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SLC26A9 is a constitutively active, CFTR-regulated anion conductance in human bronchial epithelia.

Bertrand CA, Zhang R, Pilewski JM, Frizzell RA - J. Gen. Physiol. (2009)

Bottom Line: The identity of this conductance is unknown, but SLC26A9, a member of the SLC26 family of CF transmembrane conductance regulator (CFTR)-interacting transporters, is found in the human airway and exhibits chloride channel behavior.HEK cells coexpressing SLC26A9 with DeltaF508-CFTR also failed to exhibit SLC26A9 current.We conclude that SLC26A9 functions as an anion conductance in the apical membranes of HBE cells, it contributes to transepithelial chloride currents under basal and cAMP/protein kinase A-stimulated conditions, and its activity in HBE cells requires functional CFTR.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA. cbertra@pitt.edu

ABSTRACT
Human bronchial epithelial (HBE) cells exhibit constitutive anion secretion that is absent in cells from cystic fibrosis (CF) patients. The identity of this conductance is unknown, but SLC26A9, a member of the SLC26 family of CF transmembrane conductance regulator (CFTR)-interacting transporters, is found in the human airway and exhibits chloride channel behavior. We sought differences in the properties of SLC26A9 and CFTR expressed in HEK 293 (HEK) cells as a fingerprint to identify HBE apical anion conductances. HEK cells expressing SLC26A9 displayed a constitutive chloride current that was inhibited by the CFTR blocker GlyH-101 (71 +/- 4%, 50 microM) and exhibited a near-linear current-voltage (I-V) relation during block, while GlyH-101-inhibited wild-type (wt)CFTR exhibited a strong inward-rectified (IR) I-V relation. We tested polarized HBE cells endogenously expressing either wt or DeltaF508-CFTR for similar activity. After electrical isolation of the apical membrane using basolateral alpha-toxin permeabilization, wtCFTR monolayers displayed constitutive chloride currents that were inhibited by GlyH-101 (68 +/- 6%) while maintaining a near-linear I-V relation. In the absence of blocker, the addition of forskolin stimulated a current increase having a linear I-V; GlyH-101 blocked 69 +/- 7% of the current and shifted the I-V relation IR, consistent with CFTR activation. HEK cells coexpressing SLC26A9 and wtCFTR displayed similar properties, as well as forskolin-stimulated currents that exceeded the sum of those in cells separately expressing SLC26A9 or wtCFTR, and an I-V relation during GlyH-101 inhibition that was moderately IR, indicating that SLC26A9 contributed to the stimulated current. HBE cells from CF patients expressed SLC26A9 mRNA, but no constitutive chloride currents. HEK cells coexpressing SLC26A9 with DeltaF508-CFTR also failed to exhibit SLC26A9 current. We conclude that SLC26A9 functions as an anion conductance in the apical membranes of HBE cells, it contributes to transepithelial chloride currents under basal and cAMP/protein kinase A-stimulated conditions, and its activity in HBE cells requires functional CFTR.

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HEK 293 coexpression of SLC26A9 with wtCFTR increases forskolin-stimulated chloride currents over cells expressing CFTR alone. (A) Coexpressers (gray) consistently had larger currents than CFTR alone (black) as assessed at four measurement points: the basal currents before stimulation (60 s), the transient peak in response to 10 µM forskolin (150 s on average), the current plateau in the continued presence of forskolin (240 s), and the poststimulation current observed 3 min after forskolin washout (420 s). The poststimulation current in the coexpressers did not diminish during extended recordings (extra 8 min, this case). Representative tracings of at least three experiments each are shown. I-V measurements were performed at the indicated intervals (solid diamonds). (B) Cells expressing CFTR alone displayed the classical linear I-V in response to forskolin, with minimal current before stimulation and after forskolin washout. Curves are for black trace shown in A. (C) Cells expressing SLC26A9 + CFTR also displayed a linear I-V in response to forskolin; however, the magnitudes were larger and included the constitutive and enhanced poststimulation currents (note scale doubled from B). Curves are for gray trace shown in A. (D) Summary of the responses for SLC26A9 and CFTR expressed alone and together. Representative tracings are shown in A and Fig. 2 A. Fixed measurement points are identified in A. Peak values were measured at the actual peak of each tracing, with average peak times occurring at 150 ± 7 s for SLC26A9 + wtCFTR coexpressors and 158 ± 6 s for wtCFTR alone. The value for SLC26A9 alone was measured at 150 s. The SLC26A9 + wtCFTR coexpressor currents exceed the sum of the wtCFTR and SLC26A9 individual currents during and after forskolin stimulation (see Results). All current values were normalized to cell capacitance; VH = −40 mV. *, P < 0.05 compared with CFTR alone.
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fig6: HEK 293 coexpression of SLC26A9 with wtCFTR increases forskolin-stimulated chloride currents over cells expressing CFTR alone. (A) Coexpressers (gray) consistently had larger currents than CFTR alone (black) as assessed at four measurement points: the basal currents before stimulation (60 s), the transient peak in response to 10 µM forskolin (150 s on average), the current plateau in the continued presence of forskolin (240 s), and the poststimulation current observed 3 min after forskolin washout (420 s). The poststimulation current in the coexpressers did not diminish during extended recordings (extra 8 min, this case). Representative tracings of at least three experiments each are shown. I-V measurements were performed at the indicated intervals (solid diamonds). (B) Cells expressing CFTR alone displayed the classical linear I-V in response to forskolin, with minimal current before stimulation and after forskolin washout. Curves are for black trace shown in A. (C) Cells expressing SLC26A9 + CFTR also displayed a linear I-V in response to forskolin; however, the magnitudes were larger and included the constitutive and enhanced poststimulation currents (note scale doubled from B). Curves are for gray trace shown in A. (D) Summary of the responses for SLC26A9 and CFTR expressed alone and together. Representative tracings are shown in A and Fig. 2 A. Fixed measurement points are identified in A. Peak values were measured at the actual peak of each tracing, with average peak times occurring at 150 ± 7 s for SLC26A9 + wtCFTR coexpressors and 158 ± 6 s for wtCFTR alone. The value for SLC26A9 alone was measured at 150 s. The SLC26A9 + wtCFTR coexpressor currents exceed the sum of the wtCFTR and SLC26A9 individual currents during and after forskolin stimulation (see Results). All current values were normalized to cell capacitance; VH = −40 mV. *, P < 0.05 compared with CFTR alone.

