Bl. Blazer-yost et al., Characterization and imaging of A6 epithelial cell clones expressing fluorescently labeled ENaC subunits, AM J P-CELL, 281(2), 2001, pp. C624-C632
A6 model renal epithelial cells were stably transfected with enhanced green
fluorescent protein (EGFP)-tagged alpha- or beta -subunits of the epitheli
al 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 acces
sible pool of subunits available to participate in Na+ transport. The basal
level of Na+ transport in the clonal lines was enhanced two- to fourfold r
elative 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 -trans
fected lines. In response to forskolin, EGFP-labeled channel subunits traff
ic 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 a
ldosterone stimulation, while the number of channel subunits is important f
or basal as well as cAMP-stimulated Na+ transport.