Journal
AMERICAN JOURNAL OF PHYSIOLOGY-RENAL PHYSIOLOGY
Volume 306, Issue 3, Pages F309-F320Publisher
AMER PHYSIOLOGICAL SOC
DOI: 10.1152/ajprenal.00519.2013
Keywords
protein kinase C alpha; ENaC; renal tubules; single channels; knockout mice; hypertension
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Funding
- National Institutes of Health [R37 DK037963, R01 DK89828, R01-DK067110, T32 DK07656, AHA 13POST16820072]
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The epithelial Na channel (ENaC) is negatively regulated by protein kinase C (PKC) as shown using PKC activators in a cell culture model. To determine whether PKC alpha influences ENaC activity in vivo, we examined the regulation of ENaC in renal tubules from PKC alpha(-/-) mice. Cortical collecting ducts were dissected and split open, and the exposed principal cells were subjected to cell-attached patch clamp. In the absence of PKC alpha, the open probability (P-o) of ENaC was increased three-fold vs. wild-type SV129 mice (0.52 +/- 0.04 vs. 0.17 +/- 0.02). The number of channels per patch was also increased. Using confocal microscopy, we observed an increase in membrane localization of alpha-, beta-, and gamma-subunits of ENaC in principal cells in the cortical collecting ducts of PKC alpha(-/-) mice compared with wild-type mice. To confirm this increase, one kidney from each animal was perfused with biotin, and membrane protein was pulled down with streptavidin. The nonbiotinylated kidney was used to assess total protein. While total ENaC protein did not change in PKC alpha(-/-) mice, membrane localization of all the ENaC subunits was increased. The increase in membrane ENaC could be explained by the observation that ERK1/2 phosphorylation was decreased in the knockout mice. These results imply a reduction in ENaC membrane accumulation and Po by PKC alpha in vivo. The PKC-mediated increase in ENaC activity was associated with an increase in blood pressure in knockout mice fed a high-salt diet.
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