Detection of aquaporin-2 in the plasma membranes of oocytes: A novel isolation method with improved yield and purity

Aquaporin-2 (AQP2) water channel mutations cause autosomal recessive and do minant 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 permeabil ity (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 intro duction of a tag into this loop caused misfolding and ER retardation. Membr ane-impermeable biotin analogues turned out to label intracellular AQP2 pro teins. 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 allow s for phenotype-genotype correlation studies in a wide range of channelopat hies. (C) 2001 Academic Press.