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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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Localization of endogenous hBest1, overexpressed hBest1, and overexpressed hBest1W93C in fhRPE cells. Monolayers of fhRPE were stained for hBest1 expression (green) and examined by confocal microscopy in the X-Y (A, C, E) and X-Z (B, D, F) planes. Endogenous hBest1 (A, B) was observed to be localized to the basolateral plasma membrane, as was hBest1 in monolayers overexpressing hBest1 (C, D) or hBest1W93C (E, F). Nuclei (red) were stained for positional referencing. Scale bars: 20 µM.
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f2: Localization of endogenous hBest1, overexpressed hBest1, and overexpressed hBest1W93C in fhRPE cells. Monolayers of fhRPE were stained for hBest1 expression (green) and examined by confocal microscopy in the X-Y (A, C, E) and X-Z (B, D, F) planes. Endogenous hBest1 (A, B) was observed to be localized to the basolateral plasma membrane, as was hBest1 in monolayers overexpressing hBest1 (C, D) or hBest1W93C (E, F). Nuclei (red) were stained for positional referencing. Scale bars: 20 µM.

Mentions: As we have done in previous work [32], primary cultures of fhRPE were grown on permeable supports. These cells produce monolayer cultures that normally express hBest1 (Figure 2A,B) and can generate a TER of >400 Ω x cm2 (Figure 3B) [33,34] when measured in Ringer’s solution. hBest1 and hBest1W93C were overexpressed in the monolayers using adenovirus mediated gene transfer. Controls were performed using adenovirus-mediated gene transfer with an empty “” viral vector. We have previously demonstrated that adenovirus-mediated gene transfer causes overexpression of these proteins in fhRPE cells relative to endogenous levels of Best1 [32]. Localization of overexpressed hBest1 and hBest1W93C was previously observed to be to the basolateral plasma membrane in fhRPE cells [32] and the RPE of rat eyes [31]. We confirm this here by showing immunofluorescence and confocal microscopy of endogenous Best1 (Figure 2A,B), overexpressed hBest1 (Figure 2C,D), and overexpressed hBest1W93C (Figure 2E,F).


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)

Localization of endogenous hBest1, overexpressed hBest1, and overexpressed hBest1W93C in fhRPE cells. Monolayers of fhRPE were stained for hBest1 expression (green) and examined by confocal microscopy in the X-Y (A, C, E) and X-Z (B, D, F) planes. Endogenous hBest1 (A, B) was observed to be localized to the basolateral plasma membrane, as was hBest1 in monolayers overexpressing hBest1 (C, D) or hBest1W93C (E, F). Nuclei (red) were stained for positional referencing. Scale bars: 20 µM.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Localization of endogenous hBest1, overexpressed hBest1, and overexpressed hBest1W93C in fhRPE cells. Monolayers of fhRPE were stained for hBest1 expression (green) and examined by confocal microscopy in the X-Y (A, C, E) and X-Z (B, D, F) planes. Endogenous hBest1 (A, B) was observed to be localized to the basolateral plasma membrane, as was hBest1 in monolayers overexpressing hBest1 (C, D) or hBest1W93C (E, F). Nuclei (red) were stained for positional referencing. Scale bars: 20 µM.
Mentions: As we have done in previous work [32], primary cultures of fhRPE were grown on permeable supports. These cells produce monolayer cultures that normally express hBest1 (Figure 2A,B) and can generate a TER of >400 Ω x cm2 (Figure 3B) [33,34] when measured in Ringer’s solution. hBest1 and hBest1W93C were overexpressed in the monolayers using adenovirus mediated gene transfer. Controls were performed using adenovirus-mediated gene transfer with an empty “” viral vector. We have previously demonstrated that adenovirus-mediated gene transfer causes overexpression of these proteins in fhRPE cells relative to endogenous levels of Best1 [32]. Localization of overexpressed hBest1 and hBest1W93C was previously observed to be to the basolateral plasma membrane in fhRPE cells [32] and the RPE of rat eyes [31]. We confirm this here by showing immunofluorescence and confocal microscopy of endogenous Best1 (Figure 2A,B), overexpressed hBest1 (Figure 2C,D), and overexpressed hBest1W93C (Figure 2E,F).

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