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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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Summary of effects of mutations and sulfhydryl modification. The percent change was calculated using the following equation: (IMTS − Ibefore) / Ibefore × 100% for steady-state VRAC currents measured at 100 (A) and −100 mV (B), respectively. The effect of MTSES− is shown in open bars, the peak stimulation after applying MTSET+ is shown as MTSETpeak, and the stabilized level in MTSET+ is shown as MTSETstable. (C) Effect of MTS treatment on Erev. The change in Erev is the difference between Erev before and after MTS treatment. The reversal potential of F81E currents (without MTS treatment) is shown with a cross-hatched bar. (D) Rectification ratio of native, F81C, and F81E VRAC currents. The rectification ratio was calculated as the absolute value of the VRAC current at +100 mV divided by the current at −100 mV for native, F81C, and F81E currents. Data are represented in mean ± SEM. **, significantly different at P < 0.01 level.
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fig3: Summary of effects of mutations and sulfhydryl modification. The percent change was calculated using the following equation: (IMTS − Ibefore) / Ibefore × 100% for steady-state VRAC currents measured at 100 (A) and −100 mV (B), respectively. The effect of MTSES− is shown in open bars, the peak stimulation after applying MTSET+ is shown as MTSETpeak, and the stabilized level in MTSET+ is shown as MTSETstable. (C) Effect of MTS treatment on Erev. The change in Erev is the difference between Erev before and after MTS treatment. The reversal potential of F81E currents (without MTS treatment) is shown with a cross-hatched bar. (D) Rectification ratio of native, F81C, and F81E VRAC currents. The rectification ratio was calculated as the absolute value of the VRAC current at +100 mV divided by the current at −100 mV for native, F81C, and F81E currents. Data are represented in mean ± SEM. **, significantly different at P < 0.01 level.

Mentions: To test if dBest1 is the VRAC in Drosophila S2 cells and not simply a regulator of an endogenous VRAC, we examined whether VRAC could be rescued by expressing a mutant dBest1 that had altered biophysical properties. To do this, endogenous dBest1 was first knocked down by double-stranded interfering RNA to a portion of the 5′ UTR of dBest1, as described previously (Chien and Hartzell, 2007). To verify that the dBest1 RNAi was effective in knocking down the current, RNAi-treated cells were patch clamped in each experiment (Fig. 1 B). Then, dBest1 (wild-type, F81C, F81E, or F81L) was expressed by transient transfection. Both the native and rescued VRAC current was <300 pA under isosmotic conditions (Fig. 1 A) but was activated when the extracellular solution was 40 mosmol kg−1 hyposmotic relative to the internal solution (I340/E300) (Fig. 1 B). As expected, the dBest1-F81C current had different properties than the native VRAC current. The native VRAC current had an outwardly rectifying, S-shaped I-V curve, whereas the F81C current was inwardly rectifying (Figs. 1 B and 3 D). This inwardly rectifying I-V of F81C was similar to the Ca2+-activated dBest1-F81C current expressed heterologously in HEK cells (Chien et al., 2006). Transfection with the F81L mutant produced no current (67.0 ±26.3 pA; n = 5). In contrast, F81C currents increased with time coordinately with cell swelling over several minutes. Immediately after patch break, the F81C currents were 0.4 ± 0.1 nA at +100 mV. The currents then activated slowly to a mean plateau amplitude of 1.0 ± 0.3 nA (n = 7) with an average half-time of ∼1.5 min (Fig. 1 C). The activation of the F81C current was coupled to cell swelling. On average, F81C cells swelled 33.6 ± 0.7% (n = 7) when their currents were fully activated with Δ40 mosmol kg−1 osmotic pressure (Fig. 1 D). F81C currents were volume sensitive because the current failed to activate under isosmotic conditions (Δ0 mosmol kg−1, E300/I300) and remained <0.3 nA (n = 6) throughout ∼5 min of recording. Cell swelling was not observed with these cells in isosmotic solutions. Instead, their volume decreased 13.8 ± 2.7% by the end of the recording.


