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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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α-Toxin–permeabilized HBE monolayers exhibit constitutive and forskolin-stimulated chloride currents and voltage-dependent blocker effects. (A) Short-circuit current measurements performed on three separate filters demonstrate GlyH-101 and glibenclamide inhibition properties. With a mucosal to serosal chloride gradient of 48 mM (EqCl = −13 mV), constitutive chloride currents from mucosa to serosa were evident after permeabilization, and forskolin stimulation tripled the current. GlyH-101 rapidly inhibited the stimulated current (blue trace), whereas glibenclamide inhibition was slower and less effective (black trace). GlyH-101 was equally effective at inhibiting the constitutive current before forskolin stimulation (red trace). The forskolin-stimulated current after GlyH-101 block was small, but forskolin still significantly shifted the RIV, consistent with activation of CFTR (see Results). Bars below the traces indicate additions to the basolateral chamber, whereas those above indicate additions to the apical chamber. Permeabilization with α-toxin required ∼15 min; ouabain was added to inhibit Na-K-ATPase, whereas ATP was added to replenish intracellular stores (Supplemental text). Solutions were bicarbonate free, and recordings were started within 2 min of filter submersion. Representative tracings of at least four filters per blocker are shown. (B) Same tracings as in A but including the I-V measurements, consisting of bipolar pulses from ±10 to ±60 mV in 10-mV steps. Pulses were simultaneously applied to two filters for the traces shown. I-V measurements were performed after permeabilization (basal), forskolin stimulation (fsk), and blocker inhibition (fsk+blocker). Although the I-V responses overlapped for the basal and fsk measurements, differences in the voltage dependence of the two blockers were evident in the third measurement. Cation channel blockers (A) were added to minimize contributions from ENaC and potassium channels to the I-V response. The permeabilization phase of the recording was removed for clarity. (C) The I-V curves measured for the forskolin-stimulated monolayer treated with glibenclamide (black) demonstrate the shift from linear (open symbols) to OR on blocker addition (solid triangles), whereas the I-V curves measured for the stimulated monolayer treated with GlyH-101 (blue) demonstrate the shift from linear (open symbols) to IR on blocker addition (blue triangles).
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fig10: α-Toxin–permeabilized HBE monolayers exhibit constitutive and forskolin-stimulated chloride currents and voltage-dependent blocker effects. (A) Short-circuit current measurements performed on three separate filters demonstrate GlyH-101 and glibenclamide inhibition properties. With a mucosal to serosal chloride gradient of 48 mM (EqCl = −13 mV), constitutive chloride currents from mucosa to serosa were evident after permeabilization, and forskolin stimulation tripled the current. GlyH-101 rapidly inhibited the stimulated current (blue trace), whereas glibenclamide inhibition was slower and less effective (black trace). GlyH-101 was equally effective at inhibiting the constitutive current before forskolin stimulation (red trace). The forskolin-stimulated current after GlyH-101 block was small, but forskolin still significantly shifted the RIV, consistent with activation of CFTR (see Results). Bars below the traces indicate additions to the basolateral chamber, whereas those above indicate additions to the apical chamber. Permeabilization with α-toxin required ∼15 min; ouabain was added to inhibit Na-K-ATPase, whereas ATP was added to replenish intracellular stores (Supplemental text). Solutions were bicarbonate free, and recordings were started within 2 min of filter submersion. Representative tracings of at least four filters per blocker are shown. (B) Same tracings as in A but including the I-V measurements, consisting of bipolar pulses from ±10 to ±60 mV in 10-mV steps. Pulses were simultaneously applied to two filters for the traces shown. I-V measurements were performed after permeabilization (basal), forskolin stimulation (fsk), and blocker inhibition (fsk+blocker). Although the I-V responses overlapped for the basal and fsk measurements, differences in the voltage dependence of the two blockers were evident in the third measurement. Cation channel blockers (A) were added to minimize contributions from ENaC and potassium channels to the I-V response. The permeabilization phase of the recording was removed for clarity. (C) The I-V curves measured for the forskolin-stimulated monolayer treated with glibenclamide (black) demonstrate the shift from linear (open symbols) to OR on blocker addition (solid triangles), whereas the I-V curves measured for the stimulated monolayer treated with GlyH-101 (blue) demonstrate the shift from linear (open symbols) to IR on blocker addition (blue triangles).

Mentions: α-Toxin has been used to permeabilize membranes and isolate the CFTR anion conductance in several systems (Ostedgaard et al., 1992; Hallows et al., 2003; Reddy and Quinton, 2003). It has the disadvantage of creating relatively large pores (1.5 nm; Bhakdi et al., 1993) that also permit the flux of divalent cations and ATP, which requires attention to bath solution composition. After testing several different doses of α-toxin and the impact of ATP loss on the forskolin response of permeabilized monolayers (Supplemental text), we adopted a permeabilization protocol that included a relatively low dose of α-toxin, inhibition of the Na-K-ATPase with 100 µM ouabain (to limit energy use), and addition of 1 mM Na-ATP, all applied to the basolateral bath. As shown in Fig. 10 A, complete permeabilization with α-toxin required ∼15 min, as judged by the shift in current. Sequential addition of ouabain and ATP after the initial evidence of permeabilization did not alter the short-circuit current from that seen in their absence (Supplemental text). Apical addition of the cation channel blockers amiloride and barium chloride had no impact on Isc, as expected in the absence of bath solution cation gradients.


