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Dive into the research topics where Vladimir M. Milenkovic is active.

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Featured researches published by Vladimir M. Milenkovic.


The Journal of Neuroscience | 2009

TMEM16B, A Novel Protein with Calcium-Dependent Chloride Channel Activity, Associates with a Presynaptic Protein Complex in Photoreceptor Terminals

Heidi Stöhr; Julia B. Heisig; Peter M. Benz; Simon Schöberl; Vladimir M. Milenkovic; Olaf Strauss; Wendy M. Aartsen; Jan Wijnholds; Bernhard H. F. Weber; Heidi L. Schulz

Photoreceptor ribbon synapses release glutamate in response to graded changes in membrane potential evoked by vast, logarithmically scalable light intensities. Neurotransmitter release is modulated by intracellular calcium levels. Large Ca2+-dependent chloride currents are important regulators of synaptic transmission from photoreceptors to second-order neurons; the molecular basis underlying these currents is unclear. We cloned human and mouse TMEM16B, a member of the TMEM16 family of transmembrane proteins, and show that it is abundantly present in the photoreceptor synaptic terminals in mouse retina. TMEM16B colocalizes with adaptor proteins PSD95, VELI3, and MPP4 at the ribbon synapses and contains a consensus PDZ class I binding motif capable of interacting with PDZ domains of PSD95. Furthermore, TMEM16B is lost from photoreceptor membranes of MPP4-deficient mice. This suggests that TMEM16B is a novel component of a presynaptic protein complex recruited to specialized plasma membrane domains of photoreceptors. TMEM16B confers Ca2+-dependent chloride currents when overexpressed in mammalian cells as measured by halide sensitive fluorescent protein assays and whole-cell patch-clamp recordings. The compartmentalized localization and the electrophysiological properties suggest TMEM16B to be a strong candidate for the long sought-after Ca2+-dependent chloride channel in the photoreceptor synapse.


Journal of Biological Chemistry | 2007

Insertion and Topology of Normal and Mutant Bestrophin-1 in the Endoplasmic Reticulum Membrane

Vladimir M. Milenkovic; Andrea Rivera; Franziska Horling; Bernhard H. F. Weber

The vitelliform macular dystrophy type 2 (VMD2) gene mutated in Best macular dystrophy encodes a 585-amino acid putative transmembrane protein termed bestrophin-1. The vast majority of known disease-associated alterations are of the missense type, which cluster near predicted transmembrane domains (TMDs). To investigate bestrophin-1 membrane topology and to assess consequences of point mutations on membrane integration, we have analyzed the insertion of putative TMDs into the endoplasmic reticulum (ER) membrane. Out of six potential TMDs, our data suggest a topological model of bestrophin-1 with four transmembrane-spanning segments and one large cytoplasmatic loop between putative TMD2 and TMD5. Consequently, a relatively hydrophobic segment containing putative TMD3 (aa 130-149) and TMD4 (aa 179-201) is located within the cytoplasm. Furthermore, we show that three out of 18 disease-associated alterations investigated (I73N, Y85H, F281del) reveal measurable effects on membrane insertion suggesting that defective membrane integration of bestrophin-1 may represent a potential disease mechanism for a small subset of Best macular dystrophy-related mutations.


Investigative Ophthalmology & Visual Science | 2010

Heat-sensitive TRPV channels in retinal pigment epithelial cells: regulation of VEGF-A secretion.

Sönke Cordeiro; Sebastian Seyler; Julia Stindl; Vladimir M. Milenkovic; Olaf Strauss

PURPOSE Choroidal neovascularization in age-related macular degeneration is caused, to a large extent, by increased secretion of vascular endothelial growth factor (VEGF)-A by the retinal pigment epithelium (RPE). The purpose of the study was to identify pathways that lead to increased VEGF secretion by the RPE. METHODS Ca(2+) signaling was studied in ARPE-19 and human RPE cells in primary culture by means of Ca(2+) imaging. Membrane conductance was measured in the whole-cell configuration of the patch-clamp technique. VEGF-A secretion was measured by using ELISA. RESULTS Freshly isolated RPE cells or ARPE-19 cells were shown to express TRPV1, -2, -3, and -4 channels. Increasing the temperature or stimulation by IGF-1 increased the VEGF-A secretion rate in both cell types. These effects were both sensitive to the TRPV channel blocker ruthenium red (20 μM). The heat-inducible Ca(2+) signals were blocked by the TRPV channel blockers La(3+) and ruthenium red by 68% and 52%, respectively. In contrast, high concentrations of 2-APB (3 mM) increased [Ca(2+)](i), whereas the TRPV1 channel opener capsaicin and the TRPV3 channel opener camphor had no effect. Reduction of TRPV2 expression by siRNA attenuated the heat-evoked Ca(2+) response. In addition, a heat-activated inwardly rectifying current was measured that was completely blocked by ruthenium red. IGF-1 also increased whole-cell current with a corresponding increase in [Ca(2+)](i), which was blocked by the PI3-kinase blocker LY294002. CONCLUSIONS The data strongly suggest that TRPV2 channels expressed by the RPE are involved in the Ca(2+) signaling that mediates both heat-dependent and IGF-1 (via PI3-kinase activation)-induced VEGF secretion.


