Evidence against a role of mouse, rat, and two cloned human t1alpha isoforms as a water channel or a regulator of aquaporin-type water channels.
T1alpha is a protein of unknown function that is expressed at the plasma membrane in epithelia involved in fluid transport, including type I alveolar epithelial cells, choroid plexus, and ciliary epithelium. The purpose of this study was to test the hypothesis that T1alpha functions as a water channel or a regulator of aquaporin-type water channels that are coexpressed with T1alpha. Two complementary DNAs (cDNAs) (hT1alpha-1 and hT1alpha-2) encoding human isoforms of T1alpha were cloned by homology to the rat T1alpha coding sequence. The cDNAs encoded 164 (hT1alpha-1) and 162 (hT1alpha-2) amino acid proteins with high homology to rat T1alpha in a putative membrane-spanning domain. hT1alpha-1 transcripts of 2. 6 and 1.4 kb were detected in human lung, heart, and skeletal muscle, and a single hT1alpha-2 transcript of 1.2 kb was detected in human lung. Rat and mouse T1alpha were isolated by reverse transcription-polymerase chain reaction and confirmed by DNA sequence analysis. Expression of mouse, rat, and human T1alpha isoforms in Xenopus oocytes did not increase osmotic water permeability (Pf) above that in water-injected oocytes, nor was there an effect of protein kinase A or C activation; Pf was increased > 10-fold in positive control oocytes expressing aquaporin (AQP)1 or AQP5. Coexpression of AQP1 or AQP5 with excess T1alpha gave Pf not different from that in oocytes expressing AQP1 or AQP5 alone. Oocyte plasma membrane localization of epitope-tagged T1alpha protein was confirmed and quantified by immunoprecipitation of microdissected plasma membranes. Quantitative densitometry indicated that the single-channel water permeability of T1alpha is under 2 x 10(-16) cm3/s, suggesting that T1alpha is not involved in the high transalveolar water permeability in intact lung. The cloning of hT1alpha isoforms may permit the development of an assay of type I cell antigen in airspace fluid as a marker of human lung injury.
Ma
T
T
Departments of Medicine and Physiology, Cardiovascular Research Institute, University of California, San Francisco, California, USA.
Yang
B
B
Matthay
M A
MA
Verkman
A S
AS
D000595:Amino Acid Sequence; D000818:Animals; D051398:Aquaporin 1; D051402:Aquaporin 5; D020346:Aquaporins; D001483:Base Sequence; D001789:Blood Group Antigens; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary; D006801:Humans; D007473:Ion Channels / Q000502:physiology*; D008168:Lung / Q000378:metabolism*; D008562:Membrane Glycoproteins; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D051379:Mice; D008969:Molecular Sequence Data; D009865:Oocytes / Q000378:metabolism; D010539:Permeability; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D014867:Water / Q000378:metabolism*; D014981:Xenopus; D000098853:Podoplanin
D051398:Aquaporin 1; D051402:Aquaporin 5; D020346:Aquaporins; D001789:Blood Group Antigens; D018076:DNA, Complementary; D007473:Ion Channels; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D011994:Recombinant Proteins; D014867:Water; C496020:AQP1 protein, human; C496036:AQP5 protein, human; C496021:Aqp1 protein, mouse; C496022:Aqp1 protein, rat; C496038:Aqp5 protein, mouse; C496040:Aqp5 protein, rat; C490441:Pdpn protein, mouse; D000098853:Podoplanin; C117468:PDPN protein, human; C092025:Pdpn protein, rat