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Bestrophin-1 influences transepithelial electrical properties and Ca2+ signaling in human retinal pigment epithelium.

Marmorstein AD, Kinnick TR, Stanton JB, Johnson AA, Lynch RM, Marmorstein LY - Mol. Vis. (2015)

Bottom Line: Substitution of Cl- in the bath media resulted in a significant reduction of Isc in monolayers overexpressing Best1, but no significant Isc change in monolayers expressing Best1W93C.We removed Ca2+ as a limit on transepithelial electrical properties by treating cells with ionomycin, and found that changes in Isc and TEP for monolayers expressing Best1 were absent in monolayers expressing Best1W93C.Similarly, inhibition of calcium-activated anion channels with niflumic acid reduced both Isc and TEP of control and Best1 monolayers, but did not notably affect Best1W93C monolayers.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Mayo Clinic, Rochester, MN.

ABSTRACT

Purpose: Mutations in BEST1, encoding Bestrophin-1 (Best1), cause Best vitelliform macular dystrophy (BVMD) and other inherited retinal degenerative diseases. Best1 is an integral membrane protein localized to the basolateral plasma membrane of the retinal pigment epithelium (RPE). Data from numerous in vitro and in vivo models have demonstrated that Best1 regulates intracellular Ca2+ levels. Although it is known from in vitro and crystal structure data that Best1 is also a calcium-activated anion channel, evidence for Best1 functioning as a channel in human RPE is lacking. To assess Best1-associated channel activity in the RPE, we examined the transepithelial electrical properties of fetal human RPE (fhRPE) cells, which express endogenous Best1.

Methods: Using adenovirus-mediated gene transfer, we overexpressed Best1 and the BVMD mutant Best1W93C in fhRPE cells and assessed resting transepithelial potential (TEP), transepithelial resistance, short circuit current (Isc), and intracellular Ca2+ levels. Cl- currents were directly measured in transfected HEK293 cells using whole-cell patch clamp.

Results: Best1W93C showed ablated Cl- currents and, when co-expressed, suppressed the channel activity of Best1 in HEK293 cells. In fhRPE, overexpression of Best1 increased TEP and Isc, while Best1W93C diminished TEP and Isc. Substitution of Cl- in the bath media resulted in a significant reduction of Isc in monolayers overexpressing Best1, but no significant Isc change in monolayers expressing Best1W93C. We removed Ca2+ as a limit on transepithelial electrical properties by treating cells with ionomycin, and found that changes in Isc and TEP for monolayers expressing Best1 were absent in monolayers expressing Best1W93C. Similarly, inhibition of calcium-activated anion channels with niflumic acid reduced both Isc and TEP of control and Best1 monolayers, but did not notably affect Best1W93C monolayers. Stimulation with extracellular ATP induced an increase in TEP in control monolayers that was greater than that observed in those expressing Best1(W93C). Examination of [Ca2+]i following ATP stimulation demonstrated that the expression of Best1W93C impaired intracellular Ca2+ signaling.

Conclusions: These data indicate that Best1 activity strongly influences electrophysiology and Ca2+ signaling in RPE cells, and that a common BVMD mutation disrupts both of these parameters. Our findings support the hypothesis that Best1 functions as an anion channel in human RPE.

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Related in: MedlinePlus

Whole-cell chloride currents mediated by Best1-CFP, Best1-YFP, and Best1W93C-YFP. A: Whole-cell patch-clamp recordings were performed on HEK293 cells transfected with Best1-CFP, Best1-YFP, or Best1-CFP and Best1-YFP. Cells expressing fluorescently tagged Best1 exhibited robust chloride currents compared to untransfected cells recorded from the same plate. B: In contrast, Best1W93C exhibited a significant loss of anion channel activity compared to Best1-YFP (B), and dominantly inhibited the anion channel activity of wild-type Best1-YFP when co-expressed (C).
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f1: Whole-cell chloride currents mediated by Best1-CFP, Best1-YFP, and Best1W93C-YFP. A: Whole-cell patch-clamp recordings were performed on HEK293 cells transfected with Best1-CFP, Best1-YFP, or Best1-CFP and Best1-YFP. Cells expressing fluorescently tagged Best1 exhibited robust chloride currents compared to untransfected cells recorded from the same plate. B: In contrast, Best1W93C exhibited a significant loss of anion channel activity compared to Best1-YFP (B), and dominantly inhibited the anion channel activity of wild-type Best1-YFP when co-expressed (C).

