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
Hauptverfasser: Blazer-Yost, Bonnie L, Butterworth, Michael, Hartman, Amy D, Parker, Gretchen E, Faletti, Carla J, Els, Willem J, Rhodes, Simon J
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Sprache:eng
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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
ISSN:0363-6143
1522-1563
DOI:10.1152/ajpcell.2001.281.2.c624