Expression of VAMP-2-like protein in kidney collecting duct intracellular vesicles. Colocalization with Aquaporin-2 water channels. S Nielsen, … , M Trimble, M Knepper J Clin Invest. 1995;96(4):1834-1844. https://doi.org/10.1172/JCI118229. Research Article Body water balance is controlled by vasopressin, which regulates Aquaporin-2 (AQP2) water channels in kidney collecting duct cells by vesicular trafficking between intracellular vesicles and the plasma membrane. To examine the molecular apparatus involved in vesicle trafficking and vasopressin regulation of AQP2 in collecting duct cells, we tested if targeting proteins expressed in the synaptic vesicles, namely vesicle-associated membrane proteins 1 and 2 (VAMP1 and 2), are expressed in kidney collecting duct. Immunoblotting revealed specific labeling of VAMP2 (18-kD band) but not VAMP1 in membrane fractions prepared from kidney inner medulla. Controls using preadsorbed antibody or preimmune serum were negative. Bands of identical molecular size were detected in immunoblots of brain membrane vesicles and purified synaptic vesicles. VAMP2 in kidney membranes was cleaved by tetanus toxin, revealing a tetanus toxin-sensitive VAMP homologue. Similarly, tetanus toxin cleaved VAMP2 in synaptic vesicles. In kidney inner medulla, VAMP2 was predominantly expressed in the membrane fraction enriched for intracellular vesicles, with little or no VAMP2 in the plasma membrane enriched fraction. This was confirmed by immunocytochemistry using semithin cryosections, which showed mainly vesicular labeling in collecting duct principal cells, with no labeling of intercalated cells. VAMP2 immunolabeling colocalized with AQP2 labeling in intracellular vesicles, as determined by immunoelectron microscopy after double immunolabeling of isolated vesicles. Quantitative analysis of 1,310 vesicles revealed a highly significant […] Find the latest version: https://jci.me/118229/pdf Expression of VAMP2-like Protein in Kidney Collecting Duct Intracellular Vesicles Colocalization with Aquaporin-2 Water Channels S0ren Nielsen,* David Marples,* Henrik Birn,* Mahmood Mohtashami,t Niels Ole Dalby,§ William Trimble,t and Mark Knepperil *Department of Cell Biology, Institute of Anatomy, University of Aarhus, DK-8000 Aarhus, Denmark; tDepartment of Physiology and Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, M5S IA8 Canada; §Department of Molecular Biology, University of Aarhus, DK-8000 Aarhus, Denmark; and I'Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892 Abstract words: vasopressin * vesicular trafficking * tetanus toxina immunoelectron microscopy Body water balance is controlled by vasopressin, which reg- ulates Aquaporin-2 (AQP2) water channels in kidney col- lecting duct cells by vesicular trafficking between intracellu- Introduction lar vesicles and the plasma membrane. To examine the molecular apparatus involved in vesicle trafficking and va- The antidiuretic hormone, vasopressin, controls body water bal- ance sopressin regulation of AQP2 in collecting duct cells, we by regulating Aquaporin-2 (AQP2),' the predominant va- tested if targeting proteins expressed in the synaptic vesicles, sopressin-regulated water channel in the kidney collecting duct namely vesicle-associated membrane proteins 1 and 2 (for review see reference 1). AQP2 (2) is abundant in both membrane and (VAMP1 and 2), are expressed in kidney collecting duct. plasma intracellular vesicles of collecting duct Immunoblotting revealed specific labeling of VAMP2 (18- principal cells ( 3-6 ). Mutations in the AQP2 gene cause severe kD band) but not VAMP1 in membrane fractions prepared diabetes insipidus in humans (7) and both lithium-induced diabetes from kidney inner medulla. Controls using preadsorbed an- nephrogenic insipidus (8) and central diabetes insip- in rats and are tibody or preimmune serum were negative. Bands of identi- idus (4) associated with markedly reduced levels in cal molecular size were detected in immunoblots of brain of AQP2 expression parallel with severe polyuria. A long- membrane vesicles and purified synaptic vesicles. VAMP2 in standing question has been how vasopressin acutely regulates duct water kidney membranes was cleaved by tetanus toxin, revealing a collecting permeability and body water balance. We tetanus toxin-sensitive VAMP homologue. Similarly, teta- have shown recently that acute stimulation with vasopressin induces a translocation of AQP2 water channels from intracellu- nus toxin cleaved VAMP2 in synaptic vesicles. In kidney lar vesicles to the in with an inner medulla, VAMP2 was predominantly expressed in the apical plasma membrane, parallel membrane fraction enriched for intracellular vesicles, with increase in the