Secreted Human CLCA1 Activates Calcium-Dependent Chloride Currents via Interaction with TMEM16A (Anoctamin 1)

Abstract

The calcium-activated chloride channel (CaCC) regulator (CLCA) proteins were so named because their expression leads to generation of calcium-dependent chloride currents (ICaCC) in mammalian cells; however, the molecular identity of the channel(s) mediating these currents, and the mechanisms through which CLCA1 regulates their activity, have remained unknown. In a previous study, we showed that human CLCA1 is a secreted self-cleaving zincin metalloprotease, and the resulting N-terminal fragment increases ICaCC density in HEK293T cells (J. Biol. Chem. 287, 42138-49, 2012). Here, we found that the same type of currents are activated in untransfected cells co-cultured with CLCA1-transfected cells or exposed to CLCA1-conditioned medium, indicating that secreted CLCA1 can activate ICaCC in a paracrine fashion. Because CLCA1 and the CaCC TMEM16A (Anoctamin 1) are expressed in the same tissues, such as the airway epithelia, and are upregulated in the same pathologic states, such as asthma and COPD, we tested the hypothesis that CLCA1-modulated currents are carried by TMEM16A. We observed that CLCA1-induced whole-cell ICaCC in HEK293T cells overexpressing the protein is blocked in the presence of TMEM16A-specific inhibitors T16Ainh-A01 and N-((4-methoxy)-2-naphthyl)-5-nitroanhtranilic acid, and silenced in the presence of TMEM16A siRNA. By means of live cell flow cytometry assays using a novel fluorescent CLCA probe, we demonstrate that CLCA1 and TMEM16A physically interact at the cell surface, and our real-time qPCR, immunohistochemistry and confocal microscopy data suggest that CLCA1 drives TMEM16A surface expression. These results identify CLCA1 as the first secreted direct mediator of TMEM16A activity, and suggest that CLCA1 and TMEM16A operate together to generate ICaCC in multiple tissues.