Antidiuretic hormone (ADH) stimulation of toad bladder granular cells rapidly increases the osmotic water permeability (Pf) of their apical membranes by insertion of highly selective water channels. Before ADH stimulation, these water channels are stored in large cytoplasmic vesicles called aggrephores. ADH causes aggrephores to fuse with the apical membrane. Termination of ADH stimulation results in prompt endocytosis of water channel-containing membranes via retrieval of these specialized regions of apical membrane. Protein components of the ADH water channel contained within these retrieved vesicles would be expected to be integral membrane protein(s) that span the vesicle's lipid bilayer to create narrow aqueous channels. Our previous work has identified proteins of 55 (actually a 55/53-kDa doublet), 17, 15, and 7 kDa as candidate ADH water channel components. We now have investigated these candidate ADH water channel proteins in purified retrieved vesicles. These vesicles do not contain a functional proton pump as assayed by Western blots of purified vesicle protein probed with anti-H(+)-ATPase antisera. Approximately 60% of vesicle protein is accounted for by three protein bands of 55, 53, and 46 kDa. Smaller contributions to vesicle protein are made by the 17- and 15-kDa proteins. Triton X-114-partitioning analysis shows that the 55, 53, 46, and 17 kDa are integral membrane proteins. Vectorial labeling analysis with two membrane-impermeant reagents shows that the 55-, 53-, and 46-kDa protein species span the lipid bilayer of these vesicles. Thus the 55-, 53-, and 46-kDa proteins possess characteristics expected for ADH water channel components. These data show that the 55- and 53- and perhaps the 46-, 17-, and 15-kDa proteins are likely components of aqueous transmembrane pores that constitute ADH water channels contained within these vesicles.
Screening for Influx through Membrane Proteins on the Single-Molecule Level using an Automated and Parallel Lipid Bilayer Platform
