The Experts below are selected from a list of 885 Experts worldwide ranked by ideXlab platform
Jordan Doumanov - One of the best experts on this subject based on the ideXlab platform.
-
transepithelial resistance in human Bestrophin 1 stably transfected madin darby canine kidney cells
Biotechnology & Biotechnological Equipment, 2015Co-Authors: Kirilka Mladenova, Svetla Petrova, Stoyanka Stoitsova, Irena Tabashka, Christina Chakarova, Zdravko Lalchev, Veselina Moskovadoumanova, Tanya Topouzovahristova, Jordan DoumanovAbstract:Bestrophin-1 (Best1) is a transmembrane protein, found in the basolateral plasma membrane of retinal pigmented epithelial cells. The exact structure and functions of Best1 protein are still unclear. The protein is thought to be a regulator of ion channels, or an ion channel itself: it was shown to be permeable for chloride, thiocyanate, bicarbonate, glutamate and γ-aminobutyric acid (GABA). Mutations in the gene for Best1 are leading to best vitelliform macular dystrophy (BVMD) and are found in several other types of maculopathy. In order to obtain additional information about Best1 protein, we determined cell polarization of a stably transfected Madin-Darby canine kidney cell line II (MDCK II) cell line, expressing human Best1. We measured the transepithelial resistance of transfected and non-transfected MDCK cells by voltmeter EVOM, over 10 days at 24 hour intervals. The first few days (first-fourth day) both cell lines showed the same or similar values of transmembrane resistance. As expected, on the fifth day the non-transfected cells showed maximum value of epithelial resistance, corresponding to the forming of monolayer. The transfected cells showed maximum value of transepithelial resistance on the ninth day of their cultivation. Phalloidin staining of actin demonstrated the difference in actin arrangements between transfected and non-transfected cells due to Best1. As a consequence of actin rearrangement, Best1 strongly affects the transepithelial resistance of polarizing stably transfected MDCK cells. Our results suggest that Best1 protein has an effect on transepithelial resistance and actin rearrangements of polarized stably transfected MDCK cells.
-
ARTICLE; MEDICAL BIOTECHNOLOGY Transepithelial resistance in human Bestrophin-1 stably transfected MadinDarby canine kidney cells
2015Co-Authors: Kirilka Mladenova, Svetla Petrova, Veselina Moskova-doumanova, Tanya Topouzova-hristova, Stoyanka Stoitsova, Irena Tabashka, Christina Chakarova, Zdravko Lalchev, Jordan DoumanovAbstract:Kirilka Mladenova, Svetla Petrova, Veselina Moskova-Doumanova, Tanya Topouzova-Hristova, StoyankaStoitsova, Irena Tabashka, Christina Chakarova, Zdravko Lalchev & Jordan Doumanov (2014): Transepithelial resistance inhuman Bestrophin-1 stably transfected Madin–Darby canine kidney cells, Biotechnology & Biotechnological Equipment, DOI:10.1080/13102818.2014.988078
-
Transepithelial resistance in human Bestrophin-1 stably transfected Madin–Darby canine kidney cells
Biotechnology & Biotechnological Equipment, 2014Co-Authors: Kirilka Mladenova, Svetla Petrova, Veselina Moskova-doumanova, Tanya Topouzova-hristova, Stoyanka Stoitsova, Irena Tabashka, Christina Chakarova, Zdravko Lalchev, Jordan DoumanovAbstract:Bestrophin-1 (Best1) is a transmembrane protein, found in the basolateral plasma membrane of retinal pigmented epithelial cells. The exact structure and functions of Best1 protein are still unclear. The protein is thought to be a regulator of ion channels, or an ion channel itself: it was shown to be permeable for chloride, thiocyanate, bicarbonate, glutamate and γ-aminobutyric acid (GABA). Mutations in the gene for Best1 are leading to best vitelliform macular dystrophy (BVMD) and are found in several other types of maculopathy. In order to obtain additional information about Best1 protein, we determined cell polarization of a stably transfected Madin-Darby canine kidney cell line II (MDCK II) cell line, expressing human Best1. We measured the transepithelial resistance of transfected and non-transfected MDCK cells by voltmeter EVOM, over 10 days at 24 hour intervals. The first few days (first-fourth day) both cell lines showed the same or similar values of transmembrane resistance. As expected, on the fifth day the non-transfected cells showed maximum value of epithelial resistance, corresponding to the forming of monolayer. The transfected cells showed maximum value of transepithelial resistance on the ninth day of their cultivation. Phalloidin staining of actin demonstrated the difference in actin arrangements between transfected and non-transfected cells due to Best1. As a consequence of actin rearrangement, Best1 strongly affects the transepithelial resistance of polarizing stably transfected MDCK cells. Our results suggest that Best1 protein has an effect on transepithelial resistance and actin rearrangements of polarized stably transfected MDCK cells.
