Limits...
Investigating CFTR and KCa3.1 Protein/Protein Interactions.

Klein H, Abu-Arish A, Trinh NT, Luo Y, Wiseman PW, Hanrahan JW, Brochiero E, Sauvé R - PLoS ONE (2016)

Bottom Line: 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.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.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+.

View Article: PubMed Central - PubMed

Affiliation: Département de Physiologie moléculaire et intégrative and Membrane Protein Research Group, Université de Montréal, Montréal, QC, Canada, H3C 3J7.

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+.

No MeSH data available.


Related in: MedlinePlus

Co-immunoprecipitation of endogenous CFTR and KCa3.1 proteins extracted from CFBE airway cells.Immunoblots showing CFTR and KCa3.1 proteins extracted from CFBE bronchial cells expressing wt-CFTR (A, B) and F508del-CFTR (C, D). Membranes were blotted with anti-CFTR (mAb 596 from CFFT, 1:1000, A, C) and anti-KCa3.1 (Alomone, 1:300, B, D) antibodies. Endogenous expression of CFTR and KCa3.1 proteins in the CFBE-wt and CFBE-ΔF508 cell lysates are presented in lane “Total Lysate”. Immunoprecipitation of endogenous CFTR using anti-CFTR antibody followed by co-immunoprecipitation of KCa3.1 is illustrated in lane IP CFTR (B, D), while immunoprecipitation of endogenous KCa3.1 (using anti-KCa3.1 antibody) followed by co-immunoprecitation of CFTR is shown in lane IP KCa3.1 (A, C). Note that the same lysate and IP samples were used in the upper and lower parts of the membranes, blotted with CFTR and KCa3.1 antibodies, respectively.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4836752&req=5

pone.0153665.g001: Co-immunoprecipitation of endogenous CFTR and KCa3.1 proteins extracted from CFBE airway cells.Immunoblots showing CFTR and KCa3.1 proteins extracted from CFBE bronchial cells expressing wt-CFTR (A, B) and F508del-CFTR (C, D). Membranes were blotted with anti-CFTR (mAb 596 from CFFT, 1:1000, A, C) and anti-KCa3.1 (Alomone, 1:300, B, D) antibodies. Endogenous expression of CFTR and KCa3.1 proteins in the CFBE-wt and CFBE-ΔF508 cell lysates are presented in lane “Total Lysate”. Immunoprecipitation of endogenous CFTR using anti-CFTR antibody followed by co-immunoprecipitation of KCa3.1 is illustrated in lane IP CFTR (B, D), while immunoprecipitation of endogenous KCa3.1 (using anti-KCa3.1 antibody) followed by co-immunoprecitation of CFTR is shown in lane IP KCa3.1 (A, C). Note that the same lysate and IP samples were used in the upper and lower parts of the membranes, blotted with CFTR and KCa3.1 antibodies, respectively.

Mentions: Protein/protein interactions between CFTR and KCa3.1 were next evaluated in co-immunoprecipitation experiments in airway epithelial CFBE cells expressing either the wt-CFTR (CFBE-wt) or the F508del-CFTR (CFBE-ΔF508) channels (Fig 1). We first verified that both mature band C and immature band B of wt-CFTR (Fig 1A, lane 1) were detected in CFBE-wt cells, whereas the immature band B of F508del-CFTR only (Fig 1C, lane 1) was present in CFBE-ΔF508 cell lysates, as expected. Immunoblot evidence for endogenous expression of KCa3.1 in both CFBE-wt and CFBE-ΔF508 cells are also presented in Fig 1B and 1D, respectively. More importantly, CFTR C and B bands could be detected by immunoblotting after immnoprecipitation of KCa3.1 (Fig 1A, lane 2), indicating that CFTR and KCa3.1 channels can form immunoprecipitable complexes in CFBE-wt cells. Interestingly, the immature band B of CFTR was also detected after KCa3.1 immunoprecipitation (Fig 1C, lane 2) from CFBE-ΔF508 extracts. Complementary experiments finally confirmed that it was possible to detect the KCa3.1 protein by immunoblot after CFTR protein immunoprecipitation from both CFBE-wt (Fig 1B, lane 2) and CFBE-ΔF508 (Fig 1D, lane 2) extracts, providing clear evidence of a possible physical interaction between CFTR and KCa3.1 in CFBE cells. Since the F508del- CFTR is found almost exclusively in the endoplasmic reticulum, its co-immunoprecipitation with KCa3.1 suggests the physical interaction can occur early during the biogenesis of both proteins.


