Home › Biochemistry › Demonstration of Proteolytic Activation of the Epithelial Sodium Channel (ENaC) by Combining Current Measurements with Detection of Cleavage Fragments
Biochemistry JoVE (Open Access) Citable · DOI

Demonstration of Proteolytic Activation of the Epithelial Sodium Channel (ENaC) by Combining Current Measurements with Detection of Cleavage Fragments

DOI: 10.3791/51582-v
What you'll learn
  • ✓ Perform two-electrode voltage-clamp recordings on Xenopus oocytes expressing ENaC
  • ✓ Measure amiloride-sensitive sodium currents to assess channel function
  • ✓ Detect proteolytic cleavage fragments using biotinylation and Western blot
  • ✓ Identify functionally important cleavage sites via site-directed mutagenesis
Protocol

Proteolytic activation of the epithelial sodium channel (ENaC) heterologously expressed in Xenopus laevis oocytes can be demonstrated by combining current measurements with a biotinylation approach to investigate the appearance of ion channel cleavage products at the cell surface. Functionally important cleavage sites can be identified by using site-directed mutagenesis.

Difficulty
advanced
Total time
~3-4 days (oocyte preparation 1 day, voltage-clamp experiments 1-2 days, biotinylation and Western blot 1 day)
Model organism
Xenopus laevis (oocytes)
Biosafety
BSL-1

Steps

1
Perform two-electrode voltage-clamp experiments

Set up and conduct two-electrode voltage-clamp recordings on Xenopus laevis oocytes expressing heterologous ENaC to measure ion channel activity and establish baseline electrical properties.

▶ 01:11
2
Measure amiloride-sensitive whole-cell currents

Record amiloride-sensitive sodium currents from ENaC-expressing oocytes to quantify channel function and identify proteolytic activation effects on conductance.

▶ 02:41
3
Perform biotinylation assay and detect cleavage fragments

Apply surface biotinylation to oocytes, then use Western blot analysis to identify and quantify ENaC cleavage products at the cell surface, correlating fragments with channel activation.

▶ 04:17
4
Analyze proteolytic activation and results

Interpret combined current and cleavage fragment data to demonstrate proteolytic activation mechanisms and validate functionally important cleavage sites identified by mutagenesis.

▶ 07:08
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