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Rescue of volume-regulated anion current by bestrophin mutants with altered charge selectivity.

Chien LT, Hartzell HC - J. Gen. Physiol. (2008)

Bottom Line: The F81E mutant was 1.3 times more permeable to Cs(+) than Cl(-).The finding that VRAC was rescued by F81C and F81E mutants with different biophysical properties shows that bestrophin-1 is a VRAC in S2 cells and not simply a regulator or an auxiliary subunit.F81C overexpressed in HEK293 cells also exhibits a shift of ionic selectivity after MTSES(-) treatment, although the effect is quantitatively smaller than in S2 cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Center for Neurodegenerative Disease, Emory University School of Medicine, Atlanta, GA 30322, USA.

ABSTRACT
Mutations in human bestrophin-1 are linked to various kinds of retinal degeneration. Although it has been proposed that bestrophins are Ca(2+)-activated Cl(-) channels, definitive proof is lacking partly because mice with the bestrophin-1 gene deleted have normal Ca(2+)-activated Cl(-) currents. Here, we provide compelling evidence to support the idea that bestrophin-1 is the pore-forming subunit of a cell volume-regulated anion channel (VRAC) in Drosophila S2 cells. VRAC was abolished by treatment with RNAi to Drosophila bestrophin-1. VRAC was rescued by overexpressing bestrophin-1 mutants with altered biophysical properties and responsiveness to sulfhydryl reagents. In particular, the ionic selectivity of the F81C mutant changed from anionic to cationic when the channel was treated with the sulfhydryl reagent, sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES(-)) (P(Cs)/P(Cl) = 0.25 for native and 2.38 for F81C). The F81E mutant was 1.3 times more permeable to Cs(+) than Cl(-). The finding that VRAC was rescued by F81C and F81E mutants with different biophysical properties shows that bestrophin-1 is a VRAC in S2 cells and not simply a regulator or an auxiliary subunit. F81C overexpressed in HEK293 cells also exhibits a shift of ionic selectivity after MTSES(-) treatment, although the effect is quantitatively smaller than in S2 cells. To test whether bestrophins are VRACs in mammalian cells, we compared VRACs in peritoneal macrophages from wild-type mice and mice with both bestrophin-1 and bestrophin-2 disrupted (best1(-/-)/best2(-/-)). VRACs were identical in wild-type and best1(-/-)/best2(-/-) mice, showing that bestrophins are unlikely to be the classical VRAC in mammalian cells.

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MTSES− increases the cation permeability of F81C currents. Current–voltage relationships from a typical native S2 cell (A) and a S2 cell rescued with F81C (B) before and after MTSES− modification. Currents were activated with Δ40 mosmol kg−1 hyposmotic solutions. The arrow in B points out the reversal potential of F81C current after MTSES− treatment. (C) Changes in Erev of F81C currents under different ionic conditions. The record begins after the dBest1 current had stabilized under hyposmotic solutions (I340/E300, Δ40 mosmol kg−1). MTSES− was then applied in the bath. Extracellular solution containing symmetrical Cl− and Na+ as the major cation (E300) was then replaced by solutions with Cs+ or NMDG+ as the major cations as indicated. The osmolality of all the extracellular solutions was 300 mosmol kg−1.
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fig4: MTSES− increases the cation permeability of F81C currents. Current–voltage relationships from a typical native S2 cell (A) and a S2 cell rescued with F81C (B) before and after MTSES− modification. Currents were activated with Δ40 mosmol kg−1 hyposmotic solutions. The arrow in B points out the reversal potential of F81C current after MTSES− treatment. (C) Changes in Erev of F81C currents under different ionic conditions. The record begins after the dBest1 current had stabilized under hyposmotic solutions (I340/E300, Δ40 mosmol kg−1). MTSES− was then applied in the bath. Extracellular solution containing symmetrical Cl− and Na+ as the major cation (E300) was then replaced by solutions with Cs+ or NMDG+ as the major cations as indicated. The osmolality of all the extracellular solutions was 300 mosmol kg−1.

Mentions: The effect of MTSES− on the I-V relationships of native and F81C-rescued cells is shown in Fig. 4 (A and B). F81C currents activated by hyposomotic solutions were reduced ∼60% by MTSES−, compared with an ∼20% reduction for native dBest1 (Fig. 3, A and B). More importantly, MTSES− consistently produced an Erev shift of −19.9 ± 1.2 mV (n = 12) in F81C-rescued cells but not in native cells (Fig. 3 C). This negative shift in Erev could be explained by a changed ionic selectivity of the channel. In these experiments, [Cl−] was the same on both sides of the membrane, so a pure Cl− current would have an expected Erev = 0 mV. The internal solution contained primarily Cs+ and the external solution contained primarily Na+. Therefore, the MTSES−-induced negative shift in Erev could be explained by an increased permeability to cations with PCs > PNa. This hypothesis was qualitatively tested by replacing extracellular Na+ with either Cs+ or NMDG+ in the E300 solution while maintaining Cs+ as the major intracellular cation (I340). The result from a typical F81C-rescued cell is shown in Fig. 4 C. Initially, F81C VRAC currents recorded with symmetrical Cl− and Cs+ inside and Na+ outside had an Erev of ∼0 mV, as would be predicted if the F81C current were selectively carried by Cl−. After MTSES− was applied, Erev shifted to −25.1 mV. When extracellular Na+ was replaced with Cs+, Erev changed to 1.7 mV. Replacement of extracellular Cs+ with the impermeant NMDG+ produced an Erev shift to −38 mV. These observations suggested that the F81C current had become more permeable to Cs+ and, less so, to Na+ after MTSES− modification.


