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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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Effect of hBest1 and hBest1W93C expression on Ca2+-stimulated changes in transepithelial electrical properties. A: fhRPE cells were treated with 500 nM ionomycin, which caused a biphasic increase in TEP. Each phase of the response was measured at the indicated point. When the TEP reached a plateau, 100 µM NFA was added to the bathing solution to inhibit calcium-activated anion channel activity. Representative recordings of responses are shown in (B), demonstrating that overexpression of either hBest1 or hBest1W93C resulted in a suppression of the overall response, and that the response to the addition of NFA was similar in control and monolayers overexpressing hBest1. Isc (C) was determined from TEP (D) and TER (E) for P0, P1, P2, and NFA. Responses were elevated at P0 for hBest1-overexpressing monolayers compared to controls and monolayers overexpressing hBest1W93C. However, at P1, P2, and NFA, Isc was not significantly different between controls and monolayers overexpressing hBest1. Neither ionomycin nor NFA had a significant effect on the transepithelial electrical properties of fhRPE monolayers expressing hBest1W93C. Compared to control and hBest1-overexpressing monolayers, TEP (D) and Isc (C) were significantly reduced for hBest1W93C monolayers at P0, P1, P2, and NFA. For both control and hBest1-overexpressing monolayers, the differences in TEP (D) and Isc (C) between different time points were significant (p<0.05). Data are mean ± SD, n=9 for control, n=5 for hBest1, and n=6 for W93C.
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f4: Effect of hBest1 and hBest1W93C expression on Ca2+-stimulated changes in transepithelial electrical properties. A: fhRPE cells were treated with 500 nM ionomycin, which caused a biphasic increase in TEP. Each phase of the response was measured at the indicated point. When the TEP reached a plateau, 100 µM NFA was added to the bathing solution to inhibit calcium-activated anion channel activity. Representative recordings of responses are shown in (B), demonstrating that overexpression of either hBest1 or hBest1W93C resulted in a suppression of the overall response, and that the response to the addition of NFA was similar in control and monolayers overexpressing hBest1. Isc (C) was determined from TEP (D) and TER (E) for P0, P1, P2, and NFA. Responses were elevated at P0 for hBest1-overexpressing monolayers compared to controls and monolayers overexpressing hBest1W93C. However, at P1, P2, and NFA, Isc was not significantly different between controls and monolayers overexpressing hBest1. Neither ionomycin nor NFA had a significant effect on the transepithelial electrical properties of fhRPE monolayers expressing hBest1W93C. Compared to control and hBest1-overexpressing monolayers, TEP (D) and Isc (C) were significantly reduced for hBest1W93C monolayers at P0, P1, P2, and NFA. For both control and hBest1-overexpressing monolayers, the differences in TEP (D) and Isc (C) between different time points were significant (p<0.05). Data are mean ± SD, n=9 for control, n=5 for hBest1, and n=6 for W93C.

Mentions: We have previously shown that, following ATP stimulation, Ca2+ signaling is suppressed in RPE from mice heterozygous or homozygous for the W93C mutation [21]. To assess whether the diminished TEP (Figure 3A) and Isc (Figure 3C) observed in monolayers overexpressing hBest1W93C was due to altered Ca2+ signaling, we eliminated Ca2+ influx as a limit by treating monolayer cultures with the Ca2+ ionophore ionomycin (Figure 4). This would be predicted to have the effect of overcoming any suppressive effects hBest1 could exert on [Ca2+]i and, as such, any observed changes would reflect its activity as an anion channel.


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)

Effect of hBest1 and hBest1W93C expression on Ca2+-stimulated changes in transepithelial electrical properties. A: fhRPE cells were treated with 500 nM ionomycin, which caused a biphasic increase in TEP. Each phase of the response was measured at the indicated point. When the TEP reached a plateau, 100 µM NFA was added to the bathing solution to inhibit calcium-activated anion channel activity. Representative recordings of responses are shown in (B), demonstrating that overexpression of either hBest1 or hBest1W93C resulted in a suppression of the overall response, and that the response to the addition of NFA was similar in control and monolayers overexpressing hBest1. Isc (C) was determined from TEP (D) and TER (E) for P0, P1, P2, and NFA. Responses were elevated at P0 for hBest1-overexpressing monolayers compared to controls and monolayers overexpressing hBest1W93C. However, at P1, P2, and NFA, Isc was not significantly different between controls and monolayers overexpressing hBest1. Neither ionomycin nor NFA had a significant effect on the transepithelial electrical properties of fhRPE monolayers expressing hBest1W93C. Compared to control and hBest1-overexpressing monolayers, TEP (D) and Isc (C) were significantly reduced for hBest1W93C monolayers at P0, P1, P2, and NFA. For both control and hBest1-overexpressing monolayers, the differences in TEP (D) and Isc (C) between different time points were significant (p<0.05). Data are mean ± SD, n=9 for control, n=5 for hBest1, and n=6 for W93C.
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f4: Effect of hBest1 and hBest1W93C expression on Ca2+-stimulated changes in transepithelial electrical properties. A: fhRPE cells were treated with 500 nM ionomycin, which caused a biphasic increase in TEP. Each phase of the response was measured at the indicated point. When the TEP reached a plateau, 100 µM NFA was added to the bathing solution to inhibit calcium-activated anion channel activity. Representative recordings of responses are shown in (B), demonstrating that overexpression of either hBest1 or hBest1W93C resulted in a suppression of the overall response, and that the response to the addition of NFA was similar in control and monolayers overexpressing hBest1. Isc (C) was determined from TEP (D) and TER (E) for P0, P1, P2, and NFA. Responses were elevated at P0 for hBest1-overexpressing monolayers compared to controls and monolayers overexpressing hBest1W93C. However, at P1, P2, and NFA, Isc was not significantly different between controls and monolayers overexpressing hBest1. Neither ionomycin nor NFA had a significant effect on the transepithelial electrical properties of fhRPE monolayers expressing hBest1W93C. Compared to control and hBest1-overexpressing monolayers, TEP (D) and Isc (C) were significantly reduced for hBest1W93C monolayers at P0, P1, P2, and NFA. For both control and hBest1-overexpressing monolayers, the differences in TEP (D) and Isc (C) between different time points were significant (p<0.05). Data are mean ± SD, n=9 for control, n=5 for hBest1, and n=6 for W93C.
Mentions: We have previously shown that, following ATP stimulation, Ca2+ signaling is suppressed in RPE from mice heterozygous or homozygous for the W93C mutation [21]. To assess whether the diminished TEP (Figure 3A) and Isc (Figure 3C) observed in monolayers overexpressing hBest1W93C was due to altered Ca2+ signaling, we eliminated Ca2+ influx as a limit by treating monolayer cultures with the Ca2+ ionophore ionomycin (Figure 4). This would be predicted to have the effect of overcoming any suppressive effects hBest1 could exert on [Ca2+]i and, as such, any observed changes would reflect its activity as an anion channel.

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