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 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.
Kamsteeg
E J
EJ
Department of Cell Physiology, University Medical Center Nijmegen, Nijmegen, 6500HB, The Netherlands.
Deen
P M
PM
D000818:Animals; D051399:Aquaporin 2; D051403:Aquaporin 6; D020346:Aquaporins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D001483:Base Sequence; D001710:Biotin / Q000031:analogs & derivatives; D002458:Cell Fractionation / Q000379:methods; D002462:Cell Membrane / Q000737:chemistry; D002463:Cell Membrane Permeability; D017931:DNA Primers / Q000235:genetics; D018500:Diabetes Insipidus, Nephrogenic / Q000235:genetics / Q000378:metabolism; D000939:Epitopes / Q000737:chemistry; D005260:Female; D005838:Genotype; D006801:Humans; D007120:Immunochemistry; D066298:In Vitro Techniques; D009865:Oocytes / Q000737:chemistry*; D010641:Phenotype; D017354:Point Mutation; D012822:Silicon Dioxide; D014982:Xenopus laevis
C496023:AQP2 protein, human; D051399:Aquaporin 2; D051403:Aquaporin 6; D020346:Aquaporins; D017931:DNA Primers; D000939:Epitopes; D001710:Biotin; D012822:Silicon Dioxide