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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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RT-PCR indicates that HBE monolayers express SLC26A9 mRNA. (A) RNA harvested from either wt or ΔF508/ΔF508 CF HBE monolayers grown at air liquid interface for at least 2 wk demonstrates endogenous mRNA for both SLC26A9 (1,100-bp product) and CFTR (237-bp product); β-actin was amplified as a control (353-bp product). Apparent differences in SLC26A9 mRNA between wt and CF HBE monolayers were not quantified. (B) HEK 293 cells were transfected with GFP alone (as control), or GFP and SLC26A9 ± wtCFTR, as indicated in Materials and methods and at the bottom of the figure. Control HEK cells do not express endogenous SLC26A9. Molecular weights and markers are the same as in A (n ≥ 3).
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fig9: RT-PCR indicates that HBE monolayers express SLC26A9 mRNA. (A) RNA harvested from either wt or ΔF508/ΔF508 CF HBE monolayers grown at air liquid interface for at least 2 wk demonstrates endogenous mRNA for both SLC26A9 (1,100-bp product) and CFTR (237-bp product); β-actin was amplified as a control (353-bp product). Apparent differences in SLC26A9 mRNA between wt and CF HBE monolayers were not quantified. (B) HEK 293 cells were transfected with GFP alone (as control), or GFP and SLC26A9 ± wtCFTR, as indicated in Materials and methods and at the bottom of the figure. Control HEK cells do not express endogenous SLC26A9. Molecular weights and markers are the same as in A (n ≥ 3).

Mentions: Lohi et al. (2002) have reported the expression of SLC26A9 in airway surface epithelia. We used RT-PCR to confirm that the primary HBE cells used in these protocols expressed SLC26A9, as well as to examine its expression in HBE cells from CF patients. RNA was harvested from HBE filters grown at an air–liquid interface; the filters used were from the same groups used for transepithelial current measurements. RNA was also harvested from HEK 293 cells transfected with the different constructs used for whole cell electrophysiology as controls. The RNA was reverse transcribed, and then probed for CFTR and SLC26A9 mRNA expression using PCR. As shown in Fig. 9, both wt and CF HBE cells demonstrated PCR products for both SLC26A9 and CFTR. Transfected HEK 293 cells were positive for SLC26A9 and CFTR, whereas GFP-transfected control HEK 293 cells were not.


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)

RT-PCR indicates that HBE monolayers express SLC26A9 mRNA. (A) RNA harvested from either wt or ΔF508/ΔF508 CF HBE monolayers grown at air liquid interface for at least 2 wk demonstrates endogenous mRNA for both SLC26A9 (1,100-bp product) and CFTR (237-bp product); β-actin was amplified as a control (353-bp product). Apparent differences in SLC26A9 mRNA between wt and CF HBE monolayers were not quantified. (B) HEK 293 cells were transfected with GFP alone (as control), or GFP and SLC26A9 ± wtCFTR, as indicated in Materials and methods and at the bottom of the figure. Control HEK cells do not express endogenous SLC26A9. Molecular weights and markers are the same as in A (n ≥ 3).
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2664976&req=5

fig9: RT-PCR indicates that HBE monolayers express SLC26A9 mRNA. (A) RNA harvested from either wt or ΔF508/ΔF508 CF HBE monolayers grown at air liquid interface for at least 2 wk demonstrates endogenous mRNA for both SLC26A9 (1,100-bp product) and CFTR (237-bp product); β-actin was amplified as a control (353-bp product). Apparent differences in SLC26A9 mRNA between wt and CF HBE monolayers were not quantified. (B) HEK 293 cells were transfected with GFP alone (as control), or GFP and SLC26A9 ± wtCFTR, as indicated in Materials and methods and at the bottom of the figure. Control HEK cells do not express endogenous SLC26A9. Molecular weights and markers are the same as in A (n ≥ 3).
Mentions: Lohi et al. (2002) have reported the expression of SLC26A9 in airway surface epithelia. We used RT-PCR to confirm that the primary HBE cells used in these protocols expressed SLC26A9, as well as to examine its expression in HBE cells from CF patients. RNA was harvested from HBE filters grown at an air–liquid interface; the filters used were from the same groups used for transepithelial current measurements. RNA was also harvested from HEK 293 cells transfected with the different constructs used for whole cell electrophysiology as controls. The RNA was reverse transcribed, and then probed for CFTR and SLC26A9 mRNA expression using PCR. As shown in Fig. 9, both wt and CF HBE cells demonstrated PCR products for both SLC26A9 and CFTR. Transfected HEK 293 cells were positive for SLC26A9 and CFTR, whereas GFP-transfected control HEK 293 cells were not.

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