osmotic water permeability in isolated perfused ducts This the first direct evidence that little or no VAMP2 in the plasma membrane enriched frac- collecting (6). provided of water channels represents the tion. This was confirmed by immunocytochemistry using trafficking AQP2 regulatory semithin cryosections, which showed mainly vesicular label- mechanism for the hydroosmotic action of vasopressin. We have confirmed this in vivo studies Based on ing in collecting duct principal cells, with no labeling of subsequently by (5). intercalated cells. VAMP2 immunolabeling colocalized with these studies it became important to define the molecular appa- ratus involved in the of tar- AQP2 labeling in intracellular vesicles, as determined by specific targeting, and regulation immunoelectron microscopy after double immunolabeling geting, of vesicles carrying AQP2 water channels. Such targeted of isolated vesicles. Quantitative analysis of 1,310 vesicles delivery requires a set of signals to ensure that vesicles con- are and fuse revealed a highly significant association of both AQP2 and taining AQP2 transported to, specifically with, the membrane. the process VAMP2 in the same vesicles (P < 0.0001). Furthermore, apical plasma Furthermore, delivery must be so that the presence of AQP2 in vesicles immunoisolated with anti- regulated, vesicle fusion, and hence AQP2 into the occurs when the cells VAMP2 antibodies was confirmed by im munoblotting. In insertion plasma membrane, only are to the hormonal conclusion, VAMP2, a component of the neuronal SNARE exposed trigger. complex, is expressed in vesicles carrying AQP2, suggesting Recently the SNARE hypothesis, describing a protein as- was a role in vasopressin-regulated vesicle trafficking of AQP2 sembly-disassembly pathway, proposed for vesicle traf- water channels. (J. Clin. Invest. 1995. 96:1834-1844.) Key ficking including the sequential steps of synaptic vesicle dock- ing, activation, and fusion. The SNARE hypothesis (9, 10) proposes that vesicles and their target plasma membrane are Address correspondence to S0ren Nielsen, M.D., Ph.D., Department of Cell Biology, Institute of Anatomy, University of Aarhus, DK-8000 Aarhus C, Denmark. Phone: 45-8942-3046; FAX: 45-8619-8664; E- 1. Abbreviations used in this paper: AQP2, Aquaporin-2 water channel mail: [email protected] protein; NSF, N-ethylmaleimide-sensitive fusion protein; SNAP, solu- Receivedfor publication 22 February 1995 and accepted in revised ble NSF attachment protein; SNAP25, synaptosomal-associated protein form 16 June 1995. of 25 kD; SNARE, SNAP receptor; TeTx L, tetanus toxin light chain 1; t-SNARE, SNAP receptor on the cognate target membrane; VAMP, The Journal of Clinical Investigation, Inc. vesicle-associated membrane protein; v-SNARE, vesicle-associated Volume 96, October 1995, 1834-1844 SNARE. 1834 Nielsen et al. each associated specifically with one member of a pair of mem- Preparation of membrane vesicles and subcellular brane proteins, the soluble N-ethylmaleimide-sensitive fusion fractionation protein (NSF) attachment protein (SNAP) receptors. The vesi- Crude membrane fraction of kidney inner medulla. The inner medulla cle-associated SNAP receptor (v-SNARE) is thought to pair was dissected from each kidney, minced finely, and homogenized in 10 with a SNAP receptor on the cognate target membrane (t- ml of dissecting buffer (0.3 M sucrose, 25 mM imidazole, 1 mM EDTA, SNARE) before assembly with SNAPs and NSF into a fusion pH 7.2, and containing the following protease inhibitors: 8.5 tsM leupep- particle (10). This process is thought to mediate the specific tin and 1 mM PMSF), with five strokes of a motor-driven Potter- targeting and fusion of the vesicle to its acceptor membrane. Elvehjem homogenizer at 1,250 rpm. This homogenate was centrifuged Three synapse membrane proteins have been extensively char- (L8M centrifuge; Beckman Instruments, Inc., Fullerton, CA) at 4,000 acterized and later were found to be SNAREs (9). The vesicle- g for 15 min at 40C. The pellet was rehomogenized with three to five associated membrane protein (VAMP), or synaptobrevin ( 11- strokes, and centrifugation was repeated to increase yields. The superna- tants were pooled and centrifuged at 200,000 g for 1 h. The resultant 13), is a v-SNARE, whereas syntaxin (14, 15) and SNAP25 pellet was resuspended in - 100 ul of dissecting buffer and assayed ( 16) are t-SNAREs (9). Three mammalian isoforms of VAMP for protein concentration using the method of Lowry (see reference 25). have been characterized and sequenced, VAMPI (11), VAMP2 Subcellular fractionation of kidney inner medulla. Membrane vesi- (11, 12, 17), and cellubrevin (18). VAMP2 and cellubrevin cle fractions were prepared