CLONING AND EXPRESSION OF AQP3, A WATER CHANNEL FROM THE MEDULLARY COLLECTING DUCT OF RAT-KIDNEY

The terminal part of the inner medullary collecting duct exhibits a hi gh degree of water permeability that is independent of increased intra cellular cAMP and not accounted for by the activity of the known renal epithelial water channels CHIP28 (28-kDa channel-forming integral pro tein) and WCH-CD (collecting duct water channel protein). Starting wit h rat kidney papilla mRNA, reverse transcription PCR was performed wit h degenerate primers assuming that the putative channel would be a mem ber of the major intrinsic protein (MIP) family of proteins. A cDNA fr agment was identified and used to screen a rat kidney cDNA library. A 1.9-kb cDNA clone was isolated. The open reading frame of 876 bp coded for a protein of 292 amino acids (M(r) 31,431). Aquaporin 3 (AQP3; 31 .4-kDa water channel protein) is a newly discovered member of the MIP family. Northern blot analysis showed a single transcript for AQP3 of approximate to 1.9 kb present in the renal medulla, predominantly in t he inner medulla. With in situ hybridization, abundant message was fou nd in the cells of the medullary collecting ducts. Injection of the co mplementary RNA of AQP3 into Xenopus oocytes markedly increased the os motic water permeability. This permeability had an energy of activatio n of 3.0 kcal/mol (1 cal = 4.184 J), it was fully blocked by 1 mM p-ch loromercuriphenylsulfonate, and this inhibition was reversed by 5 mM d ithiothreitol. cAMP did not increase this water permeability. AQP3 did not permit passage of monovalent ions (Na, K, Cl); however, it is sli ghtly permeable to urea. The present study demonstrates the existence of an additional water channel, AQP3, in epithelial cells of the medul lary collecting duct.