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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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Current and blocker properties in HEK 293 cells coexpressing SLC26A9 + wtCFTR are no different before forskolin stimulation from cells expressing SLC26A9 alone. The number of experiments for each case is included on the bar groups. All currents were normalized to their respective basal current at break-in. *, P < 0.05 compared with bath alone.
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fig5: Current and blocker properties in HEK 293 cells coexpressing SLC26A9 + wtCFTR are no different before forskolin stimulation from cells expressing SLC26A9 alone. The number of experiments for each case is included on the bar groups. All currents were normalized to their respective basal current at break-in. *, P < 0.05 compared with bath alone.

Mentions: Cells coexpressing SLC26A9 + wtCFTR had a basal current of −21 ± 3 pA/pF with an Rm of 218 ± 21 MΩ (n = 56) on establishment of the whole cell mode; these values were not statistically different from cells expressing SLC26A9 alone (above). To further assess whether an interaction between the channels was occurring in this initial unstimulated state, we monitored the current rundown, as well as the effects of a 3-min exposure to GlyH-101 or glibenclamide, in the absence of forskolin. As shown in Fig. 5, there was no difference in the rundown or action of the blockers before stimulation whether SLC26A9 was expressed alone or with wtCFTR. Furthermore, the I-V ratios were essentially unchanged from those measured for SLC26A9 alone: GlyH-101 induced a mild IR RIV (0.84 ± 0.04; n = 3), whereas the glibenclamide ratio remained linear (1.03 ± 0.07; n = 6). Neither ratio was statistically different from its paired, pre-blocked value or from the ratios measured for SLC26A9 alone.


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)

Current and blocker properties in HEK 293 cells coexpressing SLC26A9 + wtCFTR are no different before forskolin stimulation from cells expressing SLC26A9 alone. The number of experiments for each case is included on the bar groups. All currents were normalized to their respective basal current at break-in. *, P < 0.05 compared with bath alone.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig5: Current and blocker properties in HEK 293 cells coexpressing SLC26A9 + wtCFTR are no different before forskolin stimulation from cells expressing SLC26A9 alone. The number of experiments for each case is included on the bar groups. All currents were normalized to their respective basal current at break-in. *, P < 0.05 compared with bath alone.
Mentions: Cells coexpressing SLC26A9 + wtCFTR had a basal current of −21 ± 3 pA/pF with an Rm of 218 ± 21 MΩ (n = 56) on establishment of the whole cell mode; these values were not statistically different from cells expressing SLC26A9 alone (above). To further assess whether an interaction between the channels was occurring in this initial unstimulated state, we monitored the current rundown, as well as the effects of a 3-min exposure to GlyH-101 or glibenclamide, in the absence of forskolin. As shown in Fig. 5, there was no difference in the rundown or action of the blockers before stimulation whether SLC26A9 was expressed alone or with wtCFTR. Furthermore, the I-V ratios were essentially unchanged from those measured for SLC26A9 alone: GlyH-101 induced a mild IR RIV (0.84 ± 0.04; n = 3), whereas the glibenclamide ratio remained linear (1.03 ± 0.07; n = 6). Neither ratio was statistically different from its paired, pre-blocked value or from the ratios measured for SLC26A9 alone.

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