Investigating CFTR and KCa3.1 Protein/Protein Interactions.

Klein H, Abu-Arish A, Trinh NT, Luo Y, Wiseman PW, Hanrahan JW, Brochiero E, Sauvé R - PLoS ONE (2016)

Co-immunoprecipitation of endogenous CFTR and KCa3.1 proteins extracted from CFBE airway cells.Immunoblots showing CFTR and KCa3.1 proteins extracted from CFBE bronchial cells expressing wt-CFTR (A, B) and F508del-CFTR (C, D). Membranes were blotted with anti-CFTR (mAb 596 from CFFT, 1:1000, A, C) and anti-KCa3.1 (Alomone, 1:300, B, D) antibodies. Endogenous expression of CFTR and KCa3.1 proteins in the CFBE-wt and CFBE-ΔF508 cell lysates are presented in lane “Total Lysate”. Immunoprecipitation of endogenous CFTR using anti-CFTR antibody followed by co-immunoprecipitation of KCa3.1 is illustrated in lane IP CFTR (B, D), while immunoprecipitation of endogenous KCa3.1 (using anti-KCa3.1 antibody) followed by co-immunoprecitation of CFTR is shown in lane IP KCa3.1 (A, C). Note that the same lysate and IP samples were used in the upper and lower parts of the membranes, blotted with CFTR and KCa3.1 antibodies, respectively.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4836752&req=5

pone.0153665.g001: Co-immunoprecipitation of endogenous CFTR and KCa3.1 proteins extracted from CFBE airway cells.Immunoblots showing CFTR and KCa3.1 proteins extracted from CFBE bronchial cells expressing wt-CFTR (A, B) and F508del-CFTR (C, D). Membranes were blotted with anti-CFTR (mAb 596 from CFFT, 1:1000, A, C) and anti-KCa3.1 (Alomone, 1:300, B, D) antibodies. Endogenous expression of CFTR and KCa3.1 proteins in the CFBE-wt and CFBE-ΔF508 cell lysates are presented in lane “Total Lysate”. Immunoprecipitation of endogenous CFTR using anti-CFTR antibody followed by co-immunoprecipitation of KCa3.1 is illustrated in lane IP CFTR (B, D), while immunoprecipitation of endogenous KCa3.1 (using anti-KCa3.1 antibody) followed by co-immunoprecitation of CFTR is shown in lane IP KCa3.1 (A, C). Note that the same lysate and IP samples were used in the upper and lower parts of the membranes, blotted with CFTR and KCa3.1 antibodies, respectively.
Mentions: Protein/protein interactions between CFTR and KCa3.1 were next evaluated in co-immunoprecipitation experiments in airway epithelial CFBE cells expressing either the wt-CFTR (CFBE-wt) or the F508del-CFTR (CFBE-ΔF508) channels (Fig 1). We first verified that both mature band C and immature band B of wt-CFTR (Fig 1A, lane 1) were detected in CFBE-wt cells, whereas the immature band B of F508del-CFTR only (Fig 1C, lane 1) was present in CFBE-ΔF508 cell lysates, as expected. Immunoblot evidence for endogenous expression of KCa3.1 in both CFBE-wt and CFBE-ΔF508 cells are also presented in Fig 1B and 1D, respectively. More importantly, CFTR C and B bands could be detected by immunoblotting after immnoprecipitation of KCa3.1 (Fig 1A, lane 2), indicating that CFTR and KCa3.1 channels can form immunoprecipitable complexes in CFBE-wt cells. Interestingly, the immature band B of CFTR was also detected after KCa3.1 immunoprecipitation (Fig 1C, lane 2) from CFBE-ΔF508 extracts. Complementary experiments finally confirmed that it was possible to detect the KCa3.1 protein by immunoblot after CFTR protein immunoprecipitation from both CFBE-wt (Fig 1B, lane 2) and CFBE-ΔF508 (Fig 1D, lane 2) extracts, providing clear evidence of a possible physical interaction between CFTR and KCa3.1 in CFBE cells. Since the F508del- CFTR is found almost exclusively in the endoplasmic reticulum, its co-immunoprecipitation with KCa3.1 suggests the physical interaction can occur early during the biogenesis of both proteins.

Bottom Line: 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.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.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+.

View Article: PubMed Central - PubMed

Affiliation: Département de Physiologie moléculaire et intégrative and Membrane Protein Research Group, Université de Montréal, Montréal, QC, Canada, H3C 3J7.

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+.

No MeSH data available.


Related in: MedlinePlus