Mentions: To date, all disease-causing mutants of hBest1 tested have exhibited diminished anion channel activity [17,18]. We confirmed this for hBest1W93C by performing a whole-cell patch clamp of HEK293 cells heterologously expressing hBest1 fused with YFP or CFP (Best1-YFP, Best1-CFP), Best1W93C-YFP, or YFP alone. We have previously shown that the addition of a CFP or YFP tag to the C-terminus of hBest1 does not mitigate its ability to function as an anion channel [35]. As shown in Figure 1A, Best1-YFP and Best1-CFP produce significant Cl- currents, which are nearly absent from control cells. Cells transfected with Best1-YFP and Best1-CFP produced currents with amplitudes between those of Best1-YFP and Best1-CFP (Figure 1A). In contrast, Best1W93C-YFP produced little current in comparison to Best1-YFP (Figure 1B). In cells expressing both Best1W93C-YFP and Best1-CFP, Cl- currents were substantially diminished relative to cells expressing Best1-YFP and Best1-CFP (Figure 1C). This is the first time that differently tagged forms of fluorescent Best1 have been co-expressed together for whole-cell patch-clamp analysis, and the first time that such an analysis was done where WT and mutant Best1 could both be confirmed to be co-expressed. This is particularly significant, as it was not uncommon to observe cells in co-transfected populations only expressing one of the two Best1 variants (e.g., Best1W93C-YFP only or Best1-CFP only).


Bestrophin-1 influences transepithelial electrical properties and Ca2+ signaling in human retinal pigment epithelium.

Marmorstein AD, Kinnick TR, Stanton JB, Johnson AA, Lynch RM, Marmorstein LY - Mol. Vis. (2015)

Whole-cell chloride currents mediated by Best1-CFP, Best1-YFP, and Best1W93C-YFP. A: Whole-cell patch-clamp recordings were performed on HEK293 cells transfected with Best1-CFP, Best1-YFP, or Best1-CFP and Best1-YFP. Cells expressing fluorescently tagged Best1 exhibited robust chloride currents compared to untransfected cells recorded from the same plate. B: In contrast, Best1W93C exhibited a significant loss of anion channel activity compared to Best1-YFP (B), and dominantly inhibited the anion channel activity of wild-type Best1-YFP when co-expressed (C).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4390793&req=5

f1: Whole-cell chloride currents mediated by Best1-CFP, Best1-YFP, and Best1W93C-YFP. A: Whole-cell patch-clamp recordings were performed on HEK293 cells transfected with Best1-CFP, Best1-YFP, or Best1-CFP and Best1-YFP. Cells expressing fluorescently tagged Best1 exhibited robust chloride currents compared to untransfected cells recorded from the same plate. B: In contrast, Best1W93C exhibited a significant loss of anion channel activity compared to Best1-YFP (B), and dominantly inhibited the anion channel activity of wild-type Best1-YFP when co-expressed (C).
Mentions: To date, all disease-causing mutants of hBest1 tested have exhibited diminished anion channel activity [17,18]. We confirmed this for hBest1W93C by performing a whole-cell patch clamp of HEK293 cells heterologously expressing hBest1 fused with YFP or CFP (Best1-YFP, Best1-CFP), Best1W93C-YFP, or YFP alone. We have previously shown that the addition of a CFP or YFP tag to the C-terminus of hBest1 does not mitigate its ability to function as an anion channel [35]. As shown in Figure 1A, Best1-YFP and Best1-CFP produce significant Cl- currents, which are nearly absent from control cells. Cells transfected with Best1-YFP and Best1-CFP produced currents with amplitudes between those of Best1-YFP and Best1-CFP (Figure 1A). In contrast, Best1W93C-YFP produced little current in comparison to Best1-YFP (Figure 1B). In cells expressing both Best1W93C-YFP and Best1-CFP, Cl- currents were substantially diminished relative to cells expressing Best1-YFP and Best1-CFP (Figure 1C). This is the first time that differently tagged forms of fluorescent Best1 have been co-expressed together for whole-cell patch-clamp analysis, and the first time that such an analysis was done where WT and mutant Best1 could both be confirmed to be co-expressed. This is particularly significant, as it was not uncommon to observe cells in co-transfected populations only expressing one of the two Best1 variants (e.g., Best1W93C-YFP only or Best1-CFP only).

