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

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Abstract
A6 model renal epithelial cells were stably transfected with enhanced green fluorescent protein (EGFP)-tagged α- or β-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 α- and β-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.