM inhibitors and crippled CaM on the Pf of AQP0 suggest that when CaM binds to AQP0 the Pf is low, and when CaM is released upon Ca2+ modulation the Pf is high. Phosphorylations of Ser229 and Ser231 result in a high Pf channel, possibly because of decreased calmodulin binding.

Keywords: cell membrane/membrane specializations • ion channels • cataract 
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