Mentions: As shown in Fig. 6 A, cells expressing CFTR + SLC26A9 or CFTR alone responded to forskolin within 30 s of addition and reached peak currents in 90 ± 7 s (n = 11) or 98 ± 6 s (n = 21), respectively. However, coexpression of wtCFTR with SLC26A9 significantly modified the typical response of CFTR to stimulation. Although the essential shape of the response was similar, cells coexpressing SLC26A9 and wtCFTR exhibited larger currents that did not return to baseline after washout of forskolin. This poststimulation current was not due to a delay in CFTR recovery; as shown in Fig. 6 A, it persisted for 8 min after CFTR alone had returned to its prestimulation baseline. Coexpressors maintained linear I-V characteristics before, during, and after stimulation, and cells expressing CFTR alone demonstrated linear I-V relations during and also after forskolin stimulation (Fig. 6, B and C).


SLC26A9 is a constitutively active, CFTR-regulated anion conductance in human bronchial epithelia.

Bertrand CA, Zhang R, Pilewski JM, Frizzell RA - J. Gen. Physiol. (2009)

HEK 293 coexpression of SLC26A9 with wtCFTR increases forskolin-stimulated chloride currents over cells expressing CFTR alone. (A) Coexpressers (gray) consistently had larger currents than CFTR alone (black) as assessed at four measurement points: the basal currents before stimulation (60 s), the transient peak in response to 10 µM forskolin (150 s on average), the current plateau in the continued presence of forskolin (240 s), and the poststimulation current observed 3 min after forskolin washout (420 s). The poststimulation current in the coexpressers did not diminish during extended recordings (extra 8 min, this case). Representative tracings of at least three experiments each are shown. I-V measurements were performed at the indicated intervals (solid diamonds). (B) Cells expressing CFTR alone displayed the classical linear I-V in response to forskolin, with minimal current before stimulation and after forskolin washout. Curves are for black trace shown in A. (C) Cells expressing SLC26A9 + CFTR also displayed a linear I-V in response to forskolin; however, the magnitudes were larger and included the constitutive and enhanced poststimulation currents (note scale doubled from B). Curves are for gray trace shown in A. (D) Summary of the responses for SLC26A9 and CFTR expressed alone and together. Representative tracings are shown in A and Fig. 2 A. Fixed measurement points are identified in A. Peak values were measured at the actual peak of each tracing, with average peak times occurring at 150 ± 7 s for SLC26A9 + wtCFTR coexpressors and 158 ± 6 s for wtCFTR alone. The value for SLC26A9 alone was measured at 150 s. The SLC26A9 + wtCFTR coexpressor currents exceed the sum of the wtCFTR and SLC26A9 individual currents during and after forskolin stimulation (see Results). All current values were normalized to cell capacitance; VH = −40 mV. *, P < 0.05 compared with CFTR alone.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2664976&req=5

