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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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Short-circuit current measurements in HBE monolayers reveal GlyH-101 inhibition of basal anion secretion. After amiloride inhibition of ENaC current, a basal anion current was evident. Application of 50 µM GlyH-101 to the apical chamber inhibited the basal current (gray trace) and attenuated the subsequent response to forskolin. In the absence of GlyH-101 (black trace), forskolin stimulated additional anion secretion. Basolateral application of bumetanide inhibited the forskolin-stimulated current in both GlyH-101–treated and untreated epithelia; only the latter were sensitive to basolateral DIDS (see Results). Traces representative of four filters from each condition are shown.
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fig1: Short-circuit current measurements in HBE monolayers reveal GlyH-101 inhibition of basal anion secretion. After amiloride inhibition of ENaC current, a basal anion current was evident. Application of 50 µM GlyH-101 to the apical chamber inhibited the basal current (gray trace) and attenuated the subsequent response to forskolin. In the absence of GlyH-101 (black trace), forskolin stimulated additional anion secretion. Basolateral application of bumetanide inhibited the forskolin-stimulated current in both GlyH-101–treated and untreated epithelia; only the latter were sensitive to basolateral DIDS (see Results). Traces representative of four filters from each condition are shown.

Mentions: We initially tested whether the constitutive currents observed across our polarized, well-differentiated HBE monolayers after amiloride inhibition of sodium transport were sensitive to GlyH-101 inhibition. These experiments were performed using the short-circuit technique under standard conditions for HBE monolayers that included a bicarbonate-buffered Ringer's solution in both mucosal and serosal compartments and continuous aeration with a CO2/O2 mix to maintain pH. After amiloride inhibition of ENaC currents, HBE monolayers exhibited an average basal current of −13.7 ± 1.2 μA/cm2 (n = 8; Fig. 1). Apical addition of 50 µM GlyH-101 rapidly inhibited 44 ± 4% (n = 4) of this basal current (gray curve). The addition of forskolin to the GlyH-101–blocked monolayer produced a small increase in Cl− secretion (ΔIsc = −4.1 ± 1.5 μA/cm2) that was completely inhibited by basolateral addition of bumetanide; basolateral addition of 4,4′-diisothiocyanatostilbene-2,2' disulfonic acid (DIDS) had little or no effect on the residual current. By comparison (black curve), forskolin stimulated a larger Cl− secretory current (ΔIsc = −17.7 ± 5.6 μA/cm2; n = 4) in the absence of GlyH-101 that was substantially inhibited by basolateral addition of bumetanide. Unlike the GlyH-101–blocked monolayer, however, basolateral addition of DIDS blocked a portion of the current remaining after bumetanide inhibition, reducing the final current to a level comparable to GlyH-101–inhibited monolayers. These results indicated that GlyH-101 significantly inhibited the constitutive current present before forskolin addition, confirming prior observations (Wang et al., 2005). Additionally, they suggested that a portion of the anion secretory response in HBE monolayers after NKCC1 block by bumetanide may be due to bicarbonate secretion, as basolateral DIDS is expected to inhibit multiple basolateral bicarbonate entry processes (Devor et al., 1999; Fischer and Widdicombe, 2006).


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)

Short-circuit current measurements in HBE monolayers reveal GlyH-101 inhibition of basal anion secretion. After amiloride inhibition of ENaC current, a basal anion current was evident. Application of 50 µM GlyH-101 to the apical chamber inhibited the basal current (gray trace) and attenuated the subsequent response to forskolin. In the absence of GlyH-101 (black trace), forskolin stimulated additional anion secretion. Basolateral application of bumetanide inhibited the forskolin-stimulated current in both GlyH-101–treated and untreated epithelia; only the latter were sensitive to basolateral DIDS (see Results). Traces representative of four filters from each condition are shown.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig1: Short-circuit current measurements in HBE monolayers reveal GlyH-101 inhibition of basal anion secretion. After amiloride inhibition of ENaC current, a basal anion current was evident. Application of 50 µM GlyH-101 to the apical chamber inhibited the basal current (gray trace) and attenuated the subsequent response to forskolin. In the absence of GlyH-101 (black trace), forskolin stimulated additional anion secretion. Basolateral application of bumetanide inhibited the forskolin-stimulated current in both GlyH-101–treated and untreated epithelia; only the latter were sensitive to basolateral DIDS (see Results). Traces representative of four filters from each condition are shown.
Mentions: We initially tested whether the constitutive currents observed across our polarized, well-differentiated HBE monolayers after amiloride inhibition of sodium transport were sensitive to GlyH-101 inhibition. These experiments were performed using the short-circuit technique under standard conditions for HBE monolayers that included a bicarbonate-buffered Ringer's solution in both mucosal and serosal compartments and continuous aeration with a CO2/O2 mix to maintain pH. After amiloride inhibition of ENaC currents, HBE monolayers exhibited an average basal current of −13.7 ± 1.2 μA/cm2 (n = 8; Fig. 1). Apical addition of 50 µM GlyH-101 rapidly inhibited 44 ± 4% (n = 4) of this basal current (gray curve). The addition of forskolin to the GlyH-101–blocked monolayer produced a small increase in Cl− secretion (ΔIsc = −4.1 ± 1.5 μA/cm2) that was completely inhibited by basolateral addition of bumetanide; basolateral addition of 4,4′-diisothiocyanatostilbene-2,2' disulfonic acid (DIDS) had little or no effect on the residual current. By comparison (black curve), forskolin stimulated a larger Cl− secretory current (ΔIsc = −17.7 ± 5.6 μA/cm2; n = 4) in the absence of GlyH-101 that was substantially inhibited by basolateral addition of bumetanide. Unlike the GlyH-101–blocked monolayer, however, basolateral addition of DIDS blocked a portion of the current remaining after bumetanide inhibition, reducing the final current to a level comparable to GlyH-101–inhibited monolayers. These results indicated that GlyH-101 significantly inhibited the constitutive current present before forskolin addition, confirming prior observations (Wang et al., 2005). Additionally, they suggested that a portion of the anion secretory response in HBE monolayers after NKCC1 block by bumetanide may be due to bicarbonate secretion, as basolateral DIDS is expected to inhibit multiple basolateral bicarbonate entry processes (Devor et al., 1999; Fischer and Widdicombe, 2006).

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