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Biochem Biophys Res Commun. 2001 Apr 6;282(3):683-90.

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

Author information

1
Department of Cell Physiology, University Medical Center Nijmegen, Nijmegen, 6500HB, The Netherlands.

Abstract

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.

PMID:
11401515
DOI:
10.1006/bbrc.2001.4629
[Indexed for MEDLINE]

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