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)

α-Toxin–permeabilized HBE monolayers exhibit constitutive and forskolin-stimulated chloride currents and voltage-dependent blocker effects. (A) Short-circuit current measurements performed on three separate filters demonstrate GlyH-101 and glibenclamide inhibition properties. With a mucosal to serosal chloride gradient of 48 mM (EqCl = −13 mV), constitutive chloride currents from mucosa to serosa were evident after permeabilization, and forskolin stimulation tripled the current. GlyH-101 rapidly inhibited the stimulated current (blue trace), whereas glibenclamide inhibition was slower and less effective (black trace). GlyH-101 was equally effective at inhibiting the constitutive current before forskolin stimulation (red trace). The forskolin-stimulated current after GlyH-101 block was small, but forskolin still significantly shifted the RIV, consistent with activation of CFTR (see Results). Bars below the traces indicate additions to the basolateral chamber, whereas those above indicate additions to the apical chamber. Permeabilization with α-toxin required ∼15 min; ouabain was added to inhibit Na-K-ATPase, whereas ATP was added to replenish intracellular stores (Supplemental text). Solutions were bicarbonate free, and recordings were started within 2 min of filter submersion. Representative tracings of at least four filters per blocker are shown. (B) Same tracings as in A but including the I-V measurements, consisting of bipolar pulses from ±10 to ±60 mV in 10-mV steps. Pulses were simultaneously applied to two filters for the traces shown. I-V measurements were performed after permeabilization (basal), forskolin stimulation (fsk), and blocker inhibition (fsk+blocker). Although the I-V responses overlapped for the basal and fsk measurements, differences in the voltage dependence of the two blockers were evident in the third measurement. Cation channel blockers (A) were added to minimize contributions from ENaC and potassium channels to the I-V response. The permeabilization phase of the recording was removed for clarity. (C) The I-V curves measured for the forskolin-stimulated monolayer treated with glibenclamide (black) demonstrate the shift from linear (open symbols) to OR on blocker addition (solid triangles), whereas the I-V curves measured for the stimulated monolayer treated with GlyH-101 (blue) demonstrate the shift from linear (open symbols) to IR on blocker addition (blue triangles).
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig10: α-Toxin–permeabilized HBE monolayers exhibit constitutive and forskolin-stimulated chloride currents and voltage-dependent blocker effects. (A) Short-circuit current measurements performed on three separate filters demonstrate GlyH-101 and glibenclamide inhibition properties. With a mucosal to serosal chloride gradient of 48 mM (EqCl = −13 mV), constitutive chloride currents from mucosa to serosa were evident after permeabilization, and forskolin stimulation tripled the current. GlyH-101 rapidly inhibited the stimulated current (blue trace), whereas glibenclamide inhibition was slower and less effective (black trace). GlyH-101 was equally effective at inhibiting the constitutive current before forskolin stimulation (red trace). The forskolin-stimulated current after GlyH-101 block was small, but forskolin still significantly shifted the RIV, consistent with activation of CFTR (see Results). Bars below the traces indicate additions to the basolateral chamber, whereas those above indicate additions to the apical chamber. Permeabilization with α-toxin required ∼15 min; ouabain was added to inhibit Na-K-ATPase, whereas ATP was added to replenish intracellular stores (Supplemental text). Solutions were bicarbonate free, and recordings were started within 2 min of filter submersion. Representative tracings of at least four filters per blocker are shown. (B) Same tracings as in A but including the I-V measurements, consisting of bipolar pulses from ±10 to ±60 mV in 10-mV steps. Pulses were simultaneously applied to two filters for the traces shown. I-V measurements were performed after permeabilization (basal), forskolin stimulation (fsk), and blocker inhibition (fsk+blocker). Although the I-V responses overlapped for the basal and fsk measurements, differences in the voltage dependence of the two blockers were evident in the third measurement. Cation channel blockers (A) were added to minimize contributions from ENaC and potassium channels to the I-V response. The permeabilization phase of the recording was removed for clarity. (C) The I-V curves measured for the forskolin-stimulated monolayer treated with glibenclamide (black) demonstrate the shift from linear (open symbols) to OR on blocker addition (solid triangles), whereas the I-V curves measured for the stimulated monolayer treated with GlyH-101 (blue) demonstrate the shift from linear (open symbols) to IR on blocker addition (blue triangles).
Mentions: α-Toxin has been used to permeabilize membranes and isolate the CFTR anion conductance in several systems (Ostedgaard et al., 1992; Hallows et al., 2003; Reddy and Quinton, 2003). It has the disadvantage of creating relatively large pores (1.5 nm; Bhakdi et al., 1993) that also permit the flux of divalent cations and ATP, which requires attention to bath solution composition. After testing several different doses of α-toxin and the impact of ATP loss on the forskolin response of permeabilized monolayers (Supplemental text), we adopted a permeabilization protocol that included a relatively low dose of α-toxin, inhibition of the Na-K-ATPase with 100 µM ouabain (to limit energy use), and addition of 1 mM Na-ATP, all applied to the basolateral bath. As shown in Fig. 10 A, complete permeabilization with α-toxin required ∼15 min, as judged by the shift in current. Sequential addition of ouabain and ATP after the initial evidence of permeabilization did not alter the short-circuit current from that seen in their absence (Supplemental text). Apical addition of the cation channel blockers amiloride and barium chloride had no impact on Isc, as expected in the absence of bath solution cation gradients.

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