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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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VRAC in peritoneal macrophages from wild-type and mBest1−/−-mBest2−/− mice. (A, C, and E) Wild-type mice. (B, D, and F) mBest1−/−-mBest2−/− mice. The intracellular solution of all recordings was held constant at 306 mosmol kg−1, whereas the osmolality of the extracellular solution was altered to achieve different osmotic pressures. (A and B) Current traces under isosmotic conditions. (C and D) Current traces in Δ40 mosmol kg−1 hyposmotic conditions (266 mosmol kg−1 extracellular). (E and F) Average current–voltage relationships under Δ70 mosmol kg−1 hyposmotic (open symbols; 234 mosmol kg−1extracellular) and −Δ20 mosmol kg−1 hyperosmotic (filled symbols; 326 mosmol kg−1 extracellular) conditions.
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fig7: VRAC in peritoneal macrophages from wild-type and mBest1−/−-mBest2−/− mice. (A, C, and E) Wild-type mice. (B, D, and F) mBest1−/−-mBest2−/− mice. The intracellular solution of all recordings was held constant at 306 mosmol kg−1, whereas the osmolality of the extracellular solution was altered to achieve different osmotic pressures. (A and B) Current traces under isosmotic conditions. (C and D) Current traces in Δ40 mosmol kg−1 hyposmotic conditions (266 mosmol kg−1 extracellular). (E and F) Average current–voltage relationships under Δ70 mosmol kg−1 hyposmotic (open symbols; 234 mosmol kg−1extracellular) and −Δ20 mosmol kg−1 hyperosmotic (filled symbols; 326 mosmol kg−1 extracellular) conditions.

Mentions: To test whether bestrophins are responsible for VRAC in mammals, we measured VRAC in peritoneal macrophages from wild-type mice and mice that had both mBest1 and mBest2 disrupted (Fig. 7). VRAC in these cells was activated by changing extracellular osmolality from either 306 mosmol kg−1 (isosmotic) or 326 mosmol kg−1 (Δ20 hyperosmotic) to 266 mosmol kg−1 (Δ40 hyposmotic) or 234 mosmol kg−1 (Δ70 hyposmotic). The amplitude of VRAC in the wild-type and knockout animals was statistically the same. These results show that mBest1 and mBest2 are not required for VRAC in peritoneal macrophages and suggest that other kinds of channels are responsible for VRAC in mammalian cells.


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

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

VRAC in peritoneal macrophages from wild-type and mBest1−/−-mBest2−/− mice. (A, C, and E) Wild-type mice. (B, D, and F) mBest1−/−-mBest2−/− mice. The intracellular solution of all recordings was held constant at 306 mosmol kg−1, whereas the osmolality of the extracellular solution was altered to achieve different osmotic pressures. (A and B) Current traces under isosmotic conditions. (C and D) Current traces in Δ40 mosmol kg−1 hyposmotic conditions (266 mosmol kg−1 extracellular). (E and F) Average current–voltage relationships under Δ70 mosmol kg−1 hyposmotic (open symbols; 234 mosmol kg−1extracellular) and −Δ20 mosmol kg−1 hyperosmotic (filled symbols; 326 mosmol kg−1 extracellular) conditions.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2571971&req=5

fig7: VRAC in peritoneal macrophages from wild-type and mBest1−/−-mBest2−/− mice. (A, C, and E) Wild-type mice. (B, D, and F) mBest1−/−-mBest2−/− mice. The intracellular solution of all recordings was held constant at 306 mosmol kg−1, whereas the osmolality of the extracellular solution was altered to achieve different osmotic pressures. (A and B) Current traces under isosmotic conditions. (C and D) Current traces in Δ40 mosmol kg−1 hyposmotic conditions (266 mosmol kg−1 extracellular). (E and F) Average current–voltage relationships under Δ70 mosmol kg−1 hyposmotic (open symbols; 234 mosmol kg−1extracellular) and −Δ20 mosmol kg−1 hyperosmotic (filled symbols; 326 mosmol kg−1 extracellular) conditions.
Mentions: To test whether bestrophins are responsible for VRAC in mammals, we measured VRAC in peritoneal macrophages from wild-type mice and mice that had both mBest1 and mBest2 disrupted (Fig. 7). VRAC in these cells was activated by changing extracellular osmolality from either 306 mosmol kg−1 (isosmotic) or 326 mosmol kg−1 (Δ20 hyperosmotic) to 266 mosmol kg−1 (Δ40 hyposmotic) or 234 mosmol kg−1 (Δ70 hyposmotic). The amplitude of VRAC in the wild-type and knockout animals was statistically the same. These results show that mBest1 and mBest2 are not required for VRAC in peritoneal macrophages and suggest that other kinds of channels are responsible for VRAC in mammalian cells.

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