title: Regulation of the water channel aquaporin-1: isolation and reconstitution of the regulatory complex.
abstract: Aquaporins (AQP) are involved in rapid and active gating of water across biological membranes. The molecular regulation of AQP is unknown. Here we report the isolation, identification and reconstitution of the regulatory complex of AQP-1. AQP-1 and Galphai3 have been implicated in GTP-induced gating of water in zymogen granules (ZG), the secretory vesicles in exocrine pancreas. In the present study, detergent-solubilized ZGs immunoprecipitated with monoclonal AQP-1 antibody, co-isolates AQP-1, PLA2, Galphai3, potassium channel IRK-8, and the chloride channel ClC-2. Exposure of ZGs to either the potassium channel blocker glyburide, or the PLA2 inhibitor ONO-RS-082, blocked GTP-induced ZG swelling. RBC known to possess AQP-1 at the plasma membrane, swell on exposure to the Galphai-agonist mastoparan, and respond similarly to ONO-RS-082 and glyburide, as ZGs. Liposomes reconstituted with the AQP-1 immunoisolated complex from solubilized ZG, also swell in response to GTP. Glyburide or ONO-RS-082 abolished the GTP effect. Immunoisolate-reconstituted planar lipid bilayers demonstrate conductance, which is sensitive to glyburide and an AQP-1 specific antibody. Our results demonstrate a Galphai3-PLA2 mediated pathway and potassium channel involvement in AQP-1 regulation.
authors.affiliation: Department of Physiology, Wayne State University School of Medicine, 5239 Scott Hall, 540 E. Canfield Avenue, Detroit, MI 48201-4177, USA.
authors.forename: Rania
authors.initials: R
authors.lastname: Abu-Hamdah
authors.forename: Won-Jin
authors.initials: WJ
authors.lastname: Cho
authors.forename: Sang-Joon
authors.initials: SJ
authors.lastname: Cho
authors.forename: Aleksandar
authors.initials: A
authors.lastname: Jeremic
authors.forename: Marie
authors.initials: M
authors.lastname: Kelly
authors.forename: Alina Elena
authors.initials: AE
authors.lastname: Ilie
authors.forename: Bhanu P
authors.initials: BP
authors.lastname: Jena
mesh_terms: D062365:Aminobenzoates / Q000494:pharmacology; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D051398:Aquaporin 1; D020346:Aquaporins / Q000302:isolation & purification* / Q000502:physiology*; D001834:Body Water / Q000378:metabolism; D000090007:CLC-2 Chloride Channels; D018118:Chloride Channels / Q000032:analysis; D002723:Chlorobenzoates; D002934:Cinnamates / Q000494:pharmacology; D004594:Electrophysiology; D004912:Erythrocytes / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000032:analysis / Q000378:metabolism*; D005905:Glyburide / Q000494:pharmacology; D054501:Group II Phospholipases A2; D007004:Hypoglycemic Agents / Q000494:pharmacology; D036341:Intercellular Signaling Peptides and Proteins; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism; D008081:Liposomes / Q000037:antagonists & inhibitors; D018625:Microscopy, Atomic Force; D010455:Peptides; D010741:Phospholipases A / Q000032:analysis / Q000378:metabolism*; D054467:Phospholipases A2; D024661:Potassium Channels, Inwardly Rectifying / Q000032:analysis; D051381:Rats; D022142:Secretory Vesicles / Q000737:chemistry / Q000378:metabolism*; D015398:Signal Transduction; D012835:Silver Nitrate / Q000494:pharmacology; D014862:Wasp Venoms / Q000494:pharmacology; D062367:ortho-Aminobenzoates
chemical_list: D062365:Aminobenzoates; D000911:Antibodies, Monoclonal; C496022:Aqp1 protein, rat; D020346:Aquaporins; D000090007:CLC-2 Chloride Channels; D018118:Chloride Channels; D002723:Chlorobenzoates; D002934:Cinnamates; D007004:Hypoglycemic Agents; D036341:Intercellular Signaling Peptides and Proteins; D008051:Lipid Bilayers; D008081:Liposomes; D010455:Peptides; D024661:Potassium Channels, Inwardly Rectifying; D014862:Wasp Venoms; D062367:ortho-Aminobenzoates; D051398:Aquaporin 1; C037500:mastoparan; D012835:Silver Nitrate; C060048:2-(4-amylcinnamoyl)amino-4-chlorobenzoic acid; D010741:Phospholipases A; D054501:Group II Phospholipases A2; D054467:Phospholipases A2; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; C498820:Gnai3 protein, rat; D005905:Glyburide