Sculpting ion channel functional expression with engineered ubiquitin ligases

Abstract
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Abstract

The functional repertoire of surface ion channels is sustained by dynamic processes of trafficking, sorting, and degradation. Dysregulation of these processes underlies diverse ion channelopathies including cardiac arrhythmias and cystic fibrosis. Ubiquitination powerfully regulates multiple steps in the channel lifecycle, yet basic mechanistic understanding is confounded by promiscuity among E3 ligase/substrate interactions and ubiquitin code complexity. Here we targeted the catalytic domain of E3 ligase, CHIP, to YFP-tagged KCNQ1±KCNE1 subunits with a GFP-nanobody to selectively manipulate this channel complex in heterologous cells and adult rat cardiomyocytes. Engineered CHIP enhanced KCNQ1 ubiquitination, eliminated KCNQ1 surface-density, and abolished reconstituted K⁺ currents without affecting protein expression. A chemo-genetic variation enabling chemical control of ubiquitination revealed KCNQ1 surface-density declined with a ~3.5-hr t_1/2 by impaired forward trafficking. The results illustrate utility of engineered E3 ligases to elucidate mechanisms underlying ubiquitin regulation of membrane proteins, and to achieve effective post-translational functional knockdown of ion channels.

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Scott A Kanner

Doctoral Program in Neurobiology and Behavior, Columbia University, New York, United States

Competing interests
The authors declare that no competing interests exist.
Travis Morgenstern

Department of Pharmacology, Columbia University, New York, United States

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0003-2634-8470
Henry M Colecraft

Doctoral Program in Neurobiology and Behavior, Columbia University, New York, United States

For correspondence
hc2405@cumc.columbia.edu

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0002-2340-8899

Funding

National Heart, Lung, and Blood Institute (RO1-HL121253)

Henry M Colecraft

National Heart, Lung, and Blood Institute (1RO1-HL122421)

Henry M Colecraft

National Institutes of Health (T32 GM007367)

Scott A Kanner

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Ethics

Animal experimentation: Primary cultures of adult rat heart ventricular cells were prepared as previously described (Colecraft et al., 2002; Subramanyam et al., 2013), in accordance with the guidelines of Columbia University Animal Care and Use Committee. All of the animals were handled according to approved institutional animal care and use committee (IACUC) protocols (# AC-AAAS2515).

Copyright

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.