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Using Phage Display to Develop Ubiquitin Variant Modulators for E3 Ligases
Published on: August 27, 2021
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Sculpting ion channel functional expression with engineered ubiquitin ligases
Scott A Kanner1, Travis Morgenstern2, Henry M Colecraft1,2,3
1Doctoral Program in Neurobiology and Behavior, Columbia University College of Physicians and Surgeons, New York, United States.
Elife
|December 20, 2017
Summary
Engineered E3 ligases precisely control ion channel ubiquitination, reducing surface KCNQ1 channels and K+ currents. This method offers a novel way to study and functionally silence ion channels.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Ion channel function relies on trafficking, sorting, and degradation.
- Dysregulation of these processes causes ion channelopathies like cardiac arrhythmias.
- Ubiquitination regulates ion channel lifecycle, but its mechanisms are complex.
Purpose of the Study:
- To investigate the role of E3 ligase CHIP in KCNQ1 channel ubiquitination and surface expression.
- To develop a method for selective manipulation of ion channel ubiquitination.
- To elucidate the impact of ubiquitination on KCNQ1 channel trafficking and function.
Main Methods:
- Targeting the E3 ligase CHIP to YFP-tagged KCNQ1 ± KCNE1 subunits using a GFP-nanobody.
- Utilizing engineered CHIP in heterologous cells and adult rat cardiomyocytes.
- Employing a chemo-genetic approach for chemical control of ubiquitination.
Main Results:
- Engineered CHIP enhanced KCNQ1 ubiquitination and reduced surface KCNQ1 density.
- KCNQ1 surface-density declined with a ~3.5 hr t1/2 due to impaired forward trafficking.
- Reconstituted K+ currents were abolished without affecting overall protein expression.
Conclusions:
- Engineered E3 ligases are useful tools to study ubiquitin regulation of membrane proteins.
- This approach enables effective post-translational functional knockdown of ion channels.
- Targeting CHIP provides mechanistic insights into KCNQ1 channel regulation and potential therapeutic strategies for channelopathies.
