Abstract
Calcium-activated chloride channels (CaCCs) are key players in transepithelial ion transport and fluid secretion, smooth muscle constriction, neuronal excitability, and cell proliferation. The CaCC regulator 1 (CLCA1) modulates the activity of the CaCC TMEM16A/Anoctamin 1 (ANO1) by directly engaging the channel at the cell surface, but the exact mechanism is unknown. Here we demonstrate that the von Willebrand factor type A (VWA) domain within the cleaved CLCA1 N-terminal fragment is necessary and sufficient for this interaction. TMEM16A protein levels on the cell surface were increased in HEK293T cells transfected with CLCA1 constructs containing the VWA domain, and TMEM16A-like currents were activated. Similar currents were evoked in cells exposed to secreted VWA domain alone, and these currents were significantly knocked down by TMEM16A siRNA. VWA-dependent TMEM16A modulation was not modified by the S357N mutation, a VWA domain polymorphism associated with more severe meconium ileus in cystic fibrosis patients. VWA-activated currents were significantly reduced in the absence of extracellular Mg2+, and mutation of residues within the conserved metal ion-dependent adhesion site motif impaired the ability of VWA to potentiate TMEM16A activity, suggesting that CLCA1-TMEM16A interactions are Mg2+- and metal ion-dependent adhesion site-dependent. Increase in TMEM16A activity occurred within minutes of exposure to CLCA1 or after a short treatment with nocodazole, consistent with the hypothesis that CLCA1 stabilizes TMEM16A at the cell surface by preventing its internalization. Our study hints at the therapeutic potential of the selective activation of TMEM16A by the CLCA1 VWA domain in loss-of-function chloride channelopathies such as cystic fibrosis.
Highlights
Calcium-activated chloride channels (CaCCs) are key players in transepithelial ion transport and fluid secretion, smooth muscle constriction, neuronal excitability, and cell proliferation
Modestly outward-rectifying currents were activated in cells expressing full-length (CLCA1) or N-CLCA1 (Fig. 1, C and D), and we have demonstrated that these currents are carried by TMEM16A [13]
In cells transfected with constructs containing the von Willebrand factor type A (VWA) domain, currents with the same biophysical characteristics and comparable density as those evoked in CLCA1- or N-CLCA1-transfected cells were activated (Fig. 1, C and D), and this was not affected by the absence of the  sheet-rich domain in the CAT ϩ VWA construct, suggesting that this domain is not required for CLCA1-dependent TMEM16A regulation
Summary
CaCC, calcium-activated chloride channel; CF, cystic fibrosis; CLCA, calcium-activated chloride channel regulator; ICaCC, calcium-dependent chloride current; VWA, von Willebrand factor type A; MIDAS, metal ion-dependent adhesion site; CaV, voltage-gated Ca2ϩ channel; GPI, glycosylphosphatidylinositol; FnIII, fibronectin type III; CFTR, cystic fibrosis transmembrane conductance regulator; WGA, wheat germ agglutinin; pF, picofarad; ANOVA, analysis of variance; CAT, metalloprotease catalytic domain; F, F test statistic. Our findings provide novel insights into CLCA1 and TMEM16A as a cooperative pair and have direct implications for the targeting of CLCA1-TMEM16A interactions in airway disease
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