Pflügers Archiv: European Journal of Physiology | 2010

The presence of bestrophin-1 modulates the Ca2+ recruitment from Ca2+ stores in the ER

Rudgar Neussert; Claudia A. Müller; Vladimir M. Milenkovic; Olaf Strauß

Bestrophin-1, mainly analyzed in overexpression experiments, functions as Ca2+-dependent Cl– channel. Analysis of endogenously expressed bestrophin-1 suggested an influence on intracellular Ca2+. The aim of the study is to analyze the influence of endogenously expressed bestrophin-1 on Ca2+ homeostasis. Primary cultures of retinal pigment epithelial (RPE) cells were established from wild-type and bestrophin-1-deficient mice. Intracellular free Ca2+ ([Ca2+]i) was recorded by Ca2+ imaging; through immunocytochemistry and differential centrifugation, subcellular localization of bestrophin-1 was analyzed. RPE cells of bestrophin-1-deficient mice showed higher levels of resting [Ca2+]i than cells from wild-type mice. In cells from knockout mice and wild-type mice, ATP led to increases in [Ca2+]i subsequent to phospholipase C activation. ATP-induced Ca2+ in bestrophin-1-deficient mice rose faster and decayed slower. In cells from wild-type mice, ATP led to [Ca2+]i increase via depletion of Ca2+ from thapsigargin-sensitive stores. In cells from bestrophin-1-deficient mice, ATP-dependent increase in [Ca2+]i resulted in 40% of cells from depletion of bafilomycin-sensitive and in 60% from thapsigargin-sensitive Ca2+ stores. After differential centrifugation, bestrophin-1 was found in fractions enriched of ClC-3 Cl channel and myosin-7A. Co-localization analysis of bestrophin-1, with β-catenin or pan-cadherin, in fresh sections of porcine retina, revealed bestrophin-1 in the basolateral membrane. A portion of endogenously expressed bestrophin-1,localized in the endoplasmic reticulum, influenced uptake of Ca2+ into Ca2+ stores. Therefore, bestrophin-1 possibly conducts Cl– as counter ion for Ca2+ uptake into cytosolic Ca2+ stores.


Proceedings of the National Academy of Sciences of the United States of America | 2015

Bestrophin 1 is indispensable for volume regulation in human retinal pigment epithelium cells

Andrea Milenkovic; Caroline Brandl; Vladimir M. Milenkovic; Thomas Jendryke; Lalida Sirianant; Potchanart Wanitchakool; Stephanie Zimmermann; Charlotte M. Reiff; Franziska Horling; Heinrich Schrewe; Rainer Schreiber; Karl Kunzelmann; Christian H. Wetzel; Bernhard H. F. Weber

Significance First insight into the molecular identity of volume-regulated anion channel (VRAC) emerged only recently by demonstrating a role for leucine-rich repeats containing 8A (LRRC8A) in channel activity. Our results now expand on VRAC biology, suggesting a model where VRAC subunit composition is cell type- or tissue-specific rather than a single ubiquitous channel formed solely by LRRC8A. Here, we show that bestrophin 1 (BEST1), but not LRCC8A, is crucial in cell volume regulation in retinal pigment epithelium (RPE) cells differentiated from human-induced pluripotent stem cells (hiPSCs). VRAC-mediated currents were strongly reduced in hiPSC-RPE from macular dystrophy patients with pathologic BEST1 mutations. Our model is further supported by in vivo effects of Best1 deficiency in the mouse that manifest as severe subfertility phenotype due to enhanced abnormal sperm morphology related to impaired volume regulation. In response to cell swelling, volume-regulated anion channels (VRACs) participate in a process known as regulatory volume decrease (RVD). Only recently, first insight into the molecular identity of mammalian VRACs was obtained by the discovery of the leucine-rich repeats containing 8A (LRRC8A) gene. Here, we show that bestrophin 1 (BEST1) but not LRRC8A is crucial for volume regulation in human retinal pigment epithelium (RPE) cells. Whole-cell patch-clamp recordings in RPE derived from human-induced pluripotent stem cells (hiPSC) exhibit an outwardly rectifying chloride current with characteristic functional properties of VRACs. This current is severely reduced in hiPSC-RPE cells derived from macular dystrophy patients with pathologic BEST1 mutations. Disruption of the orthologous mouse gene (Best1−/−) does not result in obvious retinal pathology but leads to a severe subfertility phenotype in agreement with minor endogenous expression of Best1 in murine RPE but highly abundant expression in mouse testis. Sperm from Best1−/− mice showed reduced motility and abnormal sperm morphology, indicating an inability in RVD. Together, our data suggest that the molecular identity of VRACs is more complex—that is, instead of a single ubiquitous channel, VRACs could be formed by cell type- or tissue-specific subunit composition. Our findings provide the basis to further examine VRAC diversity in normal and diseased cell physiology, which is key to exploring novel therapeutic approaches in VRAC-associated pathologies.


