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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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Summary of SLC26A9 current time course and blocker effects. (A) HEK 293 cells treated with 50 µM GlyH-101 were significantly inhibited after 3 min and exhibited a partial recovery after 3 min of blocker washout. The effect of 100 µM glibenclamide was not statistically different from the 20% rundown in constitutive current normally observed in untreated cells. (B) The addition of forskolin during the blocker protocols did not significantly affect either the rundown or blocker efficacy. All currents are normalized to their respective basal current (see Results) at break-in. *, P < 0.05 compared against the same time point from unblocked cells and for paired t test with prior time point. Number of experiments for each case is indicated on the bar groups. A representative time course and protocol are shown in Fig. 2 A.
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fig3: Summary of SLC26A9 current time course and blocker effects. (A) HEK 293 cells treated with 50 µM GlyH-101 were significantly inhibited after 3 min and exhibited a partial recovery after 3 min of blocker washout. The effect of 100 µM glibenclamide was not statistically different from the 20% rundown in constitutive current normally observed in untreated cells. (B) The addition of forskolin during the blocker protocols did not significantly affect either the rundown or blocker efficacy. All currents are normalized to their respective basal current (see Results) at break-in. *, P < 0.05 compared against the same time point from unblocked cells and for paired t test with prior time point. Number of experiments for each case is indicated on the bar groups. A representative time course and protocol are shown in Fig. 2 A.

Mentions: As summarized in Fig. 3, SLC26A9-transfected cells demonstrated a rundown in current to 80% of initial levels within 3 min of break-in, which then stabilized for the duration of the recordings. The current rundown and stabilization was unaffected by the application of forskolin (Fig. 3 B). Strikingly, we found that GlyH-101 was also a potent inhibitor of the constitutive activity of SLC26A9 at a concentration (50 µM) typically used to inhibit CFTR, blocking 71 ± 4% (n = 4) of the constitutive current. This inhibition of SLC26A9 exhibited a rapid initial phase followed by a slower inhibition, and the block was partially reversed, again with two kinetic components, during a 3-min washout (Fig. 4 A). Unlike the result with GlyH-101, the addition of 100 µM glibenclamide to SLC26A9-transfected cells had no significant effect on Im (Figs. 3 and 4), in agreement with the observations of Dorwart et al. (2007).


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)

Summary of SLC26A9 current time course and blocker effects. (A) HEK 293 cells treated with 50 µM GlyH-101 were significantly inhibited after 3 min and exhibited a partial recovery after 3 min of blocker washout. The effect of 100 µM glibenclamide was not statistically different from the 20% rundown in constitutive current normally observed in untreated cells. (B) The addition of forskolin during the blocker protocols did not significantly affect either the rundown or blocker efficacy. All currents are normalized to their respective basal current (see Results) at break-in. *, P < 0.05 compared against the same time point from unblocked cells and for paired t test with prior time point. Number of experiments for each case is indicated on the bar groups. A representative time course and protocol are shown in Fig. 2 A.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig3: Summary of SLC26A9 current time course and blocker effects. (A) HEK 293 cells treated with 50 µM GlyH-101 were significantly inhibited after 3 min and exhibited a partial recovery after 3 min of blocker washout. The effect of 100 µM glibenclamide was not statistically different from the 20% rundown in constitutive current normally observed in untreated cells. (B) The addition of forskolin during the blocker protocols did not significantly affect either the rundown or blocker efficacy. All currents are normalized to their respective basal current (see Results) at break-in. *, P < 0.05 compared against the same time point from unblocked cells and for paired t test with prior time point. Number of experiments for each case is indicated on the bar groups. A representative time course and protocol are shown in Fig. 2 A.
Mentions: As summarized in Fig. 3, SLC26A9-transfected cells demonstrated a rundown in current to 80% of initial levels within 3 min of break-in, which then stabilized for the duration of the recordings. The current rundown and stabilization was unaffected by the application of forskolin (Fig. 3 B). Strikingly, we found that GlyH-101 was also a potent inhibitor of the constitutive activity of SLC26A9 at a concentration (50 µM) typically used to inhibit CFTR, blocking 71 ± 4% (n = 4) of the constitutive current. This inhibition of SLC26A9 exhibited a rapid initial phase followed by a slower inhibition, and the block was partially reversed, again with two kinetic components, during a 3-min washout (Fig. 4 A). Unlike the result with GlyH-101, the addition of 100 µM glibenclamide to SLC26A9-transfected cells had no significant effect on Im (Figs. 3 and 4), in agreement with the observations of Dorwart et al. (2007).

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