Investigating CFTR and KCa3.1 Protein/Protein Interactions.
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+.
Département de Physiologie moléculaire et intégrative and Membrane Protein Research Group, Université de Montréal, Montréal, QC, Canada, H3C 3J7.
Hélène
H
Klein
Department of Physiology, McGill University, Montréal, QC, Canada, H3G 1Y6.
Asmahan
A
Abu-Arish
Centre de recherche du Centre hospitalier de l'Université de Montréal (CRCHUM), Tour Viger, 900 rue Saint-Denis, Montréal, QC, Canada, H2X 0A9.|Department of Medicine, Université de Montréal, Montréal, Canada, H3C 3J7.
Nguyen Thu Ngan
NT
Trinh
Department of Physiology, McGill University, Montréal, QC, Canada, H3G 1Y6.
Yishan
Y
Luo
Chemistry & Physics, McGill University, Montreal, QC, Canada, H3G 1Y6.
Paul W
PW
Wiseman
Department of Physiology, McGill University, Montréal, QC, Canada, H3G 1Y6.
John W
JW
Hanrahan
Centre de recherche du Centre hospitalier de l'Université de Montréal (CRCHUM), Tour Viger, 900 rue Saint-Denis, Montréal, QC, Canada, H2X 0A9.|Department of Medicine, Université de Montréal, Montréal, Canada, H3C 3J7.
Emmanuelle
E
Brochiero
Département de Physiologie moléculaire et intégrative and Membrane Protein Research Group, Université de Montréal, Montréal, QC, Canada, H3C 3J7.
Rémy
R
Sauvé
D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D018118:Chloride Channels / Q000378:metabolism; D003550:Cystic Fibrosis / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000378:metabolism*; D004847:Epithelial Cells / Q000378:metabolism; D004848:Epithelium / Q000378:metabolism; D006801:Humans; D051660:Intermediate-Conductance Calcium-Activated Potassium Channels / Q000378:metabolism*; D017136:Ion Transport / Q000502:physiology; D009154:Mutation / Q000235:genetics; D011188:Potassium / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D060066:Protein Interaction Maps / Q000502:physiology*
C505032:CFTR protein, human; D018118:Chloride Channels; D051660:Intermediate-Conductance Calcium-Activated Potassium Channels; C496923:KCNN4 protein, human; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D011188:Potassium; D002118:Calcium