Rat Kidney Papilla Contains Abundant Synaptobrevin Protein that Participates in the Fusion of Antidiuretic Hormone-regulated Water Channel-containing Endosomes In Vitro

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Title: Rat Kidney Papilla Contains Abundant Synaptobrevin Protein that Participates in the Fusion of Antidiuretic Hormone-regulated Water Channel-containing Endosomes In Vitro
Authors: Harris, H. William; Amendt-Raduege, Amy M.; Majewski, Rebecca R.; Hammond, Timothy G.; Jo, Inho
Publisher: Proceedings of the National Academy of Sciences of the United States of America
Date Published: March 14, 1995
Reference Number: 264
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Antidiuretic hormone (ADH) regulates renal water excretion by altering the permeability of the collecting duct to water. ADH-responsive epithelial cells are the major cell type lining kidney tubules in the inner medulla and papilla. ADH modulates apical membrane water permeability by the insertion and removal of vesicles containing aquaporin collecting duct water channel protein (now termed AQP-2). To identify and characterize proteins responsible for trafficking of AQP-2-containing vesicles, we utilized antibody and cDNA probes to synaptobrevin b (also termed VAMP-2, for vesicle-associated membrane protein 2), a protein that mediates synaptic vesicle exocytosis in the brain and whose structural homologs are now considered to be components of a complex responsible for intracellular vesicle fusion in all cells. We now report that rat kidney inner medulla and papilla contain abundant synaptobrevin protein. Only light endosomes, one of two types of purified papillary AQP-2-containing endosomes, possess synaptobrevin. Light endosomes fuse in vitro by means of an ATP-dependent process that is significantly inhibited when endosomes are preincubated with either anti-synaptobrevin antibody or tetanus toxin. These data define a functional role for a synaptobrevin protein in the fusion of endosomes in vitro. The presence of abundant synaptobrevin proteins in endosomes containing AQP-2 water channels, as well as insulin-sensitive glucose transporters [Cain, C. C., Trimble, W. S. & Lienhard, G. E. (1992) J. Biol. Chem. 267, 11681-11684], and in cells of Malpighian tubules responsible for urine formation in insects [Chin, A. S., Burgess, R. W., Wong, B. R., Schwartz, T. L. & Scheller, R. H. (1993) Gene 131, 175-181] suggests a specialized role for synaptobrevin in vesicle-mediated membrane transport modulated by peptide hormones.
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This translation by the NDI Foundation is to assist the lay reader. To provide a clear, accessible interpretation of the original article, we eliminated or simplified some technical detail and complicated scientific language. We concentrated our translation on those aspects of the article dealing directly with NDI. The NDI Foundation thanks the researchers for their work toward understanding and more effectively treating this disorder.
© Copyright NDI Foundation 2007 (JC)

Antidiuretic hormone (ADH) regulates the water permeability of the apical membranes of the principal cells of the human kidney collecting duct. It does this by signaling water transporting proteins called aquaporin-2 (AQP2) to travel to and insert themselves into the apical membranes. There they act as channels through which water can travel through the cell membrane. When ADH absents itself from the cell, the AQP2s are retrieved from the apical membrane and taken back into the interior of the cell.

AQP2s are encased in small sacs called vesicles. It is these AQP2-containing vesicles that carry the AQP2s to the apical cell membrane and fuse with it so the AQP2s may be inserted in the membrane. Jo, et al., sought to identify the proteins responsible for taking and fusing the AQP2-containing vesicles to the apical membrane.

They thought that the protein, synaptobrevin, might be the protein that performs this function. First located in the central nervous system and related tissues (such as brain tissues), recent research has revealed that synaptobrevin and its homologues (proteins such as cellubrevin that are similar in form and function to synaptobrevin) are widespread in yeasts, flies and mammals where they act as parts of a complex of structures that are responsible for fusing intracellular vesicles to intracellular membranes.

Researchers have used a tetanus toxin on live experimental animals which specifically degrades both synaptobrevin and cellubrevin to provide support that these proteins do help endosomes (vesicles that have lost their coating of a protein called clathrin after they have returned to the cell from the cell membrane) travel to and fuse with membranes. But, as yet, researchers had not provided a direct demonstration of a functional role for synaptobrevin or its homologues in the fusion of endosomes in laboratory cell cultures.

In their paper, Jo, et al., demonstrate that the innermost part of the rat kidney, purified and made into a cell culture, contains abundant synaptobrevin protein that fuse the endosomes which they are in to rat kidney cell membranes. The authors infused rats with flourescein-conjugated dextran. This was absorbed by the apical endosomes in the rat kidney. This allowed the researchers to track the movement and function of the inner kidney endosomes after they homogenized, purified and recovered them.

The authors found two types of AQP2 containing endosomes: light endosomes (LE) and heavy endosomes (HE). Although both contained abundant AQP2, only LE possessed synaptobrevin. LE were tested to see if they fused with intracellular membranes in the laboratory cell culture, and about 50% of them did.

To determine if synaptobrevin protein in LE was an important part of the fusion process, the researchers pretreated LE with substances known to inhibit the function of synaptobrevin. Then they tested LE fusion and found it to be greatly inhibited. This data provides strong support for the involvement of synaptobrevin proteins in fusing AQP2-containing vesicles to the apical membrane.