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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

Effect of ATP on transepithelial electrical properties of fhRPE monolayers. Representative recordings from monolayers expressing endogenous hBest1 (control) or overexpressing hBest1 or hBest1W93C (W93C) in response to a 50 µM ATP stimulus are shown in A. TEP (B) increased in controls and cells expressing hBest1W93C in response to ATP, but diminished significantly in cells overexpressing hBest1. Vertical bars in the recordings in A correspond to the voltage deflection induced by a 10 µA bipolar current pulse used to determine TER (C), which diminished precipitously in monolayers overexpressing hBest1. Isc (D) was calculated from TEP and TER. Data are representative of ≥4 experiments.
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f5: Effect of ATP on transepithelial electrical properties of fhRPE monolayers. Representative recordings from monolayers expressing endogenous hBest1 (control) or overexpressing hBest1 or hBest1W93C (W93C) in response to a 50 µM ATP stimulus are shown in A. TEP (B) increased in controls and cells expressing hBest1W93C in response to ATP, but diminished significantly in cells overexpressing hBest1. Vertical bars in the recordings in A correspond to the voltage deflection induced by a 10 µA bipolar current pulse used to determine TER (C), which diminished precipitously in monolayers overexpressing hBest1. Isc (D) was calculated from TEP and TER. Data are representative of ≥4 experiments.

Mentions: We next examined the effects of hBest1 and hBest1W93C overexpression on the responses of fhRPE cells to a physiologic stimulus that would raise [Ca2+]i, extracellular ATP. ATP has been demonstrated to increase intracellular Ca2+ in the RPE [29], a response that is impaired in Best1W93C knock-in mice [21]. Furthermore, ATP is a candidate for the LP substance [16] and is known to activate RPE Cl- conductances [29]. Figure 5 shows that stimulation of control fhRPE monolayers with 50 µM ATP results in a rapid increase in TEP (Figure 5A,B) and a modest but slower decrease in TER (Figure 5C). Isc in control monolayers was increased, reaching a plateau within 6 min of stimulus (Figure 5D). In contrast, cells overexpressing hBest1 exhibited a rapid decrease in TEP (Figure 5A,B) and TER (Figure 5C), as well as a smaller increase in Isc compared to controls (Figure 5D). The increase in Isc (Figure 5D) in cells overexpressing hBest1W93C stimulated with extracellular ATP was smaller and slower than both control and hBest1-overexpressing monolayers. This was due primarily to a smaller, slower increase in TEP (Figure 5A,B) in cells overexpressing hBest1W93C compared to controls, although the change in TER (Figure 5C) was comparable. The very large, very rapid decrease in TER (Figure 5C) in monolayers overexpressing hBest1 suggested that the tight junctions of the fhRPE had been compromised. As a result, although we show TEP (Figure 5A,B) and calculated Isc (Figure 5D) for these monolayers, these data cannot be interpreted as representing events occurring at the plasma membrane.


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 ATP on transepithelial electrical properties of fhRPE monolayers. Representative recordings from monolayers expressing endogenous hBest1 (control) or overexpressing hBest1 or hBest1W93C (W93C) in response to a 50 µM ATP stimulus are shown in A. TEP (B) increased in controls and cells expressing hBest1W93C in response to ATP, but diminished significantly in cells overexpressing hBest1. Vertical bars in the recordings in A correspond to the voltage deflection induced by a 10 µA bipolar current pulse used to determine TER (C), which diminished precipitously in monolayers overexpressing hBest1. Isc (D) was calculated from TEP and TER. Data are representative of ≥4 experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: Effect of ATP on transepithelial electrical properties of fhRPE monolayers. Representative recordings from monolayers expressing endogenous hBest1 (control) or overexpressing hBest1 or hBest1W93C (W93C) in response to a 50 µM ATP stimulus are shown in A. TEP (B) increased in controls and cells expressing hBest1W93C in response to ATP, but diminished significantly in cells overexpressing hBest1. Vertical bars in the recordings in A correspond to the voltage deflection induced by a 10 µA bipolar current pulse used to determine TER (C), which diminished precipitously in monolayers overexpressing hBest1. Isc (D) was calculated from TEP and TER. Data are representative of ≥4 experiments.
Mentions: We next examined the effects of hBest1 and hBest1W93C overexpression on the responses of fhRPE cells to a physiologic stimulus that would raise [Ca2+]i, extracellular ATP. ATP has been demonstrated to increase intracellular Ca2+ in the RPE [29], a response that is impaired in Best1W93C knock-in mice [21]. Furthermore, ATP is a candidate for the LP substance [16] and is known to activate RPE Cl- conductances [29]. Figure 5 shows that stimulation of control fhRPE monolayers with 50 µM ATP results in a rapid increase in TEP (Figure 5A,B) and a modest but slower decrease in TER (Figure 5C). Isc in control monolayers was increased, reaching a plateau within 6 min of stimulus (Figure 5D). In contrast, cells overexpressing hBest1 exhibited a rapid decrease in TEP (Figure 5A,B) and TER (Figure 5C), as well as a smaller increase in Isc compared to controls (Figure 5D). The increase in Isc (Figure 5D) in cells overexpressing hBest1W93C stimulated with extracellular ATP was smaller and slower than both control and hBest1-overexpressing monolayers. This was due primarily to a smaller, slower increase in TEP (Figure 5A,B) in cells overexpressing hBest1W93C compared to controls, although the change in TER (Figure 5C) was comparable. The very large, very rapid decrease in TER (Figure 5C) in monolayers overexpressing hBest1 suggested that the tight junctions of the fhRPE had been compromised. As a result, although we show TEP (Figure 5A,B) and calculated Isc (Figure 5D) for these monolayers, these data cannot be interpreted as representing events occurring at the plasma membrane.

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