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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

The temporal autocorrelations and the cross-correlation functions of EGFP-CFTR and KCa3.1-dsRed.A: Examples of confocal microscopy images obtained after excitation of EGFP-CFTR and KCa3.1-dsRed at 488 nm and 561 nm respectively, in low Ca2+ conditions. Merged figures support colocalization of EGFP-CFTR and KCa3.1-dsRed at the membrane (orange). B: Examples of confocal microscopy images obtained for EGFP-CFTR and KCa3.1-dsRed following CPA pretreatment in zero Ca2+ and addition of external Ca2+ to initiate Ca2+ influx. Merged figures (orange) showed the formation of larger KCa3.1 clusters after internal Ca2+ increase. C: Evidence for CFTR/KCa3.1 interactions provided by cross-correlation measurements (see Eq 1b in Materials and Methods section). CFTR-KCa3.1 interactions on the plasma membrane yielded a non-zero cross-correlation function (black) at the slow time scales of the correlation function. This strongly suggests that only the slow populations of CFTR and KCa3.1 interact.
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pone.0153665.g003: The temporal autocorrelations and the cross-correlation functions of EGFP-CFTR and KCa3.1-dsRed.A: Examples of confocal microscopy images obtained after excitation of EGFP-CFTR and KCa3.1-dsRed at 488 nm and 561 nm respectively, in low Ca2+ conditions. Merged figures support colocalization of EGFP-CFTR and KCa3.1-dsRed at the membrane (orange). B: Examples of confocal microscopy images obtained for EGFP-CFTR and KCa3.1-dsRed following CPA pretreatment in zero Ca2+ and addition of external Ca2+ to initiate Ca2+ influx. Merged figures (orange) showed the formation of larger KCa3.1 clusters after internal Ca2+ increase. C: Evidence for CFTR/KCa3.1 interactions provided by cross-correlation measurements (see Eq 1b in Materials and Methods section). CFTR-KCa3.1 interactions on the plasma membrane yielded a non-zero cross-correlation function (black) at the slow time scales of the correlation function. This strongly suggests that only the slow populations of CFTR and KCa3.1 interact.

Mentions: Examples of confocal microscopy images obtained after excitation of EGFP-CFTR and KCa3.1-dsRed in low (A) and high (B) internal Ca2+ conditions are presented in Fig 3. In both cases merged figures support colocalization of EGFP-CFTR and KCa3.1-dsRed at the membrane (orange). Additional examples are available within S1 Fig. To assess the average transport dynamics and density of a population of fluorescently labeled KCa3.1 and CFTR channels, fluorescence confocal microscopy image time series were used to calculate the temporal correlation function for CFTR, KCa3.1 and the combination of KCa3.1-CFTR. Fig 3C shows the average auto-correlation functions of CFTR (green) and KCa3.1 (red), and the average cross-correlation function of the interacting population CFTR-KCa3.1 (black) in the plasma membrane of unpolarized, live CFBE cells under resting conditions (control, Ctr). A non-zero cross-correlation function indicates the presence of an interacting population of CFTR and KCa3.1 channels. Both autocorrelation functions exhibited two component decays indicating the presence of two populations with dynamics occurring on different time scales (referred to as fast and slow) for each protein. A model for two diffusing species was used to fit the autocorrelation functions and extract decay times (τd) and amplitudes (g(0,0,0)) and c of the fast, slow and immobile populations for each protein, as indicated in the Materials and Methods section. By contrast, the cross-correlation function exhibited one slow decay component indicating the presence of a single, slowly moving population of CFTR-KCa3.1 interacting in a common complex. This strongly suggests that only the slow populations of CFTR and KCa3.1 interact.


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)

The temporal autocorrelations and the cross-correlation functions of EGFP-CFTR and KCa3.1-dsRed.A: Examples of confocal microscopy images obtained after excitation of EGFP-CFTR and KCa3.1-dsRed at 488 nm and 561 nm respectively, in low Ca2+ conditions. Merged figures support colocalization of EGFP-CFTR and KCa3.1-dsRed at the membrane (orange). B: Examples of confocal microscopy images obtained for EGFP-CFTR and KCa3.1-dsRed following CPA pretreatment in zero Ca2+ and addition of external Ca2+ to initiate Ca2+ influx. Merged figures (orange) showed the formation of larger KCa3.1 clusters after internal Ca2+ increase. C: Evidence for CFTR/KCa3.1 interactions provided by cross-correlation measurements (see Eq 1b in Materials and Methods section). CFTR-KCa3.1 interactions on the plasma membrane yielded a non-zero cross-correlation function (black) at the slow time scales of the correlation function. This strongly suggests that only the slow populations of CFTR and KCa3.1 interact.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0153665.g003: The temporal autocorrelations and the cross-correlation functions of EGFP-CFTR and KCa3.1-dsRed.A: Examples of confocal microscopy images obtained after excitation of EGFP-CFTR and KCa3.1-dsRed at 488 nm and 561 nm respectively, in low Ca2+ conditions. Merged figures support colocalization of EGFP-CFTR and KCa3.1-dsRed at the membrane (orange). B: Examples of confocal microscopy images obtained for EGFP-CFTR and KCa3.1-dsRed following CPA pretreatment in zero Ca2+ and addition of external Ca2+ to initiate Ca2+ influx. Merged figures (orange) showed the formation of larger KCa3.1 clusters after internal Ca2+ increase. C: Evidence for CFTR/KCa3.1 interactions provided by cross-correlation measurements (see Eq 1b in Materials and Methods section). CFTR-KCa3.1 interactions on the plasma membrane yielded a non-zero cross-correlation function (black) at the slow time scales of the correlation function. This strongly suggests that only the slow populations of CFTR and KCa3.1 interact.
Mentions: Examples of confocal microscopy images obtained after excitation of EGFP-CFTR and KCa3.1-dsRed in low (A) and high (B) internal Ca2+ conditions are presented in Fig 3. In both cases merged figures support colocalization of EGFP-CFTR and KCa3.1-dsRed at the membrane (orange). Additional examples are available within S1 Fig. To assess the average transport dynamics and density of a population of fluorescently labeled KCa3.1 and CFTR channels, fluorescence confocal microscopy image time series were used to calculate the temporal correlation function for CFTR, KCa3.1 and the combination of KCa3.1-CFTR. Fig 3C shows the average auto-correlation functions of CFTR (green) and KCa3.1 (red), and the average cross-correlation function of the interacting population CFTR-KCa3.1 (black) in the plasma membrane of unpolarized, live CFBE cells under resting conditions (control, Ctr). A non-zero cross-correlation function indicates the presence of an interacting population of CFTR and KCa3.1 channels. Both autocorrelation functions exhibited two component decays indicating the presence of two populations with dynamics occurring on different time scales (referred to as fast and slow) for each protein. A model for two diffusing species was used to fit the autocorrelation functions and extract decay times (τd) and amplitudes (g(0,0,0)) and c of the fast, slow and immobile populations for each protein, as indicated in the Materials and Methods section. By contrast, the cross-correlation function exhibited one slow decay component indicating the presence of a single, slowly moving population of CFTR-KCa3.1 interacting in a common complex. This strongly suggests that only the slow populations of CFTR and KCa3.1 interact.

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