Am J Physiol Cell Physiol 2001 Aug 01;2812:C624-32. doi: 10.1152/ajpcell.2001.281.2.C624.

Characterization and imaging of A6 epithelial cell clones expressing fluorescently labeled ENaC subunits.

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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.

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Species referenced: Xenopus
Genes referenced: camp ins