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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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Responses of forskolin-stimulated currents to blockers in HEK 293 cells expressing wtCFTR ± SLC26A9. (A) HEK 293 cells expressing wtCFTR alone were stimulated with forskolin and allowed to reach a plateau, and then inhibited with either glibenclamide or GlyH-101; the latter rapidly and almost completely inhibited wtCFTR alone. VH = −40 mV. (B) I-V curves measured 90 s after the addition of blocker (330 s in A) demonstrate the opposite voltage dependence of the two blockers. (C) HEK 293 cells coexpressing SLC26A9 and wtCFTR were stimulated with forskolin and allowed to reach a plateau, and then inhibited with either glibenclamide or GlyH-101. VH = −40 mV. (D) The I-V curves measured 90 s after the addition of blocker (330 s in C) demonstrate the opposite voltage dependencies of the two blockers. The nonlinearity or voltage dependence of the block of wtCFTR alone (B) is greater than evidenced in the coexpressors (see Table I for mean values).
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fig7: Responses of forskolin-stimulated currents to blockers in HEK 293 cells expressing wtCFTR ± SLC26A9. (A) HEK 293 cells expressing wtCFTR alone were stimulated with forskolin and allowed to reach a plateau, and then inhibited with either glibenclamide or GlyH-101; the latter rapidly and almost completely inhibited wtCFTR alone. VH = −40 mV. (B) I-V curves measured 90 s after the addition of blocker (330 s in A) demonstrate the opposite voltage dependence of the two blockers. (C) HEK 293 cells coexpressing SLC26A9 and wtCFTR were stimulated with forskolin and allowed to reach a plateau, and then inhibited with either glibenclamide or GlyH-101. VH = −40 mV. (D) The I-V curves measured 90 s after the addition of blocker (330 s in C) demonstrate the opposite voltage dependencies of the two blockers. The nonlinearity or voltage dependence of the block of wtCFTR alone (B) is greater than evidenced in the coexpressors (see Table I for mean values).

Mentions: We tested the effect of blockers on forskolin-stimulated cells and first verified published data on the action of the blockers on cells expressing wtCFTR alone. The time course of the inhibition and corresponding I-V curves are shown in Fig. 7. We found that GlyH-101 was very effective on wtCFTR alone (Fig. 7 A), inhibiting 90 ± 2% (n = 3; P < 0.001) of the plateau current within 1 min of addition. Glibenclamide inhibition was slower to develop, most likely due to its intracellular binding site, and it was less effective, inhibiting 52 ± 10% (n = 4) of the plateau current (both 3 min after addition). The I-V relations during inhibition (Fig. 7 B) demonstrate significant differences in the voltage dependence of block. Applying the same protocol to cells coexpressing SLC26A9 and wtCFTR (Fig. 7 C), we also found that both blockers inhibited the plateau current within 2 min of addition. GlyH-101 acted more rapidly and inhibited 65 ± 3% (n = 4) of the plateau current, whereas glibenclamide inhibition was slower to develop and blocked 36 ± 11% (n = 3) of the plateau current. The I-V relations (Fig. 7 D) demonstrated the expected inward and OR profiles for GlyH-101 and glibenclamide block, respectively; however, the voltage dependence of the inhibition was decreased compared with CFTR alone (Fig. 7 B; see quantitation in Table I, discussed below).


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)

Responses of forskolin-stimulated currents to blockers in HEK 293 cells expressing wtCFTR ± SLC26A9. (A) HEK 293 cells expressing wtCFTR alone were stimulated with forskolin and allowed to reach a plateau, and then inhibited with either glibenclamide or GlyH-101; the latter rapidly and almost completely inhibited wtCFTR alone. VH = −40 mV. (B) I-V curves measured 90 s after the addition of blocker (330 s in A) demonstrate the opposite voltage dependence of the two blockers. (C) HEK 293 cells coexpressing SLC26A9 and wtCFTR were stimulated with forskolin and allowed to reach a plateau, and then inhibited with either glibenclamide or GlyH-101. VH = −40 mV. (D) The I-V curves measured 90 s after the addition of blocker (330 s in C) demonstrate the opposite voltage dependencies of the two blockers. The nonlinearity or voltage dependence of the block of wtCFTR alone (B) is greater than evidenced in the coexpressors (see Table I for mean values).
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fig7: Responses of forskolin-stimulated currents to blockers in HEK 293 cells expressing wtCFTR ± SLC26A9. (A) HEK 293 cells expressing wtCFTR alone were stimulated with forskolin and allowed to reach a plateau, and then inhibited with either glibenclamide or GlyH-101; the latter rapidly and almost completely inhibited wtCFTR alone. VH = −40 mV. (B) I-V curves measured 90 s after the addition of blocker (330 s in A) demonstrate the opposite voltage dependence of the two blockers. (C) HEK 293 cells coexpressing SLC26A9 and wtCFTR were stimulated with forskolin and allowed to reach a plateau, and then inhibited with either glibenclamide or GlyH-101. VH = −40 mV. (D) The I-V curves measured 90 s after the addition of blocker (330 s in C) demonstrate the opposite voltage dependencies of the two blockers. The nonlinearity or voltage dependence of the block of wtCFTR alone (B) is greater than evidenced in the coexpressors (see Table I for mean values).
Mentions: We tested the effect of blockers on forskolin-stimulated cells and first verified published data on the action of the blockers on cells expressing wtCFTR alone. The time course of the inhibition and corresponding I-V curves are shown in Fig. 7. We found that GlyH-101 was very effective on wtCFTR alone (Fig. 7 A), inhibiting 90 ± 2% (n = 3; P < 0.001) of the plateau current within 1 min of addition. Glibenclamide inhibition was slower to develop, most likely due to its intracellular binding site, and it was less effective, inhibiting 52 ± 10% (n = 4) of the plateau current (both 3 min after addition). The I-V relations during inhibition (Fig. 7 B) demonstrate significant differences in the voltage dependence of block. Applying the same protocol to cells coexpressing SLC26A9 and wtCFTR (Fig. 7 C), we also found that both blockers inhibited the plateau current within 2 min of addition. GlyH-101 acted more rapidly and inhibited 65 ± 3% (n = 4) of the plateau current, whereas glibenclamide inhibition was slower to develop and blocked 36 ± 11% (n = 3) of the plateau current. The I-V relations (Fig. 7 D) demonstrated the expected inward and OR profiles for GlyH-101 and glibenclamide block, respectively; however, the voltage dependence of the inhibition was decreased compared with CFTR alone (Fig. 7 B; see quantitation in Table I, discussed below).

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