Characterization and imaging of A6 epithelial cell clones expressing fluorescently labeled ENaC subunits.
A6 model renal epithelial cells were stably transfected with enhanced green fluorescent protein (EGFP)-tagged alpha- or beta-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 predominantly 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 alpha- and beta-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.
Blazer-Yost
B L
BL
Department of Biology, Indiana University-Purdue University Indianapolis, Indianapolis, Indiana 46202, USA. bblazer@iupui.edu
Butterworth
M
M
Hartman
A D
AD
Parker
G E
GE
Faletti
C J
CJ
Els
W J
WJ
Rhodes
S J
SJ
D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002999:Clone Cells; D005576:Colforsin / Q000494:pharmacology; D004847:Epithelial Cells / Q000378:metabolism* / Q000648:ultrastructure; D053503:Epithelial Sodium Channels; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D018613:Microscopy, Confocal; D020033:Protein Isoforms / Q000378:metabolism; D015222:Sodium Channels / Q000378:metabolism*; D014018:Tissue Distribution; D014981:Xenopus
D053503:Epithelial Sodium Channels; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D020033:Protein Isoforms; D015222:Sodium Channels; D049452:Green Fluorescent Proteins; D005576:Colforsin