fig6: HEK 293 coexpression of SLC26A9 with wtCFTR increases forskolin-stimulated chloride currents over cells expressing CFTR alone. (A) Coexpressers (gray) consistently had larger currents than CFTR alone (black) as assessed at four measurement points: the basal currents before stimulation (60 s), the transient peak in response to 10 µM forskolin (150 s on average), the current plateau in the continued presence of forskolin (240 s), and the poststimulation current observed 3 min after forskolin washout (420 s). The poststimulation current in the coexpressers did not diminish during extended recordings (extra 8 min, this case). Representative tracings of at least three experiments each are shown. I-V measurements were performed at the indicated intervals (solid diamonds). (B) Cells expressing CFTR alone displayed the classical linear I-V in response to forskolin, with minimal current before stimulation and after forskolin washout. Curves are for black trace shown in A. (C) Cells expressing SLC26A9 + CFTR also displayed a linear I-V in response to forskolin; however, the magnitudes were larger and included the constitutive and enhanced poststimulation currents (note scale doubled from B). Curves are for gray trace shown in A. (D) Summary of the responses for SLC26A9 and CFTR expressed alone and together. Representative tracings are shown in A and Fig. 2 A. Fixed measurement points are identified in A. Peak values were measured at the actual peak of each tracing, with average peak times occurring at 150 ± 7 s for SLC26A9 + wtCFTR coexpressors and 158 ± 6 s for wtCFTR alone. The value for SLC26A9 alone was measured at 150 s. The SLC26A9 + wtCFTR coexpressor currents exceed the sum of the wtCFTR and SLC26A9 individual currents during and after forskolin stimulation (see Results). All current values were normalized to cell capacitance; VH = −40 mV. *, P < 0.05 compared with CFTR alone.
Mentions: As shown in Fig. 6 A, cells expressing CFTR + SLC26A9 or CFTR alone responded to forskolin within 30 s of addition and reached peak currents in 90 ± 7 s (n = 11) or 98 ± 6 s (n = 21), respectively. However, coexpression of wtCFTR with SLC26A9 significantly modified the typical response of CFTR to stimulation. Although the essential shape of the response was similar, cells coexpressing SLC26A9 and wtCFTR exhibited larger currents that did not return to baseline after washout of forskolin. This poststimulation current was not due to a delay in CFTR recovery; as shown in Fig. 6 A, it persisted for 8 min after CFTR alone had returned to its prestimulation baseline. Coexpressors maintained linear I-V characteristics before, during, and after stimulation, and cells expressing CFTR alone demonstrated linear I-V relations during and also after forskolin stimulation (Fig. 6, B and C).

Bottom Line: The identity of this conductance is unknown, but SLC26A9, a member of the SLC26 family of CF transmembrane conductance regulator (CFTR)-interacting transporters, is found in the human airway and exhibits chloride channel behavior.HEK cells coexpressing SLC26A9 with DeltaF508-CFTR also failed to exhibit SLC26A9 current.We conclude that SLC26A9 functions as an anion conductance in the apical membranes of HBE cells, it contributes to transepithelial chloride currents under basal and cAMP/protein kinase A-stimulated conditions, and its activity in HBE cells requires functional CFTR.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA. cbertra@pitt.edu

ABSTRACT
Human bronchial epithelial (HBE) cells exhibit constitutive anion secretion that is absent in cells from cystic fibrosis (CF) patients. The identity of this conductance is unknown, but SLC26A9, a member of the SLC26 family of CF transmembrane conductance regulator (CFTR)-interacting transporters, is found in the human airway and exhibits chloride channel behavior. We sought differences in the properties of SLC26A9 and CFTR expressed in HEK 293 (HEK) cells as a fingerprint to identify HBE apical anion conductances. HEK cells expressing SLC26A9 displayed a constitutive chloride current that was inhibited by the CFTR blocker GlyH-101 (71 +/- 4%, 50 microM) and exhibited a near-linear current-voltage (I-V) relation during block, while GlyH-101-inhibited wild-type (wt)CFTR exhibited a strong inward-rectified (IR) I-V relation. We tested polarized HBE cells endogenously expressing either wt or DeltaF508-CFTR for similar activity. After electrical isolation of the apical membrane using basolateral alpha-toxin permeabilization, wtCFTR monolayers displayed constitutive chloride currents that were inhibited by GlyH-101 (68 +/- 6%) while maintaining a near-linear I-V relation. In the absence of blocker, the addition of forskolin stimulated a current increase having a linear I-V; GlyH-101 blocked 69 +/- 7% of the current and shifted the I-V relation IR, consistent with CFTR activation. HEK cells coexpressing SLC26A9 and wtCFTR displayed similar properties, as well as forskolin-stimulated currents that exceeded the sum of those in cells separately expressing SLC26A9 or wtCFTR, and an I-V relation during GlyH-101 inhibition that was moderately IR, indicating that SLC26A9 contributed to the stimulated current. HBE cells from CF patients expressed SLC26A9 mRNA, but no constitutive chloride currents. HEK cells coexpressing SLC26A9 with DeltaF508-CFTR also failed to exhibit SLC26A9 current. We conclude that SLC26A9 functions as an anion conductance in the apical membranes of HBE cells, it contributes to transepithelial chloride currents under basal and cAMP/protein kinase A-stimulated conditions, and its activity in HBE cells requires functional CFTR.

Show MeSH
Related in: MedlinePlus