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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 hBest1 and hBest1W93C on ATP-induced release of Ca2+ stores. A: The concentration of intracellular Ca2+ was measured using the indicator dye fura-2 in fhRPE following stimulation with 100 µM ATP. [Ca2+]i was compared between control monolayers and monolayers overexpressing hBest1 or hBest1W93C after ATP-stimulation, as well as after subsequent washout of ATP. B: Prior to ATP stimulation, mean baselines [Ca2+]i between control and overexpressing monolayers are shown. Differences between monolayers were significant (p<0.005 versus controls, n=4). Data are mean ± SEM.
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f6: Effect of hBest1 and hBest1W93C on ATP-induced release of Ca2+ stores. A: The concentration of intracellular Ca2+ was measured using the indicator dye fura-2 in fhRPE following stimulation with 100 µM ATP. [Ca2+]i was compared between control monolayers and monolayers overexpressing hBest1 or hBest1W93C after ATP-stimulation, as well as after subsequent washout of ATP. B: Prior to ATP stimulation, mean baselines [Ca2+]i between control and overexpressing monolayers are shown. Differences between monolayers were significant (p<0.005 versus controls, n=4). Data are mean ± SEM.

Mentions: The dramatic hBest1- and hBest1W93C-dependent differences observed in fhRPE monolayers in response to ATP motivated us to follow the ATP-induced changes in [Ca2+]i resulting from hBest1 or hBest1W93C overexpression using the indicator dye fura-2. Mean [Ca2+]i in fhRPE cells differed significantly between controls and cells overexpressing hBest1 or hBest1W93C, suggesting that hBest1 can affect resting [Ca2+]i in human RPE (Figure 6). Compared to controls, mean resting [Ca2+]i was substantially reduced in monolayers overexpressing hBest1 (Figure 6B). Prior to ATP stimulation, resting [Ca2+]i was elevated beyond that of controls in monolayers overexpressing hBest1W93C (Figure 6B). Measured resting [Ca2+]i was 83±3 nM for controls, 66±11 nM for hBest1, and 90±5 nM for hBest1W93C (average ± SE, n=4 experiments; Figure 6B). Following ATP stimulation, [Ca2+]i was observed to increase in controls, hBest1-overexpressing cells, and cells expressing hBest1W93C (Figure 6A). However, the increase in mean [Ca2+]i in cells overexpressing hBest1 was not sustained and [Ca2+]i returned rapidly to near resting levels. Cells expressing hBest1W93C exhibited an initial fast rise in [Ca2+]i similar to that observed in hBest1-overexpressing cells, but did not continue to increase and instead remained steady at a level approximately half of that observed in control cells (Figure 6A).


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 on ATP-induced release of Ca2+ stores. A: The concentration of intracellular Ca2+ was measured using the indicator dye fura-2 in fhRPE following stimulation with 100 µM ATP. [Ca2+]i was compared between control monolayers and monolayers overexpressing hBest1 or hBest1W93C after ATP-stimulation, as well as after subsequent washout of ATP. B: Prior to ATP stimulation, mean baselines [Ca2+]i between control and overexpressing monolayers are shown. Differences between monolayers were significant (p<0.005 versus controls, n=4). Data are mean ± SEM.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f6: Effect of hBest1 and hBest1W93C on ATP-induced release of Ca2+ stores. A: The concentration of intracellular Ca2+ was measured using the indicator dye fura-2 in fhRPE following stimulation with 100 µM ATP. [Ca2+]i was compared between control monolayers and monolayers overexpressing hBest1 or hBest1W93C after ATP-stimulation, as well as after subsequent washout of ATP. B: Prior to ATP stimulation, mean baselines [Ca2+]i between control and overexpressing monolayers are shown. Differences between monolayers were significant (p<0.005 versus controls, n=4). Data are mean ± SEM.
Mentions: The dramatic hBest1- and hBest1W93C-dependent differences observed in fhRPE monolayers in response to ATP motivated us to follow the ATP-induced changes in [Ca2+]i resulting from hBest1 or hBest1W93C overexpression using the indicator dye fura-2. Mean [Ca2+]i in fhRPE cells differed significantly between controls and cells overexpressing hBest1 or hBest1W93C, suggesting that hBest1 can affect resting [Ca2+]i in human RPE (Figure 6). Compared to controls, mean resting [Ca2+]i was substantially reduced in monolayers overexpressing hBest1 (Figure 6B). Prior to ATP stimulation, resting [Ca2+]i was elevated beyond that of controls in monolayers overexpressing hBest1W93C (Figure 6B). Measured resting [Ca2+]i was 83±3 nM for controls, 66±11 nM for hBest1, and 90±5 nM for hBest1W93C (average ± SE, n=4 experiments; Figure 6B). Following ATP stimulation, [Ca2+]i was observed to increase in controls, hBest1-overexpressing cells, and cells expressing hBest1W93C (Figure 6A). However, the increase in mean [Ca2+]i in cells overexpressing hBest1 was not sustained and [Ca2+]i returned rapidly to near resting levels. Cells expressing hBest1W93C exhibited an initial fast rise in [Ca2+]i similar to that observed in hBest1-overexpressing cells, but did not continue to increase and instead remained steady at a level approximately half of that observed in control cells (Figure 6A).

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