Abstract
In epithelia, Cl- channels play a prominent role in fluid and electrolyte transport. Of particular importance is the cAMP-dependent cystic fibrosis transmembrane conductance regulator Cl- channel (CFTR) with mutations of the CFTR encoding gene causing cystic fibrosis. The bulk transepithelial transport of Cl- ions and electrolytes needs however to be coupled to an increase in K+ conductance in order to recycle K+ and maintain an electrical driving force for anion exit across the apical membrane. In several epithelia, this K+ efflux is ensured by K+ channels, including KCa3.1, which is expressed at both the apical and basolateral membranes. We show here for the first time that CFTR and KCa3.1 can physically interact. We first performed a two-hybrid screen to identify which KCa3.1 cytosolic domains might mediate an interaction with CFTR. Our results showed that both the N-terminal fragment M1-M40 of KCa3.1 and part of the KCa3.1 calmodulin binding domain (residues L345-A400) interact with the NBD2 segment (G1237-Y1420) and C- region of CFTR (residues T1387-L1480), respectively. An association of CFTR and F508del-CFTR with KCa3.1 was further confirmed in co-immunoprecipitation experiments demonstrating the formation of immunoprecipitable CFTR/KCa3.1 complexes in CFBE cells. Co-expression of KCa3.1 and CFTR in HEK cells did not impact CFTR expression at the cell surface, and KCa3.1 trafficking appeared independent of CFTR stimulation. Finally, evidence is presented through cross-correlation spectroscopy measurements that KCa3.1 and CFTR colocalize at the plasma membrane and that KCa3.1 channels tend to aggregate consequent to an enhanced interaction with CFTR channels at the plasma membrane following an increase in intracellular Ca2+ concentration. Altogether, these results suggest 1) that the physical interaction KCa3.1/CFTR can occur early during the biogenesis of both proteins and 2) that KCa3.1 and CFTR form a dynamic complex, the formation of which depends on internal Ca2+.
Highlights
The CFTR protein is a plasma membrane cAMP-regulated Cl− channel which mediates transepithelial salt and fluid transport in the airways, intestine, kidney, vas deferens, and sweat duct [1]
We provide the first evidence for a regulated, physical interaction between CFTR and the calcium activated potassium channel of intermediate conductance KCa3.1
This proposal is supported by a two-hybrid screening analysis which showed that the N- and C- regions of KCa3.1 interact with NBD2 and C-terminal regions of CFTR respectively, coupled to results from co-immunoprecipitation experiments indicating that wt-CFTR and F508del-CFTR form an immunoprecipitable complex with KCa3.1 in native non-cystic fibrosis (CF) and CF airway epithelial CFBE cells
Summary
The CFTR protein is a plasma membrane cAMP-regulated Cl− channel which mediates transepithelial salt and fluid transport in the airways, intestine, kidney, vas deferens, and sweat duct [1]. Defective CFTR activity causes cystic fibrosis (CF), a disease characterized by an impaired Cl- secretion and unbalanced Na+∕Cl-transport that leads to airway surface liquid (ASL) volume depletion and defective mucociliary clearance [2,3,4]. Besides CFTR, other Cl- channels have been identified at the apical membrane of many epithelial cells. Studies using TMEM16A-/- mice have confirmed that the loss of TMEM16A strongly affects Ca2+ -dependent Cl- transport in airways, colonic epithelia, acinar cells from pancreas and submandibular glands [5;6]. The bulk transepithelial transport of Cl- ions needs to be coupled to an increase in
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