The cystic fibrosis transmembrane conductance regulator is an extracellular chloride sensor

Abstract

The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl− channel that governs the quantity and composition of epithelial secretions. CFTR function is normally tightly controlled as dysregulation can lead to life-threatening diseases such as secretory diarrhoea and cystic fibrosis. CFTR activity is regulated by phosphorylation of its cytosolic regulatory (R) domain, and ATP binding and hydrolysis at two nucleotide-binding domains (NBDs). Here, we report that CFTR activity is also controlled by extracellular Cl− concentration ([Cl−]o). Patch clamp current recordings show that a rise in [Cl−]o stimulates CFTR channel activity, an effect conferred by a single arginine residue, R899, in extracellular loop 4 of the protein. Using NBD mutants and ATP dose response studies in WT channels, we determined that [Cl−]o sensing was linked to changes in ATP binding energy at NBD1, which likely impacts NBD dimer stability. Biochemical measurements showed that increasing [Cl−]o decreased the intrinsic ATPase activity of CFTR mainly through a reduction in maximal ATP turnover. Our studies indicate that sensing [Cl−]o is a novel mechanism for regulating CFTR activity and suggest that the luminal ionic environment is an important physiological arbiter of CFTR function, which has significant implications for salt and fluid homeostasis in epithelial tissues.

The chloride anion as a signalling effector

“…Several channels and transporters regulate [Cl − ] i (Plans, Rickheit & Jentsch, 2009;Krall et al, 2015;Kunzelmann, 2015;Stauber, 2015;Oh & Jung, 2016), and conversely, changes in [Cl − ] i regulate their activity (Choi et al, 2001;Xie & Schafer, 2004;Plested, 2011;Yu et al, 2013;Broadbent et al, 2015).…”

“…By mutagenesis of the CFTR, they showed that alterations in residues R347 and R334 of the channel pore might be necessary for activation of ATP efflux in response to increased [Cl − ] e (Jiang et al, 1998). Subsequently, Broadbent et al (2015) found that increased [Cl − ] e stimulates CFTR activity, and similarly proposed that CFTR may act as an [Cl − ] e sensor (Broadbent et al, 2015). This characteristic was conferred by arginine residue R899, located in extracellular loop 4 of the CFTR, linking [Cl − ] e sensing to changes in the ATP binding energy at nucleotide binding domain 1 (NBD1) that affected dimerization with NBD2 and ATP turnover (Broadbent et al, 2015).…”

“…Subsequently, Broadbent et al (2015) found that increased [Cl − ] e stimulates CFTR activity, and similarly proposed that CFTR may act as an [Cl − ] e sensor (Broadbent et al, 2015). This characteristic was conferred by arginine residue R899, located in extracellular loop 4 of the CFTR, linking [Cl − ] e sensing to changes in the ATP binding energy at nucleotide binding domain 1 (NBD1) that affected dimerization with NBD2 and ATP turnover (Broadbent et al, 2015). By contrast, Shcheynikov et al (2015) described the molecular mechanism of Cl − modulation over several electrogenic Na + /HCO 3 − transporters as involving a GxxxP motif in Cl − sensing.…”

“…It also affects the function of endosomes (Miller Jr. et al, 2007;Matsuda et al, 2010), phagosomes (Painter et al, 2010;Riazanski et al, 2015), endoplasmic reticulum (ER) (Barro- Soria et al, 2010), and mitochondria (Tomaskova & Ondrias, 2010;Nunes et al, 2015). It might have even more specific roles, perhaps as a second messenger (Orlov & Hamet, 2006), affecting the activity of channels (Bekar & Walz, 1999;Bachhuber et al, 2005;Succol et al, 2012;Broadbent et al, 2015;Sirianant et al, 2016), and enzymes (Piala et al, 2014;Bazua-Valenti et al, 2015;Huang & Cheng, 2015;Terker et al, 2016). It is also involved in the regulation of specific genes (Sanders, Venema & Kok, 1997;Roessler & Muller, 2002;Miyazaki et al, 2008;Valdivieso et al, 2016;Clauzure et al, 2017).…”

Ca2+ signals mediated by bradykinin type 2 receptors in normal pancreatic stellate cells can be inhibited by specific Ca2+ channel blockade

“…The acinar Ca 2+ signals also regulate acinar fluid secretion via Ca 2+ -activated Cl − and K + channels (Petersen, 1992;Park et al 2001). However, the major component of pancreatic fluid secretion is contributed by the ducts and this is regulated by secretin-elicited intra-ductal cyclic AMP formation controlling cystic fibrosis transmembrane conductance regulator (CFTR) channels (Argent, 2006), whose openings are also regulated by the Cl − concentration in the luminal fluid (Broadbent et al 2015).…”