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.sup.+] 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.sup.+] transport. The basal level of [Na.sup.+] 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.sup.+] 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.sup.+] transport. sodium transport; amiloride; aldosterone; insulin; channel trafficking; adenosine 3',5'-cyclic monophosphate, signal transduction; epithelial sodium channel; green fluorescent protein