Cellular and subcellular immunolocalization of vasopressin-regulated water channel in rat kidney.

Abstract

(CHIP28), which is the major water-channel protein of erythrocytes (3), present in the renal proximal tubule and descending limb of the loop of Henle (4), and found in several other water-transporting tissues (5). Recently, the nucleotide sequence of a cDNA coding for an additional member of this family of proteins [referred to as the aquaporin family (2)] has been reported by Fushimi et al. (6). This collecting-duct water-channel protein (WCH-CD) is expressed chiefly, if not exclusively, in the renal collecting duct. On the basis of this localization, WCH-CD has been proposed to be the vasopressin-regulated water channel.Vasopressin binds to receptors in the basolateral plasma membrane and, acting through cAMP, increases the water permeability of the apical plasma membrane of principal and IMCD cells. The mechanism whereby the water permeability increases is unknown, although it presumably involves an increase in the number of or unit conductance of water channels in the apical membrane. The most widely accepted theory is the "shuttle" hypothesis (7), which proposes that a reservoir of water channels is contained in the membranes of intracellular vesicles and that vasopressin increases the apical plasma membrane water permeability by triggering exocytosis of these vesicles, delivering the vesicles with their water channels to the apical membrane. Studies using freezefracture electron microscopy in collecting ducts or vasopressin-responsive amphibian-bladder epithelia have supported this model by showing that discrete patches (termed clusters or aggregates) of intramembrane particles appear in the apical membrane with vasopressin exposure and disappear with vasopressin withdrawal (7-9). These clusters appear to be localized to clathrin-coated pits (8). The intramembrane particles have been proposed to contain the water channels. One item of evidence that is lacking, but is crucial for verification ofthe shuttle hypothesis, is direct localization of the water-channel protein at a subcellular level. In the present study, we have prepared polyclonal antibodies to the WCH-CD protein and have used these antibodies to carry out immunohisto-and immunocytochemistry to determine the cellular and subcellular localization of the WCH-CD channel.MATERIALS AND METHODS Preparation of Antibodies. Two peptides were synthesized by standard automated solid-phase techniques (10) based on the predicted amino acid sequence of WCH-CD (6). These were as follows: peptide 1 (P1), CEVRRRQSVELHSPQSL-PRGSKA (amino acids 250-271 of WCH-CD plus an N-terminal cysteine used for conjugation), and peptide 2 (P2), CELHSPQSLPRGSKA (amino acids 258-271 plus an N-terminal cysteine). These peptides were conjugated to keyhole Abbreviations: WCH-CD, collecting-duct water-channel protein;IMCD, inner medullary collecting duct.tTo whom reprint requests should be addressed at: Laboratory of

Identification of a novel protein containing two C2 domains selectively expressed in the rat brain and kidney

“…3C and 4D) is intriguing as it overlaps with these two vasopressinregulated proteins [21,22]. The vasopressin-mediated increases in water and urea permeability are known to be mediated by G-protein coupled V2-type vasopressin receptors (V2R) whose activation leads to an increase in cAMP and activation of pro- ) and then exposed to film for 3 days.…”

The Proteasome Is Involved in the Degradation of Different Aquaporin-2 Mutants Causing Nephrogenic Diabetes Insipidus

“…15,16 When living cells were incubated with fluorescently labeled transferrin to label endosomes ( Figure 4B) and were subsequently subjected to confocal immunofluorescence microscopy to detect AQP2 and late endosomes/lysosomes with an antibody against LAMP 1 ( Figure 4D), this triple labeling protocol provided evidence for co-distribution of wt AQP2 with a limited number (ϳ20 to 30%) of transferrin-loaded endosomes ( Figure 4C). However, the limited x-y resolution of confocal immunofluorescence (Յ200 nm) allows no statement that wt AQP2 actually co-localizes with endosomes.…”

“…12 AQP2 belongs to the large family of AQPs, 13,14 and is a 29-kd polytope membrane protein that contains a single N-glycosylation site and two phosphorylation sites, and is present in the principal cells of renal collecting ducts. 15,16 In states of hypernatremia or hypovolemia, translocation of phosphorylated AQP2 homotetramers from vesicles to the apical plasma membrane of the principal cells is triggered by a signal transduction cascade induced by arginine-vasopressin. 16 -18 Alike to normal human kidney, 19 wild-type (wt) AQP2, when expressed in Xenopus oocytes 20,21 or various mammalian cell lines, 18,22 existed as a nonglycosylated 29-kd form.…”

Immunomorphometric study of rat renal inner medulla

“…To perform immunolocalization studies, we used peptide-directed polyclonal antibodies to 1) AQP1, raised in rabbits (L266) and chickens (LC18), to the same peptide (28); 2) ClC-K (COOH-terminal peptide of ClC-K2, KKAISTLINPARK), kindly provided by Klaus Steinmeyer, that recognizes both ClC-K1 and ClC-K2 (30) or ClC-K antibody (AB5392; Chemicon, Temecula, CA); 3) von Willebrand factor (vWF; Cedarlane Labs, Ontario, PQ); and 4) AQP2 raised in guinea pigs [GP7; COOH-terminal peptide of AQP2 to the same peptide previously used to produce antibodies in rabbits (22)]. …”

“…ClC-K1 is found in ATL cells (29,30) and in ATL-type DTL segments (2,5). On the other hand, AQP2 is a water channel found in collecting duct principal cells and in inner medullary collecting duct cells (22).…”