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

Bonnie L. Blazer-Yost, Michael Butterworth, Amy D. Hartman, Gretchen E. Parker, Carla J. Faletti, Willem J. Els, Simon J. Rhodes

Research output: Contribution to journalArticle

22 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)C624-C632
JournalAmerican Journal of Physiology - Cell Physiology
Volume281
Issue number2 50-2
DOIs
StatePublished - 2001

Keywords

  • Adenosine 3′,5′-cyclic monophosphate
  • Aldosterone
  • Amiloride
  • Channel trafficking
  • Epithelial sodium channel
  • Green fluorescent protein
  • Insulin
  • Signal transduction
  • Sodium transport

ASJC Scopus subject areas

  • Clinical Biochemistry
  • Cell Biology
  • Physiology
  • Physiology (medical)

Fingerprint Dive into the research topics of 'Characterization and imaging of A6 epithelial cell clones expressing fluorescently labeled ENaC subunits'. Together they form a unique fingerprint.

  • Cite this