Experimental Eye Research | 2010

Effect of bestrophin-1 on L-type Ca2+ channel activity depends on the Ca2+ channel beta-subunit.

Nadine Reichhart; Vladimir M. Milenkovic; Claire-Amelie Halsband; Sönke Cordeiro; Olaf Strauß

Bests vitelliforme macular degeneration is an inherited retinal degeneration associated with a reduction of the light-peak in the patients electro-oculogram. Bestrophin-1, the product of the disease-promoting/forming gene can function as regulator of voltage-dependent L-type Ca(2+) channels in the retinal pigment epithelium (RPE). Since mice deficient for either β4-subunits or Ca(V)1.3 subunits show reduced light-peaks, the regulatory function of bestrophin-1 on heterologously expressed Ca(2+) channels composed of the pore-forming Ca(V)1.3 and the auxiliary β4-subunit was analyzed. Precipitation of β4-subunits led to co-precipitation with bestrophin-1 and subsequent analysis of subcellular localization showed co-localization of bestrophin-1, Ca(V)1.3 and β4-subunit in the cell membrane. Ca(V)1.3 currents in the presence of β4-subunits and bestrophin-1 showed accelerated time-dependent activation and decreased current density compared to currents measured in the absence of bestrophin-1. In the presence of the β3-subunit, which is not expressed in the RPE bestrophin-1 did not modulate Ca(V)1.3 activity. Deletion of a cluster of proline-rich motifs in the C-terminus of bestrophin-1 reduced its co-immuno precipitation with the β4-subunit and strongly reduced the Ca(V)1.3 activity. Cells co-expressing bestrophin-1 lacking the proline-rich motifs and Ca(V)1.3 subunits showed less efficient trafficking of bestrophin-1 into the cell membrane. In summary, we conclude that bestrophin-1 modulates L-type channels of the RPE via proline-rich motif-dependent interaction with β4-subunits. A disturbed interaction reduces the currents of the Ca(V)1.3 subunits. This mechanism could open new ways to understand changes in the patients electro-oculogram and functional alterations of the RPE leading to retinal degeneration.


Journal of Cell Science | 2011

Disease-associated missense mutations in bestrophin-1 affect cellular trafficking and anion conductance

Vladimir M. Milenkovic; Elena Röhrl; Bernhard H. F. Weber; Olaf Strauss

Bestrophin-1, an integral membrane protein encoded by the BEST1 gene, is localized predominantly to the basolateral membrane of the retinal pigment epithelium. Mutations in the BEST1 gene have been associated with Best vitelliforme macular dystrophy (BMD), a central retinopathy with autosomal dominant inheritance and variable penetrance. Over 120 disease-causing mutations are known, the majority of which result in amino acid substitutions within four mutational hot-spot regions in the highly conserved N-terminal half of the protein. Although initially thought to impair Cl− channel function, the molecular pathology of BEST1 mutations is still controversial. We have analyzed the subcellular localization of 13 disease-associated BEST1 mutant proteins in polarized MDCK II cells, an established model of apical to basolateral protein sorting. Immunostaining demonstrated that nine of the 13 mutant proteins failed to integrate into the cell membrane. The defective proteins were predominantly retained in the cytoplasm, whereas wild-type bestrophin-1 revealed cell membrane localization. Functional analysis of I− fluxes in HEK-293 cells showed that all mutants exhibited a significant reduction in anion conductance. Our data indicate that defective intracellular trafficking could be a common cause of BMD accompanied by impaired anion conductance, representing a loss of anion channel function that is probably due to mistargeting of mutant protein.


PLOS ONE | 2011

Interaction of bestrophin-1 and Ca2+ channel β-subunits: identification of new binding domains on the bestrophin-1 C-terminus.

