Limits...
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.

Show MeSH

Related in: MedlinePlus

The CFTR channel inhibitor GlyH-101 inhibits SLC26A9. (A) HEK 293 cells transfected with SLC26A9 and voltage clamped at −40 mV were exposed to either 50 µM GlyH-101 or 100 µM glibenclamide after a 60-s stabilization period. GlyH-101 inhibited 50% of the constitutive chloride current within seconds and an additional 20% over the course of a 3-min application. The inhibition was partially reversed after 3 min of washout, also with two phases of recovery. The minor drop in current during application of 100 µM glibenclamide was statistically insignificant; longer exposure times and/or higher doses were not explored. Representative tracings of at least three experiments each are shown. (B) Representative I-V behavior of SLC26A9 and CFTR in the presence of the indicated blockers. SLC26A9-transfected cells displayed a mild IR I-V response after 3 min of exposure to either GlyH-101 or glibenclamide. Cells transfected with wtCFTR, stimulated with 10 µM forskolin, and then inhibited with 50 µM GlyH-101 for 3 min displayed a strong IR I-V response. SLC26A9 I-V curves are for traces shown in A, measured at 240 s (solid diamond). All current values are normalized by cell capacitance.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig4: The CFTR channel inhibitor GlyH-101 inhibits SLC26A9. (A) HEK 293 cells transfected with SLC26A9 and voltage clamped at −40 mV were exposed to either 50 µM GlyH-101 or 100 µM glibenclamide after a 60-s stabilization period. GlyH-101 inhibited 50% of the constitutive chloride current within seconds and an additional 20% over the course of a 3-min application. The inhibition was partially reversed after 3 min of washout, also with two phases of recovery. The minor drop in current during application of 100 µM glibenclamide was statistically insignificant; longer exposure times and/or higher doses were not explored. Representative tracings of at least three experiments each are shown. (B) Representative I-V behavior of SLC26A9 and CFTR in the presence of the indicated blockers. SLC26A9-transfected cells displayed a mild IR I-V response after 3 min of exposure to either GlyH-101 or glibenclamide. Cells transfected with wtCFTR, stimulated with 10 µM forskolin, and then inhibited with 50 µM GlyH-101 for 3 min displayed a strong IR I-V response. SLC26A9 I-V curves are for traces shown in A, measured at 240 s (solid diamond). All current values are normalized by cell capacitance.

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)

The CFTR channel inhibitor GlyH-101 inhibits SLC26A9. (A) HEK 293 cells transfected with SLC26A9 and voltage clamped at −40 mV were exposed to either 50 µM GlyH-101 or 100 µM glibenclamide after a 60-s stabilization period. GlyH-101 inhibited 50% of the constitutive chloride current within seconds and an additional 20% over the course of a 3-min application. The inhibition was partially reversed after 3 min of washout, also with two phases of recovery. The minor drop in current during application of 100 µM glibenclamide was statistically insignificant; longer exposure times and/or higher doses were not explored. Representative tracings of at least three experiments each are shown. (B) Representative I-V behavior of SLC26A9 and CFTR in the presence of the indicated blockers. SLC26A9-transfected cells displayed a mild IR I-V response after 3 min of exposure to either GlyH-101 or glibenclamide. Cells transfected with wtCFTR, stimulated with 10 µM forskolin, and then inhibited with 50 µM GlyH-101 for 3 min displayed a strong IR I-V response. SLC26A9 I-V curves are for traces shown in A, measured at 240 s (solid diamond). All current values are normalized by cell capacitance.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig4: The CFTR channel inhibitor GlyH-101 inhibits SLC26A9. (A) HEK 293 cells transfected with SLC26A9 and voltage clamped at −40 mV were exposed to either 50 µM GlyH-101 or 100 µM glibenclamide after a 60-s stabilization period. GlyH-101 inhibited 50% of the constitutive chloride current within seconds and an additional 20% over the course of a 3-min application. The inhibition was partially reversed after 3 min of washout, also with two phases of recovery. The minor drop in current during application of 100 µM glibenclamide was statistically insignificant; longer exposure times and/or higher doses were not explored. Representative tracings of at least three experiments each are shown. (B) Representative I-V behavior of SLC26A9 and CFTR in the presence of the indicated blockers. SLC26A9-transfected cells displayed a mild IR I-V response after 3 min of exposure to either GlyH-101 or glibenclamide. Cells transfected with wtCFTR, stimulated with 10 µM forskolin, and then inhibited with 50 µM GlyH-101 for 3 min displayed a strong IR I-V response. SLC26A9 I-V curves are for traces shown in A, measured at 240 s (solid diamond). All current values are normalized by cell capacitance.
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