Bottom Line: Substitution of Cl- in the bath media resulted in a significant reduction of Isc in monolayers overexpressing Best1, but no significant Isc change in monolayers expressing Best1W93C.We removed Ca2+ as a limit on transepithelial electrical properties by treating cells with ionomycin, and found that changes in Isc and TEP for monolayers expressing Best1 were absent in monolayers expressing Best1W93C.Similarly, inhibition of calcium-activated anion channels with niflumic acid reduced both Isc and TEP of control and Best1 monolayers, but did not notably affect Best1W93C monolayers.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Mayo Clinic, Rochester, MN.

ABSTRACT

Purpose: Mutations in BEST1, encoding Bestrophin-1 (Best1), cause Best vitelliform macular dystrophy (BVMD) and other inherited retinal degenerative diseases. Best1 is an integral membrane protein localized to the basolateral plasma membrane of the retinal pigment epithelium (RPE). Data from numerous in vitro and in vivo models have demonstrated that Best1 regulates intracellular Ca2+ levels. Although it is known from in vitro and crystal structure data that Best1 is also a calcium-activated anion channel, evidence for Best1 functioning as a channel in human RPE is lacking. To assess Best1-associated channel activity in the RPE, we examined the transepithelial electrical properties of fetal human RPE (fhRPE) cells, which express endogenous Best1.

Methods: Using adenovirus-mediated gene transfer, we overexpressed Best1 and the BVMD mutant Best1W93C in fhRPE cells and assessed resting transepithelial potential (TEP), transepithelial resistance, short circuit current (Isc), and intracellular Ca2+ levels. Cl- currents were directly measured in transfected HEK293 cells using whole-cell patch clamp.

Results: Best1W93C showed ablated Cl- currents and, when co-expressed, suppressed the channel activity of Best1 in HEK293 cells. In fhRPE, overexpression of Best1 increased TEP and Isc, while Best1W93C diminished TEP and Isc. Substitution of Cl- in the bath media resulted in a significant reduction of Isc in monolayers overexpressing Best1, but no significant Isc change in monolayers expressing Best1W93C. We removed Ca2+ as a limit on transepithelial electrical properties by treating cells with ionomycin, and found that changes in Isc and TEP for monolayers expressing Best1 were absent in monolayers expressing Best1W93C. Similarly, inhibition of calcium-activated anion channels with niflumic acid reduced both Isc and TEP of control and Best1 monolayers, but did not notably affect Best1W93C monolayers. Stimulation with extracellular ATP induced an increase in TEP in control monolayers that was greater than that observed in those expressing Best1(W93C). Examination of [Ca2+]i following ATP stimulation demonstrated that the expression of Best1W93C impaired intracellular Ca2+ signaling.

Conclusions: These data indicate that Best1 activity strongly influences electrophysiology and Ca2+ signaling in RPE cells, and that a common BVMD mutation disrupts both of these parameters. Our findings support the hypothesis that Best1 functions as an anion channel in human RPE.

Show MeSH
Related in: MedlinePlus