Immunochemical localization of amiloride-sensitive sodium channels in sodium-transporting epithelia.
The localization of amiloride-sensitive Na+ channels in Na+-transporting epithelia was examined using antibodies made against amiloride-binding Na+ channel protein purified from bovine kidney. The distribution of the channel protein was determined in thick frozen sections at the light-microscopic level using indirect immunofluorescence, and at the electron-microscopic level using immunogold labelling. In the cells of both the intact bovine collecting tubule and A6 confluent monolayers, only the luminal or apical-facing surface membranes showed staining. Sodium channel protein was characteristically localized on microvillar domains of the apical plasma membrane. Little or no basolateral membrane staining was evident. Channel protein was also absent from subapical vesicles and tight junctions, and was not found in bovine renal proximal tubules, cultured human secretory sweat coils, non-epithelial Chinese hamster ovary (CHO) cells or human skin fibroblasts. Trypsinization of intact A6 monolayers prior to cell fixation abolished specific staining with antibody. Pretreatment with amiloride protected against this loss of staining. Thus, our probes are specific for amiloride-binding Na+ channel protein, and this channel protein is largely or completely confined to the apical membrane of Na+-transporting epithelia. The level and distribution of specific immunostaining in A6 cells was unchanged by aldosterone treatment, although channel activity, as measured by short-circuit current, increased threefold. This result demonstrates that Na+ channel protein is ever present at the cell surface and exists in both an active and an inactive form. We find no evidence that stimulation of Na+ uptake by aldosterone involves recruitment of new channels from a cytoplasmic pool.
Tousson
A
A
Department of Cell Biology and Anatomy, University of Alabama, Birmingham 35294.
Alley
C D
CD
Sorscher
E J
EJ
Brinkley
B R
BR
Benos
D J
DJ
D000450:Aldosterone / Q000494:pharmacology; D000584:Amiloride / Q000494:pharmacology*; D000818:Animals; D001001:Anura; D001692:Biological Transport; D002417:Cattle; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D004847:Epithelial Cells; D004848:Epithelium / Q000378:metabolism / Q000648:ultrastructure; D005260:Female; D006801:Humans; D007150:Immunohistochemistry; D007668:Kidney / Q000166:cytology / Q000378:metabolism / Q000648:ultrastructure; D008297:Male; D010053:Ovary / Q000166:cytology / Q000378:metabolism / Q000648:ultrastructure; D012867:Skin / Q000166:cytology / Q000378:metabolism / Q000648:ultrastructure; D012964:Sodium / Q000378:metabolism; D015222:Sodium Channels / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D013545:Sweat Glands / Q000166:cytology / Q000378:metabolism / Q000648:ultrastructure
D015222:Sodium Channels; D000450:Aldosterone; D000584:Amiloride; D012964:Sodium