April 2009
Volume 50, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2009
Secondary Calmodulin Binding Site in AQP0 C-terminus Results in Calcium Independent Water Permeability Regulation
Author Affiliations & Notes
  • K. Kalman
    Physiology and Biophysics, Univ of California-Irvine, Irvine, California
  • K. L. Nemeth-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.L. Nemeth-Cahalan, None; A. Froger, None; J.E. Hall, None.
  • Footnotes
    Support  NIH Grant EY5661
Investigative Ophthalmology & Visual Science April 2009, Vol.50, 3874. doi:https://doi.org/
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      K. Kalman, K. L. Nemeth-Cahalan, A. Froger, J. E. Hall; Secondary Calmodulin Binding Site in AQP0 C-terminus Results in Calcium Independent Water Permeability Regulation. Invest. Ophthalmol. Vis. Sci. 2009;50(13):3874. doi: https://doi.org/.

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

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Abstract

Purpose: : To investigate the water permeability regulation of AQP0 by calmodulin, PKA and PKC

Methods: : Xenopus oocyte water permeability assay, bovine-AQP0, site directed mutagenesis, Fluorescent Resonance Energy Transfer (FRET), immunohistochemistry.

Results: : AQP0, the major intrinsic protein of the lens fiber cells, can act as a water channel, junctional protein, and an anchor for the cytoskeleton. During fiber cell differentiation, AQP0 undergoes spatially and temporally regulated phosphorylations that may have unique physiological roles in lens fiber cells. We recently published data that shows the pattern of C-terminal phosphorylation of AQP0 creates four distinct water permeability (Pf) phenotypes. These four phenotypes are: 1. Low-Pf with 0 mM Ca2+ sensitivity; 2. Low-Pf with 5 mM Ca2+ sensitivity; 3. Low-Pf no Ca2+ sensitivity; 4. High-Pf with no Ca2+ sensitivity. The rate of water permeability is high in the absence of calmodulin and low in the presence of calmodulin, thus calmodulin is an inhibitor for AQP0 Pf. Our site-directed mutagenesis confirms that calcium sensitivity and affinity of calmodulin binding depend on the pattern of serine pseudo-phosphorylations. We have created three C-terminal mutations, S235D (pseudo-phosphorylated serine), R241A, and R233K in the bovine AQP0, that convert the 0 mM Ca2+ sensitivity into 5 mM Ca2+ sensitivity. Interestingly, PKC phosphorylation of these mutants mimics the 5 mM Ca2+ sensitivity resulting in the increase of Pf. We tested the direct interaction between calmodulin and AQP0 in wild type and mutant AQP0s when the oocytes were incubated in ND96 containing 2mM and 5 mM Ca2+. We also tested the direct interaction between calmodulin and AQP0 C-terminus when PKC enzymes were activated in the oocytes. Our experiments suggest an alternative calmodulin docking configuration exists in the S235D mutant that allows additional phosphorylation of the C-terminus by PKC, leading to the release of calmodulin and a high-Pf phenotype.

Conclusions: : S235D promotes a secondary calmodulin binding site at the C-terminus of AQP0, and PKC phosphorylation of AQP0-S235D leads to a calcium-independent Pf increase.

Keywords: phosphorylation • protein modifications-post translational • cataract 
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