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

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

Summary of effects of mutations and sulfhydryl modification. The percent change was calculated using the following equation: (IMTS − Ibefore) / Ibefore × 100% for steady-state VRAC currents measured at 100 (A) and −100 mV (B), respectively. The effect of MTSES− is shown in open bars, the peak stimulation after applying MTSET+ is shown as MTSETpeak, and the stabilized level in MTSET+ is shown as MTSETstable. (C) Effect of MTS treatment on Erev. The change in Erev is the difference between Erev before and after MTS treatment. The reversal potential of F81E currents (without MTS treatment) is shown with a cross-hatched bar. (D) Rectification ratio of native, F81C, and F81E VRAC currents. The rectification ratio was calculated as the absolute value of the VRAC current at +100 mV divided by the current at −100 mV for native, F81C, and F81E currents. Data are represented in mean ± SEM. **, significantly different at P < 0.01 level.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: Summary of effects of mutations and sulfhydryl modification. The percent change was calculated using the following equation: (IMTS − Ibefore) / Ibefore × 100% for steady-state VRAC currents measured at 100 (A) and −100 mV (B), respectively. The effect of MTSES− is shown in open bars, the peak stimulation after applying MTSET+ is shown as MTSETpeak, and the stabilized level in MTSET+ is shown as MTSETstable. (C) Effect of MTS treatment on Erev. The change in Erev is the difference between Erev before and after MTS treatment. The reversal potential of F81E currents (without MTS treatment) is shown with a cross-hatched bar. (D) Rectification ratio of native, F81C, and F81E VRAC currents. The rectification ratio was calculated as the absolute value of the VRAC current at +100 mV divided by the current at −100 mV for native, F81C, and F81E currents. Data are represented in mean ± SEM. **, significantly different at P < 0.01 level.
Mentions: To test if dBest1 is the VRAC in Drosophila S2 cells and not simply a regulator of an endogenous VRAC, we examined whether VRAC could be rescued by expressing a mutant dBest1 that had altered biophysical properties. To do this, endogenous dBest1 was first knocked down by double-stranded interfering RNA to a portion of the 5′ UTR of dBest1, as described previously (Chien and Hartzell, 2007). To verify that the dBest1 RNAi was effective in knocking down the current, RNAi-treated cells were patch clamped in each experiment (Fig. 1 B). Then, dBest1 (wild-type, F81C, F81E, or F81L) was expressed by transient transfection. Both the native and rescued VRAC current was <300 pA under isosmotic conditions (Fig. 1 A) but was activated when the extracellular solution was 40 mosmol kg−1 hyposmotic relative to the internal solution (I340/E300) (Fig. 1 B). As expected, the dBest1-F81C current had different properties than the native VRAC current. The native VRAC current had an outwardly rectifying, S-shaped I-V curve, whereas the F81C current was inwardly rectifying (Figs. 1 B and 3 D). This inwardly rectifying I-V of F81C was similar to the Ca2+-activated dBest1-F81C current expressed heterologously in HEK cells (Chien et al., 2006). Transfection with the F81L mutant produced no current (67.0 ±26.3 pA; n = 5). In contrast, F81C currents increased with time coordinately with cell swelling over several minutes. Immediately after patch break, the F81C currents were 0.4 ± 0.1 nA at +100 mV. The currents then activated slowly to a mean plateau amplitude of 1.0 ± 0.3 nA (n = 7) with an average half-time of ∼1.5 min (Fig. 1 C). The activation of the F81C current was coupled to cell swelling. On average, F81C cells swelled 33.6 ± 0.7% (n = 7) when their currents were fully activated with Δ40 mosmol kg−1 osmotic pressure (Fig. 1 D). F81C currents were volume sensitive because the current failed to activate under isosmotic conditions (Δ0 mosmol kg−1, E300/I300) and remained <0.3 nA (n = 6) throughout ∼5 min of recording. Cell swelling was not observed with these cells in isosmotic solutions. Instead, their volume decreased 13.8 ± 2.7% by the end of the recording.

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