Cloning, Immunolocalization And Characterization Of Porcine Aquaporin 1 Water Channel

Aquaporins (AQPs) are water channel proteins located on membranes of various celltypes where they create high water permeability. So far 13 members of the AQP family(AQP0-12) have been reported in human and rodents. Recent studies on human subjects withAQP gene mutations and using transgenic AQP knockout mice have indicated importantfunctions of AQPs in multi-tissue physiology and pathophysiology such as renalconcentrating mechanisms, exocrine gland secretion, fluid transport in brain and eye, brainedema formation and tumor angiogenesis.Although significant progresses have been made in molecular biology and physiology ofAQP family, identification and characterization of AQPs of mammalian species other thanhuman and rodents has been poorly studied. In mammals, the pig organs more resemble thehuman organs both structurally and physiologically than rodents, so it is more reasonableanimal model on the invastigation of AQPs gene expression and function in vivo. However,no porcine AQP member has been identified molecularly so far.In the present study, we cloned the first porcine aquaporin, pAQP1, by a PCR-basedhomologous cloning strategy from pig liver. pAQP1 consists of 271 amino acids with a highlyconserved six transmembrane domains and two NPA motifs. pAQP1 has greatest amino acididentity to dog AQP1 (94%) and high homology to human AQP1 (93%) and mouse AQP1(91%). Northern blot analysis revealed that pAQP1 is strongly expressed in pig liver, salivaryglands and intestines with a single transcript of ~3.0 kb. pAQP1 gene structure is similar tohAQP1, containing 4 exons and 3 introns. Multi-tissue RT-PCR analysis showed that pAQP1mRNA is expressed extensively in various pig tissues. Further analysis byimmunohistochemistry showed specific AQP1 localization in the endothelium of centrallacteals in villi of the small intestine and in the endothelium of microvessels in salivary glands.In the liver, AQP1 staining was mainly seen in the epithelium of small bile ducts andendothelium of some small vessels. AQP1 staining could also be seen in kidney proximaltubules, choroid plexus in the brain, endothelial layer of cornea in the eye and some neuronsin the central nervous system. Immunoblot analysis showed that an unglycosylated (28 kDa)band and a glycosylated (35-55kDa) band were determined both in red blood cells of pigs andin pAQP1-transfected CHO cells. The water-transporting function of pAQP1 was analyzedusing pig red blood cells and CHO cells stably transfected with pAQP1 and a Cl--sensitiveEYFP mutant (EYFP-H148Q-V163S). The pAQP1 created high osmotic water permeabilityin the cell membranes that was inhibitable by HgCl2, similar to the AQP1 homologs of humanand mouse in erythrocytes and in heterologous expression systems.In summary, we have successfully cloned pAQP1, the first molecularly identified porcineAQP water channel and studied its function, expression and tissue localization systematically.The cloning and characterization of pAQP1 will facilitate the studies of its regulation andfunction using pig as an in vivo model. In addition, this study provided new information tostudies on comparative biology of aquaporin water channels.