Characterization and imaging of A6 epithelial cell clones expressing fluorescently labeled ENaC subunits
1 Department of Biology, Indiana University-Purdue University Indianapolis, Indianapolis, Indiana 46202; and 2 Department of Anatomy and Cell Biology, University of Cape Town Medical School, Cape Town, South Africa A6 model renal epithelial cells were stably transfected with enhanced green fluores...
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Veröffentlicht in: | American Journal of Physiology: Cell Physiology 2001-08, Vol.281 (2), p.C624-C632 |
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Zusammenfassung: | 1 Department of Biology, Indiana University-Purdue
University Indianapolis, Indianapolis, Indiana 46202; and
2 Department of Anatomy and Cell Biology, University of Cape
Town Medical School, Cape Town, South Africa
A6 model renal epithelial
cells were stably transfected with enhanced green fluorescent protein
(EGFP)-tagged - or -subunits of the epithelial Na +
channel (ENaC). Transfected RNA and proteins were both expressed in low
abundance, similar to the endogenous levels of ENaC in native cells. In
living cells, laser scanning confocal microscopy revealed a
predominately subapical distribution of EGFP-labeled subunits,
suggesting a readily accessible pool of subunits available to
participate in Na + transport. The basal level of
Na + transport in the clonal lines was enhanced two- to
fourfold relative to the parent line. Natriferic responses to insulin
or aldosterone were similar in magnitude to the parent line, while
forskolin-stimulated Na + transport was 64% greater than
control in both the - and -transfected lines. In response to
forskolin, EGFP-labeled channel subunits traffic to the apical
membrane. These data suggest that channel regulators, not the channel
per se, form the rate-limiting step in response to insulin or
aldosterone stimulation, while the number of channel subunits is
important for basal as well as cAMP-stimulated Na + transport.
sodium transport; amiloride; aldosterone; insulin; channel
trafficking; adenosine 3',5'-cyclic monophosphate, signal transduction; epithelial sodium channel; green fluorescent protein |
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ISSN: | 0363-6143 1522-1563 |
DOI: | 10.1152/ajpcell.2001.281.2.c624 |