Detection of aquaporin-2 in the plasma membranes of oocytes: a novel isolation method with improved yield and purity.
SourceBiochemical and Biophysical Research Communications, 282, 3, (2001), pp. 683--90
Article / Letter to editor
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Biochemical and Biophysical Research Communications
SubjectRegulation of salt and water reabsorption in the renal collecting duct; Regulatie water en zouttransport in de verzamelbuis van de nier
Aquaporin-2 (AQP2) water channel mutations cause autosomal recessive and dominant nephrogenic diabetes insipidus (NDI). Expressed in oocytes, a mutant in dominant (AQP2-E258K), but not in recessive (AQP2-R187C), NDI conferred a specific dominant-negative effect on wild-type (wt) AQP2 water permeability (Pf) only at low expression levels. Since at these levels, the yield of conventional-isolated plasma membranes was too low, an improved technique to semiquantify AQP2 in the plasma membrane was needed. Antibodies against the C-loop of AQP2 were not applicable since they were unspecific and introduction of a tag into this loop caused misfolding and ER retardation. Membrane-impermeable biotin analogues turned out to label intracellular AQP2 proteins. Therefore, a method has been developed which generates a high yield of nearly pure plasma membranes, which enables semiquantification of plasma membrane proteins expressed at low levels in oocytes. Our new method allows for phenotype-genotype correlation studies in a wide range of channelopathies.
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