Vladimir M. Milenkovic; Sarka Krejcova; Nadine Reichhart; Andrea Wagner; Olaf Strauß

Bestrophin-1 modulates currents through voltage-dependent L-type Ca2+ channels by physically interacting with the β-subunits of Ca2+ channels. The main function of β-subunits is to regulate the number of pore-forming CaV-subunits in the cell membrane and modulate Ca2+ channel currents. To understand the influence of full-length bestrophin-1 on β-subunit function, we studied binding and localization of bestrophin-1 and Ca2+ channel subunits, together with modulation of CaV1.3 Ca2+ channels currents. In heterologeous expression, bestrophin-1 showed co-immunoprecipitation with either, β3-, or β4-subunits. We identified a new highly conserved cluster of proline-rich motifs on the bestrophin-1 C-terminus between amino acid position 468 and 486, which enables possible binding to SH3-domains of β-subunits. A bestrophin-1 that lacks these proline-rich motifs (ΔCT-PxxP bestrophin-1) showed reduced efficiency to co-immunoprecipitate with β3 and β4-subunits. In the presence of ΔCT-PxxP bestrophin-1, β4-subunits and CaV1.3 subunits partly lost membrane localization. Currents from CaV1.3 subunits were modified in the presence of β4-subunit and wild-type bestrophin-1: accelerated time-dependent activation and reduced current density. With ΔCTPxxP bestrophin-1, currents showed the same time-dependent activation as with wild-type bestrophin-1, but the current density was further reduced due to decreased number of Ca2+ channels proteins in the cell membrane. In summary, we described new proline-rich motifs on bestrophin-1 C-terminus, which help to maintain the ability of β-subunits to regulate surface expression of pore-forming CaV Ca2+-channel subunits.


Neuromolecular Medicine | 2014

In-Depth Characterisation of Retinal Pigment Epithelium (RPE) Cells Derived from Human Induced Pluripotent Stem Cells (hiPSC)

Caroline Brandl; Stephanie Zimmermann; Vladimir M. Milenkovic; Sibylle M. G. Rosendahl; Felix Grassmann; Andrea Milenkovic; Ute Hehr; Marianne Federlin; Christian H. Wetzel; Horst Helbig; Bernhard H. F. Weber

Induced pluripotent stem cell (iPSC)-derived retinal pigment epithelium (RPE) has widely been appreciated as a promising tool to model human ocular disease emanating from primary RPE pathology. Here, we describe the successful reprogramming of adult human dermal fibroblasts to iPSCs and their differentiation to pure expandable RPE cells with structural and functional features characteristic for native RPE. Fibroblast cultures were established from skin biopsy material and subsequently reprogrammed following polycistronic lentiviral transduction with OCT4, SOX2, KLF4 and L-Myc. Fibroblast-derived iPSCs showed typical morphology, chromosomal integrity and a distinctive stem cell marker profile. Subsequent differentiation resulted in expandable pigmented hexagonal RPE cells. The cells revealed stable RNA expression of mature RPE markers RPE65, RLBP and BEST1. Immunolabelling verified localisation of BEST1 at the basolateral plasma membrane, and scanning electron microscopy showed typical microvilli at the apical side of iPSC-derived RPE cells. Transepithelial resistance was maintained at high levels during cell culture indicating functional formation of tight junctions. Secretion capacity was demonstrated for VEGF-A. Feeding of porcine photoreceptor outer segments revealed the proper ability of these cells for phagocytosis. IPSC-derived RPE cells largely maintained these properties after cryopreservation. Together, our study underlines that adult dermal fibroblasts can serve as a valuable resource for iPSC-derived RPE with characteristics highly reminiscent of true RPE cells. This will allow its broad application to establish cellular models for RPE-related human diseases.


BMC Evolutionary Biology | 2010

Evolution and functional divergence of the anoctamin family of membrane proteins

Vladimir M. Milenkovic; Marisa Brockmann; Heidi Stöhr; Bernhard H. F. Weber; Olaf Strauss

BackgroundThe anoctamin family of transmembrane proteins are found in all eukaryotes and consists of 10 members in vertebrates. Ano1 and ano2 were observed to have Ca2+ activated Cl- channel activity. Recent findings however have revealed that ano6, and ano7 can also produce chloride currents, although with different properties. In contrast, ano9 and ano10 suppress baseline Cl- conductance when co-expressed with ano1 thus suggesting that different anoctamins can interfere with each other. In order to elucidate intrinsic functional diversity, and underlying evolutionary mechanism among anoctamins, we performed comprehensive bioinformatics analysis of anoctamin gene family.ResultsOur results show that anoctamin protein paralogs evolved from several gene duplication events followed by functional divergence of vertebrate anoctamins. Most of the amino acid replacements responsible for the functional divergence were fixed by adaptive evolution and this seem to be a common pattern in anoctamin gene family evolution. Strong purifying selection and the loss of many gene duplication products indicate rigid structure-function relationships among anoctamins.ConclusionsOur study suggests that anoctamins have evolved by series of duplication events, and that they are constrained by purifying selection. In addition we identified a number of protein domains, and amino acid residues which contribute to predicted functional divergence. Hopefully, this work will facilitate future functional characterization of the anoctamin membrane protein family.

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