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K. Kalman, K. L. Nemeth-Cahalan, D. Clemens, J. E. Hall; PKC Phosphorylation Can Completely Eliminate AQP0 Water Permeability. Invest. Ophthalmol. Vis. Sci. 2010;51(13):4600.
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© ARVO (1962-2015); The Authors (2016-present)
Investigate the effects of PKC phosphorylation on the water permeability of AQP0.
Xenopus oocyte water permeability assay, BtAQP0, site directed mutagenesis, PKC activation, PKC inhibition, Fluorescent Resonance Energy Transfer (FRET), immunohistochemistry.
AQP0, the major intrinsic protein of lens fiber cells, can act as a water channel, junctional protein, or anchor protein for the cytoskeleton and promotes the formation of micro-domains in the plasma membrane. During fiber cell differentiation AQP0 undergoes spatially and temporally regulated phosphorylations that could have unique physiological roles in lens fiber cells. The pattern of C-terminal phosphorylation of AQP0 creates four distinct water permeability (Pf) phenotypes: 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 (Kalman, K. et al. .JBC., 2008). In the inner cortex, Ser229 and Ser231 phosphorylation increases (Ball, L.E. et al. Biochem. 2004), and these modified AQP0s become calmodulin-independent high-Pf-phenotype channels. Using site-directed mutagenesis, PKC activators and inhibitors, we investigated the effects of PKC phosphorylation(s) on "group-4" Ser229Asp, Ser229Asn, Ser231Asp and Ser231Asn mutants. Our data show that DAG-activated PKC inhibits the Pf of Ser229Asp mutant and probably to closes the channel pore. This result reveals a fifth phenotype of regulation of AQP0 by phosphorylation.
PKC phosphorylation is an essential component of the regulation of AQP0 water permeability. Our observation suggests that there is a particular combination of C-terminus serine-phosphorylation which completely eliminates AQP0 water permeability. This suggests a greater range of Pf regulation then before observed.
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