A novel microscopy-based assay identifies extended synaptotagmin-1 (ESYT1) as a positive regulator of anoctamin 1 traffic

https://doi.org/10.1016/j.bbamcr.2017.11.009Get rights and content
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Highlights

  • A novel robust, cell-based assay to study Anoctamin 1 (ANO1) traffic is described.

  • The assay identified siRNAs acting both as ANO1 traffic enhancer and inhibitor.

  • ESYT1-Extended synaptotagmin-1 siRNA decreases ANO1 traffic and channel function.

  • ESYT1 may enhance ANO1 traffic by its role in coupling ER to the plasma membrane.

  • This tool allows further studies and drug discovery of disease-related Anoctamin 1.

Abstract

An attractive possibility to treat Cystic Fibrosis (CF), a severe condition caused by dysfunctional CFTR, an epithelial anion channel, is through the activation of alternative (non-CFTR) anion channels. Anoctamin 1 (ANO1) was demonstrated to be a Ca2+-activated chloride channel (CaCC) and thus of high potential to replace CFTR. Despite that ANO1 is expressed in human lung CF tissue, it is present at the cell surface at very low levels. In addition, little is known about regulation of ANO1 traffic, namely which factors promote its plasma membrane (PM) localization.

Here, we generated a novel cellular model, expressing an inducible 3HA-ANO1-eGFP construct, and validated its usage as a microscopy tool to monitor for ANO1 traffic.

We demonstrate the robustness and specificity of this cell-based assay, by the identification of siRNAs acting both as ANO1 traffic enhancer and inhibitor, targeting respectively COPB1 and ESYT1 (extended synaptotagmin-1), the latter involved in coupling of the endoplasmic reticulum to the PM at specific microdomains. We further show that knockdown of ESYT1 (and family members ESYT2 and ESYT3) significantly decreased ANO1 current density.

This ANO1 cell-based assay constitutes an important tool to be further used in high-throughput screens and drug discovery of high relevance for CF and cancer.

Abbreviations

ANO
Anoctamin
BSA
bovine serum albumin
CaCC
calcium (Ca2+)-activated Cl- channel
CF
cystic fibrosis
CFBE
cystic fibrosis bronchial epithelial (cells)
CFTR
cystic fibrosis transmembrane conductance regulator
Dox
doxycycline
ER
endoplasmic reticulum
EGFR
epidermal growth factor receptor
ERQC
ER quality control
ESYT1
extended synaptotagmin-1
HA
hemagglutinin
HTS
high throughput screening
ISC
short circuit current
PBS
phosphate buffered saline
PFA
paraformaldehyde
PM
plasma membrane
TEER
transepithelial resistance
Vte
transepithelial voltage
WB
Western blot

Keywords

Intracellular traffic
Endoplasmic reticulum
Calcium-activated chloride channels
Cystic fibrosis
Automated fluorescence microscopy

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