May 2007
Volume 48, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2007
Implication for an Intracellular Functional Role of Bestrophin-1
Author Affiliations & Notes
  • S. Krejcova
    Experimental Ophthalmology, University Hospital Hamburg - Eppendorf, Hamburg, Germany
  • R. Neussert
    Experimental Ophthalmology, University Hospital Hamburg - Eppendorf, Hamburg, Germany
  • B. Grafelmann
    Experimental Ophthalmology, University Hospital Hamburg - Eppendorf, Hamburg, Germany
  • S. Wimmers
    Experimental Ophthalmology, University Hospital Hamburg - Eppendorf, Hamburg, Germany
  • O. Strauss
    Experimental Ophthalmology, University Hospital Hamburg - Eppendorf, Hamburg, Germany
  • Footnotes
    Commercial Relationships S. Krejcova, None; R. Neussert, None; B. Grafelmann, None; S. Wimmers, None; O. Strauss, None.
  • Footnotes
    Support DFG STR480/9-1, ProRetina
Investigative Ophthalmology & Visual Science May 2007, Vol.48, 2529. doi:
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      S. Krejcova, R. Neussert, B. Grafelmann, S. Wimmers, O. Strauss; Implication for an Intracellular Functional Role of Bestrophin-1. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2529.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose:: Vitelliform macular dystrophy (VMD2) also known as Best macular dystrophy (BMD) is an autosomal dominant disorder characterized by egg-yolk macular lesions, decrease light peak in the electro-oculogram and juvenile age of onset. VMD2 gene encodes bestrophin-1 is expressed in retinal pigment epithelium (RPE) cells. Bestrophin function has been proposed as Cl- channel or to have regulatory effect on voltage dependent Ca2+ channels. We investigated the expression, subcellular localization and intracellular function of endogenous and heterologously expressed bestrophin.

Methods:: Human RPE cells were isolated from adult donor eyes. After removing the vitreous and retina the RPE cells were brushed off and grown in cell culture. Protein analysis of endogenous bestrophin-1 from freshly isolated RPE cells of adult human donor eyes, or heterologously expressed were performed with equal amounts of protein extracts from different cell compartments. Ca2+ imaging was performed on murine RPE cells isolated and held in culture for 3-4 weeks prior to measurement to obtain a confluent culture. The ATP induced pattern of intracellular free calcium concentration ([Ca2+]i) changes were monitored using the Fura-2 Ca2+ imaging method.

Results:: The subcellular distribution of endogenous bestrophin-1 in human RPE cells revealed cytoplasmic expression pattern. Detection of heterologously expressed bestrophin revealed a comparable pattern in cytoplasmic compartment using bestrophin-eYFP fusion construct. The fluorescence signal in HEK293, CHO-K1 and ARPE-19 cells were seenperinuclear. Cytosolic localization of endogenous bestrophin-1 freshly isolated from human RPE cells, and also heterologously expressed was confirmed by western blot analysis and support the data obtained from immunocytochemistry. Here, bestrophin-1 is expressed in different subcellular fraction containing cell organelles. In RPE cells from both, wt and Vmd2 deficient mice, application of ATP (100µM) resulted in a fast increase in [Ca2+]i. After depletion of cytosolic Ca2+ stores by Thapsigargin (1µM) application of ATP did not result in this rise in [Ca2+]i. However wt and Vmd2 deficient RPE cells were different in their character of ATP induced [Ca2+]i response. Vmd2 deficient cells showed a faster recovery from increase in [Ca2+]i .

Conclusions:: Large amounts bestrophin-1 protein are localized in fractions containing cell organelles. Functional data from recruitment of Ca2+ from cytosolic stores in the absence of bestrophin-1 provide further evidence of physiological implication of this localization.

Keywords: retinal pigment epithelium • calcium • ion channels 
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