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

作者：Lerias Joana R; Pinto Madalena C; Botelho Hugo M; Awatade Nikhil T; Quaresma Margarida C; Silva Iris A L; Wanitchakool Podchanart; Schreiber Rainer; Pepperkok Rainer; Kunzelmann Karl; Amaral Margarida D^*

来源：Biochimica et Biophysica Acta-Molecular Cell Research, 2018, 1865(2): 421-431.

DOI：10.1016/j.bbamcr.2017.11.009

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

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.

• 出版日期2018-2