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

Schematic representation of the effect of internal Ca2+ on KCa3.1 interactions and dynamics.Illustration of the increase in CFTR/KCa3.1 clustering in response to an internal Ca2+ rise. PM refers to plasma membrane. This scheme accounts for the decrease in KCa3.1 density in the presence of Ca2+ as most KCa3.1 channels form aggregates with CFTR.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0153665.g005: Schematic representation of the effect of internal Ca2+ on KCa3.1 interactions and dynamics.Illustration of the increase in CFTR/KCa3.1 clustering in response to an internal Ca2+ rise. PM refers to plasma membrane. This scheme accounts for the decrease in KCa3.1 density in the presence of Ca2+ as most KCa3.1 channels form aggregates with CFTR.

Mentions: Comparing the confocal images obtained in low and high Ca2+ also revealed a strong effect of internal Ca2+ on the CFTR/KCa3.1 interaction pattern. Fig 4A shows in this regard that while the molecular density of CFTR (number of molecules/μm2) remained constant before and after Ca2+ influx, KCa3.1 density was significantly reduced to < 25% of its original value after Ca2+ influx. To determine if this decrease was due to internalization of the K+ channels or their clustering with CFTR, the degree of aggregation (DA) was calculated for both proteins (Fig 4B). DA is proportional to the number of fluorescent subunits in each dynamic entity (as defined in the Materials and Methods section); i.e. the higher the DA, the larger the number of fluorescent proteins in the cluster and thus the higher the oligomerization state of the protein complex. While the degree of aggregation of CFTR was not altered by Ca2+ influx, a 4-fold increase was seen for the KCa3.1 (Fig 4B). This increase in the size of the KCa3.1 cluster is sufficient to explain the 4-fold decrease in the number density without internalization. We also calculated the interaction fractions of CFTR (Ra) and KCa3.1 (Rb) by comparing the amplitudes of the cross-correlation functions with those of the autocorrelation functions. It was concluded that about 20–25% of CFTR and KCa3.1 channels interacted in control low internal Ca2+ conditions (Fig 4C; n = 62), compared to 44±5% in high internal Ca2+ conditions (n = 21). This indicates that interaction between the two proteins is increased following a rise in intracellular Ca2+, with more of the K+ channels joining the pre-existing slow population of CFTR molecules. Finally, protein/protein interactions appeared to occur on a slow time scale (Fig 4D) and most interactions involved molecules that were immobilized on the plasma membrane (Fig 4E). The apparent lack of internalization on the time scale of these measurements was also evident from the constant average fluorescence intensity for both proteins at the cell surface before and after Ca2+ influx (data not shown). These data are summarized in the schematic model presented in Fig 5, where an increase in KCa3.1 clustering involving CFTR is seen as a result of a rise in internal Ca2+.


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)

Schematic representation of the effect of internal Ca2+ on KCa3.1 interactions and dynamics.Illustration of the increase in CFTR/KCa3.1 clustering in response to an internal Ca2+ rise. PM refers to plasma membrane. This scheme accounts for the decrease in KCa3.1 density in the presence of Ca2+ as most KCa3.1 channels form aggregates with CFTR.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0153665.g005: Schematic representation of the effect of internal Ca2+ on KCa3.1 interactions and dynamics.Illustration of the increase in CFTR/KCa3.1 clustering in response to an internal Ca2+ rise. PM refers to plasma membrane. This scheme accounts for the decrease in KCa3.1 density in the presence of Ca2+ as most KCa3.1 channels form aggregates with CFTR.
Mentions: Comparing the confocal images obtained in low and high Ca2+ also revealed a strong effect of internal Ca2+ on the CFTR/KCa3.1 interaction pattern. Fig 4A shows in this regard that while the molecular density of CFTR (number of molecules/μm2) remained constant before and after Ca2+ influx, KCa3.1 density was significantly reduced to < 25% of its original value after Ca2+ influx. To determine if this decrease was due to internalization of the K+ channels or their clustering with CFTR, the degree of aggregation (DA) was calculated for both proteins (Fig 4B). DA is proportional to the number of fluorescent subunits in each dynamic entity (as defined in the Materials and Methods section); i.e. the higher the DA, the larger the number of fluorescent proteins in the cluster and thus the higher the oligomerization state of the protein complex. While the degree of aggregation of CFTR was not altered by Ca2+ influx, a 4-fold increase was seen for the KCa3.1 (Fig 4B). This increase in the size of the KCa3.1 cluster is sufficient to explain the 4-fold decrease in the number density without internalization. We also calculated the interaction fractions of CFTR (Ra) and KCa3.1 (Rb) by comparing the amplitudes of the cross-correlation functions with those of the autocorrelation functions. It was concluded that about 20–25% of CFTR and KCa3.1 channels interacted in control low internal Ca2+ conditions (Fig 4C; n = 62), compared to 44±5% in high internal Ca2+ conditions (n = 21). This indicates that interaction between the two proteins is increased following a rise in intracellular Ca2+, with more of the K+ channels joining the pre-existing slow population of CFTR molecules. Finally, protein/protein interactions appeared to occur on a slow time scale (Fig 4D) and most interactions involved molecules that were immobilized on the plasma membrane (Fig 4E). The apparent lack of internalization on the time scale of these measurements was also evident from the constant average fluorescence intensity for both proteins at the cell surface before and after Ca2+ influx (data not shown). These data are summarized in the schematic model presented in Fig 5, where an increase in KCa3.1 clustering involving CFTR is seen as a result of a rise in internal Ca2+.

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