Secreted Human CLCA1 Activates Calcium-Dependent Chloride Currents through Direct Binding of Its VWA Domain with an Extracellular Loop of 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. Previously, we showed that 1) human CLCA1 is a secreted self-cleaving zincin metalloprotease, 2) the resulting N-terminal fragment increases ICaCC density in HEK293T cells in a paracrine fashion, 3) these ICaCC are carried by TMEM16A/Anoctamin 1, and 4) CLCA1 physically interacts with, and drives the surface expression of, TMEM16A (J. Biol. Chem. 2012;287:42138, and eLife 2015;4:e05875). Here, we demonstrate that a Von Willebrand factor type A (VWA) domain within the CLCA1 N-terminus is the minimum requirement for activation of TMEM16A-mediated currents. Thus, ICaCC with the biophysical hallmarks of TMEM16A were measured in HEK293T cells transfected with N-terminal CLCA1 (N-CLCA1) protein constructs containing the VWA domain, and in the same cells the surface expression of TMEM16A was notably increased. Those effects were not observed in mock-transfected cells, or in cells transfected with VWA-less N-CLCA1 constructs. Our live-cell flow cytometry binding assays, confocal microscopy data and whole-cell patch clamp recordings indicate that there is a rapid, direct interaction between the VWA domain of CLCA1 and the last extracellular loop of TMEM16A, and that the conserved metal-ion-dependent adhesion site (MIDAS) motif within the VWA domain, usually responsible for stabilizing protein-protein interactions, is not involved in CLCA1-TMEM16A interactions. CLCA1 is the first secreted direct mediator of TMEM16A activity, and our studies suggest that CLCA1 and TMEM16A operate together to generate ICaCC in multiple tissues.