Molecular identification and immunolocalization of the water channel protein aquaporin 1 in CBCECs

PURPOSE. Water channel proteins are important pathways for water movements across cell membranes, including those in the corneal endothelium that cont ribute to the fluid transport mechanism essential in maintaining corneal tr ansparency. This study was conducted to identify and locate the water chann el protein(s) in cultured bovine corneal endothelial cells (CBCECs). METHODS. Poly(A)(+) RNA was isolated from CBCECs, and MMLV reverse transcri ptase and random hexamer primers were used to generate a cDNA pool by rever se transcription-polymerase chain reaction (RT-PCR). Two specific degenerat e primers were synthesized based on consensus sequences from the major intr insic lens protein superfamily; a "touchdown" PCR protocol accommodated the degeneracy. Immunolocalization was performed by incubating sections of CBC ECs with an antibody against human aquaporin 1 (AQP1). Cryosections (0.85 m u m) of CBCECs were used for light microscopy, and 800-A ultrathin cryosect ions were used for electron microscopy (EM). RESULTS. ii 372-bp fragment was isolated. Its encoded amino acid sequence w as 100% identical with that of bovine AQP1 (AQP1-bovin). CBCECs reacted str ongly with the anti-AQP1 antibody, and the labeling was selectively localiz ed to the plasma membrane by light microscopy. Subcellular localization by EM revealed immunoreactivity with the inner leaflets of the plasma membrane . CONCLUSIONS. The identity of the aquaporin, its abundance, and its membrane location suggest that it is a major pathway for fluid flow across endothel ial cell membranes. This is consistent with transcellular endothelial fluid transport.