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 Citations (Scopus)

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
StatePublished - Aug 27 2001

Fingerprint

Epithelial Sodium Channels
Clone Cells
Epithelial Cells
Colforsin
Aldosterone
Imaging techniques
Insulin
Confocal microscopy
Protein Subunits
Confocal Microscopy
Cells
RNA
Membranes
Scanning
Kidney
Lasers
enhanced green fluorescent protein
Proteins

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)

Cite this

Blazer-Yost, B. L., Butterworth, M., Hartman, A. D., Parker, G. E., Faletti, C. J., Els, W. J., & Rhodes, S. J. (2001). Characterization and imaging of A6 epithelial cell clones expressing fluorescently labeled ENaC subunits. American Journal of Physiology - Cell Physiology, 281(2 50-2), C624-C632.

Characterization and imaging of A6 epithelial cell clones expressing fluorescently labeled ENaC subunits. / Blazer-Yost, Bonnie L.; Butterworth, Michael; Hartman, Amy D.; Parker, Gretchen E.; Faletti, Carla J.; Els, Willem J.; Rhodes, Simon J.

In: American Journal of Physiology - Cell Physiology, Vol. 281, No. 2 50-2, 27.08.2001, p. C624-C632.

Research output: Contribution to journalArticle

Blazer-Yost, BL, Butterworth, M, Hartman, AD, Parker, GE, Faletti, CJ, Els, WJ & Rhodes, SJ 2001, 'Characterization and imaging of A6 epithelial cell clones expressing fluorescently labeled ENaC subunits', American Journal of Physiology - Cell Physiology, vol. 281, no. 2 50-2, pp. C624-C632.
Blazer-Yost BL, Butterworth M, Hartman AD, Parker GE, Faletti CJ, Els WJ et al. Characterization and imaging of A6 epithelial cell clones expressing fluorescently labeled ENaC subunits. American Journal of Physiology - Cell Physiology. 2001 Aug 27;281(2 50-2):C624-C632.
Blazer-Yost, Bonnie L. ; Butterworth, Michael ; Hartman, Amy D. ; Parker, Gretchen E. ; Faletti, Carla J. ; Els, Willem J. ; Rhodes, Simon J. / Characterization and imaging of A6 epithelial cell clones expressing fluorescently labeled ENaC subunits. In: American Journal of Physiology - Cell Physiology. 2001 ; Vol. 281, No. 2 50-2. pp. C624-C632.
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