Anoctamin-1 (ANO1) is a Ca2+-activated chloride channel (CaCC) that plays important physiological roles in normal and cancerous tissues. However, the plasma membrane trafficking mechanisms of ANO1 remain poorly characterized. In yeast two-hybrid screening experiments, we observed direct interactions of ANO1 with beta-COP, which is a subunit of Coat Protein Complex I (COPI). This interaction was then confirmed using several in vitro and in vivo binding assays. Moreover, the cotransfection of beta-COP with ANO1 into HEK293T cells led to decreased the surface expression and the channel activity of ANO1. Accordingly, endogenous ANO1 was associated with beta-COP in U251 glioblastoma cells, and silencing of beta-COP enhanced surface expression and whole-cell currents of ANO1 in these cells. Taken together, these data suggest that beta-COP negatively regulates ANO1 surface expression. (C) 2016 Elsevier Inc. All rights reserved.