The basolateral expression of mUT-A3 in the mouse kidney.
Facilitative UT-A urea transporters play a central role in the urinary concentrating mechanism. There are three major UT-A isoforms found in the mouse kidney: mUT-A1, mUT-A2, and mUT-A3. The major aim of this study was to identify the location and function of mUT-A3. UT-A proteins were investigated using three novel mouse UT-A-targeted antibodies: ML446, MQ2, and ML194. ML446 detected mUT-A1 and mUT-A3. ML194 detected mUT-A1 and mUT-A2. Importantly, MQ2 was found to be selective for mUT-A3. MQ2 detected a 45- to 65-kDa signal in the mouse kidney inner medulla, which was deglycosylated to a 40-kDa protein band. Immunolocalization studies showed that mUT-A3 was strongly detected in the papillary tip, mainly in the basolateral regions of inner medullary collecting duct (IMCD) cells. Immunoblotting of subcellular fractions of inner medullary protein suggested that in mouse kidney mUT-A3 was present in plasma membranes. Consistent with this, immunoelectron microscopy demonstrated that mUT-A3 was predominantly localized at the basal plasma membrane domains of the IMCD cells in mouse kidney. Heterologous expression of mUT-A3-enhanced green fluorescent protein in Madin-Darby canine kidney cells showed that the protein localized to the basolateral membrane. In conclusion, our study indicates that mUT-A3 is a basolateral membrane transporter expressed in IMCD cells.
Stewart
Gavin S
GS
School of Biological Sciences, University of Manchester, M13 9PT, UK.
Fenton
Robert A
RA
Wang
Weidong
W
Kwon
Tae-Hwan
TH
White
Stanley J
SJ
Collins
Valerie M
VM
Cooper
Gordon
G
Nielsen
Søren
S
Smith
Craig P
CP
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D016764:Cell Polarity / Q000502:physiology; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism / Q000648:ultrastructure; D049452:Green Fluorescent Proteins; D006706:Homeostasis / Q000502:physiology; D015151:Immunoblotting; D007202:Indicators and Reagents / Q000378:metabolism; D007679:Kidney Medulla / Q000166:cytology / Q000378:metabolism*; D007685:Kidney Tubules, Collecting / Q000166:cytology / Q000378:metabolism*; D008164:Luminescent Proteins / Q000235:genetics; D008297:Male; D026901:Membrane Transport Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D011817:Rabbits; D014882:Water-Electrolyte Balance / Q000502:physiology; D000096982:Urea Transporters
D000906:Antibodies; D000242:Cyclic AMP; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D026901:Membrane Transport Proteins; D000096982:Urea Transporters