Rescue of volume-regulated anion current by bestrophin mutants with altered charge selectivity.

Chien LT, Hartzell HC - J. Gen. Physiol. (2008)

MTSES− increases the cation permeability of F81C currents. Current–voltage relationships from a typical native S2 cell (A) and a S2 cell rescued with F81C (B) before and after MTSES− modification. Currents were activated with Δ40 mosmol kg−1 hyposmotic solutions. The arrow in B points out the reversal potential of F81C current after MTSES− treatment. (C) Changes in Erev of F81C currents under different ionic conditions. The record begins after the dBest1 current had stabilized under hyposmotic solutions (I340/E300, Δ40 mosmol kg−1). MTSES− was then applied in the bath. Extracellular solution containing symmetrical Cl− and Na+ as the major cation (E300) was then replaced by solutions with Cs+ or NMDG+ as the major cations as indicated. The osmolality of all the extracellular solutions was 300 mosmol kg−1.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2571971&req=5

fig4: MTSES− increases the cation permeability of F81C currents. Current–voltage relationships from a typical native S2 cell (A) and a S2 cell rescued with F81C (B) before and after MTSES− modification. Currents were activated with Δ40 mosmol kg−1 hyposmotic solutions. The arrow in B points out the reversal potential of F81C current after MTSES− treatment. (C) Changes in Erev of F81C currents under different ionic conditions. The record begins after the dBest1 current had stabilized under hyposmotic solutions (I340/E300, Δ40 mosmol kg−1). MTSES− was then applied in the bath. Extracellular solution containing symmetrical Cl− and Na+ as the major cation (E300) was then replaced by solutions with Cs+ or NMDG+ as the major cations as indicated. The osmolality of all the extracellular solutions was 300 mosmol kg−1.
Mentions: The effect of MTSES− on the I-V relationships of native and F81C-rescued cells is shown in Fig. 4 (A and B). F81C currents activated by hyposomotic solutions were reduced ∼60% by MTSES−, compared with an ∼20% reduction for native dBest1 (Fig. 3, A and B). More importantly, MTSES− consistently produced an Erev shift of −19.9 ± 1.2 mV (n = 12) in F81C-rescued cells but not in native cells (Fig. 3 C). This negative shift in Erev could be explained by a changed ionic selectivity of the channel. In these experiments, [Cl−] was the same on both sides of the membrane, so a pure Cl− current would have an expected Erev = 0 mV. The internal solution contained primarily Cs+ and the external solution contained primarily Na+. Therefore, the MTSES−-induced negative shift in Erev could be explained by an increased permeability to cations with PCs > PNa. This hypothesis was qualitatively tested by replacing extracellular Na+ with either Cs+ or NMDG+ in the E300 solution while maintaining Cs+ as the major intracellular cation (I340). The result from a typical F81C-rescued cell is shown in Fig. 4 C. Initially, F81C VRAC currents recorded with symmetrical Cl− and Cs+ inside and Na+ outside had an Erev of ∼0 mV, as would be predicted if the F81C current were selectively carried by Cl−. After MTSES− was applied, Erev shifted to −25.1 mV. When extracellular Na+ was replaced with Cs+, Erev changed to 1.7 mV. Replacement of extracellular Cs+ with the impermeant NMDG+ produced an Erev shift to −38 mV. These observations suggested that the F81C current had become more permeable to Cs+ and, less so, to Na+ after MTSES− modification.

Bottom Line: The F81E mutant was 1.3 times more permeable to Cs(+) than Cl(-).The finding that VRAC was rescued by F81C and F81E mutants with different biophysical properties shows that bestrophin-1 is a VRAC in S2 cells and not simply a regulator or an auxiliary subunit.F81C overexpressed in HEK293 cells also exhibits a shift of ionic selectivity after MTSES(-) treatment, although the effect is quantitatively smaller than in S2 cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Center for Neurodegenerative Disease, Emory University School of Medicine, Atlanta, GA 30322, USA.

ABSTRACT
Mutations in human bestrophin-1 are linked to various kinds of retinal degeneration. Although it has been proposed that bestrophins are Ca(2+)-activated Cl(-) channels, definitive proof is lacking partly because mice with the bestrophin-1 gene deleted have normal Ca(2+)-activated Cl(-) currents. Here, we provide compelling evidence to support the idea that bestrophin-1 is the pore-forming subunit of a cell volume-regulated anion channel (VRAC) in Drosophila S2 cells. VRAC was abolished by treatment with RNAi to Drosophila bestrophin-1. VRAC was rescued by overexpressing bestrophin-1 mutants with altered biophysical properties and responsiveness to sulfhydryl reagents. In particular, the ionic selectivity of the F81C mutant changed from anionic to cationic when the channel was treated with the sulfhydryl reagent, sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES(-)) (P(Cs)/P(Cl) = 0.25 for native and 2.38 for F81C). The F81E mutant was 1.3 times more permeable to Cs(+) than Cl(-). The finding that VRAC was rescued by F81C and F81E mutants with different biophysical properties shows that bestrophin-1 is a VRAC in S2 cells and not simply a regulator or an auxiliary subunit. F81C overexpressed in HEK293 cells also exhibits a shift of ionic selectivity after MTSES(-) treatment, although the effect is quantitatively smaller than in S2 cells. To test whether bestrophins are VRACs in mammalian cells, we compared VRACs in peritoneal macrophages from wild-type mice and mice with both bestrophin-1 and bestrophin-2 disrupted (best1(-/-)/best2(-/-)). VRACs were identical in wild-type and best1(-/-)/best2(-/-) mice, showing that bestrophins are unlikely to be the classical VRAC in mammalian cells.

Show MeSH
Related in: MedlinePlus