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K. Varadaraj, S. S. Kumari, R. T. Mathias; Functional Characterization of Human Lens Mutant Aquaporin 0. Invest. Ophthalmol. Vis. Sci. 2007;48(13):3993.
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Two individuals in a family of European descent were identified with polymorphic autosomal dominant cataracts, which were due to a single nucleotide base deletion mutation of human lens hAQP0; the deletion resulted in a frameshift just prior to the C-terminus, with a premature stop codon (Geyer et al., Am. J. Opthal., 2006). In the wild type (WT) hAQP0, the C- and N-terminus are normally cleaved in the central fiber cells. The present study was conducted to: a) determine the trafficking, dominant negative effect and water transport properties of mutant hAQP0 (ΔhAQP0); b) determine the effect of C- and N-terminus truncation on water transport properties of hAQP0.
hAQP0 and ΔhAQP0 were cloned into appropriate vectors containing T7 promoter for cRNA expression in Xenopus oocytes, and CMV promoter for mammalian cell expression, with EGFP (Clontech) and mCherry as tags (a kind gift from Dr. R. Y. Tsien, UC San Diego, USA). Trafficking and dominant negative effect studies were conducted using Fluorescence Resonance Energy Transfer (FRET). Water permeability (Pw) was determined from the rate of volume change when the oocyte or cell was subjected to hypotonic bathing solution.
hAQP0 protein with or without EGFP tag, localized in the plasma membranes of oocytes and tissue culture cells whereas ΔhAQP0 was mostly in the cytoplasmic organelles. Deletion of 2nd to 13th amino acid at the N-terminus, or amino acids from position 243 onward at the C-terminus, caused no significant change in the amount of hAQP0 in the plasma membrane or cytoplasmic vesicles. Membrane Pw of ΔhAQP0 (18±2 µm/s) was significantly lower than that of wild type hAQP0 (32±3 µm/s). Control water injected oocyte Pw was 13±2 µm/s. When hAQP0 was co-expressed with ΔhAQP0, the Pw was also significantly lower (22±3 µm/s) than for hAQP0 alone. FRET studies showed that wild type hAQP0 partly localized with co-expressed ΔhAQP0. Incubating the cells with chemical chaperones, namely glycerol and DMSO (48-72h), to determine whether ΔhAQP0 trafficking defect is correctable, did not yield any positive results.
In exogenous expression systems, this single nucleotide base deletion mutation significantly affects the normal trafficking of hAQP0 and reduces the cell membrane Pw. Interactions of hAQP0 with ΔhAQP0 may account for the dominant negative effect. The tested N- and C-terminal deletions did not affect trafficking or Pw.
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