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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

CFTR and KCa3.1 expression in cell lysates and streptavidin pulldowns after cell-surface biotinylation.T-Rex HEK cells expressing WT-CFTR were transfected with HA-tagged KCa3.1 channels and CFTR expression induced by tetracycline (Tet). Lanes 1 and 4 show CFTR, Na+/K+-ATPase and KCa3.1-HA proteins in the lysate and pulldown (PD) after biotinylation. As controls, lanes 2 and 5 show Na+/K+-ATPAse and KCa3.1-HA proteins in the lysate and pulldown after biotinylation in absence of forskolin stimulation, and lanes 3 and 6 show CFTR in the lysate and pulldown after biotinylation without forskolin stimulation and KCa3.1 transfection. The molecular mass in kDa is indicated.
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pone.0153665.g002: CFTR and KCa3.1 expression in cell lysates and streptavidin pulldowns after cell-surface biotinylation.T-Rex HEK cells expressing WT-CFTR were transfected with HA-tagged KCa3.1 channels and CFTR expression induced by tetracycline (Tet). Lanes 1 and 4 show CFTR, Na+/K+-ATPase and KCa3.1-HA proteins in the lysate and pulldown (PD) after biotinylation. As controls, lanes 2 and 5 show Na+/K+-ATPAse and KCa3.1-HA proteins in the lysate and pulldown after biotinylation in absence of forskolin stimulation, and lanes 3 and 6 show CFTR in the lysate and pulldown after biotinylation without forskolin stimulation and KCa3.1 transfection. The molecular mass in kDa is indicated.

Mentions: Trafficking of CFTR to the plasma membrane of BHK cells has already been investigated using biotinylation and streptavidin pulldown [29]. Because KCa3.1 was found to interact with the CFTR immature form, an identical approach was used to determine if KCa3.1 expression can alter CFTR surface expression. Fig 2 shows a Western blot of T-Rex HEK CFTR-wt cells and streptavidin pulldowns from cells transfected (lanes 1, 2, 4, 5) or not (lanes 3, 6) with KCa3.1-HA tagged channel. In these experiments, CFTR expression was induced by incubating T-Rex HEK CFTR cells in tetracycline during 24h and CFTR was stimulated with 10 μM forskolin (lanes 1 and 4) or not (lanes 2, 3, 5 and 6) before biotinylation and pulldown. CFTR immunoblotting gave a strong and diffuse band of approx. 180kDa as expected after complex glycosylation (band C). This glycoform was enriched by surface biotinylation and pulldown on streptavidin beads (lanes 4, 5, 6). Our results indicated that the surface expression of CFTR did not change whether T-Rex HEK CFTR cells had been cotransfected with HA-KCa3.1 or not. In these experiments, T-Rex HEK CFTR cells non-transfected with KCa3.1 (lanes 3, 6) were used as negative control. Our results also showed that the surface expression of KCa3.1 slightly changed in forskolin CFTR-stimulated cells (lane 4) compared to non-stimulated cells (lane 5). To confirm the effectiveness of our biotinylation and pulldown procedure, we also probed blots for the membrane protein Na+/K+-ATPase α-subunit, a membrane protein with N-linked glycosylation. The Na+/K+-ATPase α-subunit was readily detected in T-Rex HEK induced CFTR cells, and its electrophoretic mobility appeared slightly slower in pulldown samples, consistent with an enrichment with the mature form. Altogether, these results show that coexpression of CFTR and KCa3.1 did not significantly change the surface expression of CFTR and that the expression of KCa3.1was not altered by stimulating CFTR.


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)

CFTR and KCa3.1 expression in cell lysates and streptavidin pulldowns after cell-surface biotinylation.T-Rex HEK cells expressing WT-CFTR were transfected with HA-tagged KCa3.1 channels and CFTR expression induced by tetracycline (Tet). Lanes 1 and 4 show CFTR, Na+/K+-ATPase and KCa3.1-HA proteins in the lysate and pulldown (PD) after biotinylation. As controls, lanes 2 and 5 show Na+/K+-ATPAse and KCa3.1-HA proteins in the lysate and pulldown after biotinylation in absence of forskolin stimulation, and lanes 3 and 6 show CFTR in the lysate and pulldown after biotinylation without forskolin stimulation and KCa3.1 transfection. The molecular mass in kDa is indicated.
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Related In: Results  -  Collection

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

pone.0153665.g002: CFTR and KCa3.1 expression in cell lysates and streptavidin pulldowns after cell-surface biotinylation.T-Rex HEK cells expressing WT-CFTR were transfected with HA-tagged KCa3.1 channels and CFTR expression induced by tetracycline (Tet). Lanes 1 and 4 show CFTR, Na+/K+-ATPase and KCa3.1-HA proteins in the lysate and pulldown (PD) after biotinylation. As controls, lanes 2 and 5 show Na+/K+-ATPAse and KCa3.1-HA proteins in the lysate and pulldown after biotinylation in absence of forskolin stimulation, and lanes 3 and 6 show CFTR in the lysate and pulldown after biotinylation without forskolin stimulation and KCa3.1 transfection. The molecular mass in kDa is indicated.
Mentions: Trafficking of CFTR to the plasma membrane of BHK cells has already been investigated using biotinylation and streptavidin pulldown [29]. Because KCa3.1 was found to interact with the CFTR immature form, an identical approach was used to determine if KCa3.1 expression can alter CFTR surface expression. Fig 2 shows a Western blot of T-Rex HEK CFTR-wt cells and streptavidin pulldowns from cells transfected (lanes 1, 2, 4, 5) or not (lanes 3, 6) with KCa3.1-HA tagged channel. In these experiments, CFTR expression was induced by incubating T-Rex HEK CFTR cells in tetracycline during 24h and CFTR was stimulated with 10 μM forskolin (lanes 1 and 4) or not (lanes 2, 3, 5 and 6) before biotinylation and pulldown. CFTR immunoblotting gave a strong and diffuse band of approx. 180kDa as expected after complex glycosylation (band C). This glycoform was enriched by surface biotinylation and pulldown on streptavidin beads (lanes 4, 5, 6). Our results indicated that the surface expression of CFTR did not change whether T-Rex HEK CFTR cells had been cotransfected with HA-KCa3.1 or not. In these experiments, T-Rex HEK CFTR cells non-transfected with KCa3.1 (lanes 3, 6) were used as negative control. Our results also showed that the surface expression of KCa3.1 slightly changed in forskolin CFTR-stimulated cells (lane 4) compared to non-stimulated cells (lane 5). To confirm the effectiveness of our biotinylation and pulldown procedure, we also probed blots for the membrane protein Na+/K+-ATPase α-subunit, a membrane protein with N-linked glycosylation. The Na+/K+-ATPase α-subunit was readily detected in T-Rex HEK induced CFTR cells, and its electrophoretic mobility appeared slightly slower in pulldown samples, consistent with an enrichment with the mature form. Altogether, these results show that coexpression of CFTR and KCa3.1 did not significantly change the surface expression of CFTR and that the expression of KCa3.1was not altered by stimulating CFTR.

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