-
Interaction of Bestrophin-1 with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) in surface films.
Colloids and Surfaces B: Biointerfaces, 2014Co-Authors: Kirilka Mladenova, Svetla Petrova, Veselina Moskova-doumanova, Zdravko Lalchev, Georgi Georgiev, Jordan DoumanovAbstract:Abstract Human Bestrophin-1 (hBest1) is a transmembrane channel protein, predominantly expressed in the membrane of retinal pigment epithelium (RPE) cells. Although it is clear that hBest1's interactions with lipids are crucial for its function such studies were not performed as the protein was not purified. Here we describe an effective purification of hBest1 from Madin-Darby Canine Kidney (MDCK) cells via simple gel-filtration and affinity chromatographic steps, which makes possible to probe the protein interplay with lipids. The interaction of the purified hBest1 with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was studied in Langmuir monolayers. The surface pressure (π)–area (A) isotherms and compression/expansion isocycles of POPC monolayer were recorded in absence and presence of hBest1 in the subphase. The π(A) isotherms were analyzed in terms of surface compressional modulus and via two-dimensional virial equation of state. The dilatational rheological properties of the surface films and their surface potential were also measured. The morphology of the films was observed by Brewster angle microscopy. The inclusion of the protein in the film subphase does not lead to in-depth penetration of hBest1 but interaction takes place in the headgroup region of the monolayer. The hBest1/POPC interaction resulted in formation of more condensed films, which rheological properties and lateral structure differed significantly from the pure POPC monolayers. Our study sheds light on the still unclear question how hBest1 gets in touch with biomembrane phospholipids of eukaryotic cells that might be of key importance for the proper structure and function of RPE biomembranes.
-
Disease-Causing Mutations in BEST1 Gene Are Associated with Altered Sorting of Bestrophin-1 Protein
International Journal of Molecular Sciences, 2013Co-Authors: Jordan Doumanov, Veselina Moskova-doumanova, Christina Zeitz, Paloma Dominguez Gimenez, Isabelle Audo, Abhay Krishna, Giovanna Alfano, Maria Diaz Bellido Diaz, Marie-elise Lancelot, José-alain SahelAbstract:Mutations in BEST1 gene, encoding the Bestrophin-1 (Best1) protein are associated with macular dystrophies. Best1 is predominantly expressed in the retinal pigment epithelium (RPE), and is inserted in its basolateral membrane. We investigated the cellular localization in polarized MDCKII cells of disease-associated Best1 mutant proteins to study specific sorting motifs of Best1. Real-time PCR and western blots for endogenous expression of BEST1 in MDCK cells were performed. Best1 mutant constructs were generated using site-directed mutagenesis and transfected in MDCK cells. For protein sorting, confocal microscopy studies, biotinylation assays and statistical methods for quantification of mislocalization were used. Analysis of endogenous expression of BEST1 in MDCK cells revealed the presence of BEST1 transcript but no protein. Confocal microscopy and quantitative analyses indicate that transfected normal human Best1 displays a basolateral localization in MDCK cells, while cell sorting of several Best1 mutants (Y85H, Q96R, L100R, Y227N, Y227E) was altered. In contrast to constitutively active Y227E, constitutively inactive Y227F Best1 mutant localized basolaterally similar to the normal Best1 protein. Our data suggest that at least three basolateral sorting motifs might be implicated in proper Best1 basolateral localization. In addition, non-phosphorylated tyrosine 227 could play a role for basolateral delivery.
Olaf Strauß - One of the best experts on this subject based on the ideXlab platform.
-
Inhibition of Ca2+ channel surface expression by mutant Bestrophin-1 in RPE cells.
The FASEB Journal, 2020Co-Authors: Magdalena Cordes, Nadine Reichhart, Piotr Bucichowski, Ahmad S. Alfaar, Stephen H. Tsang, Seba Almedawar, Olaf StraußAbstract:The BEST1 gene product Bestrophin-1, a Ca2+ -dependent anion channel, interacts with CaV 1.3 Ca2+ channels in the retinal pigment epithelium (RPE). BEST1 mutations lead to Best vitelliform macular dystrophy. A common functional defect of these mutations is reduced trafficking of Bestrophin-1 into the plasma membrane. We hypothesized that this defect affects the interaction partner CaV 1.3 channel affecting Ca2+ signaling and altered RPE function. Thus, we investigated the protein interaction between CaV 1.3 channels and Bestrophin-1 by immunoprecipitation, CaV 1.3 activity in the presence of mutant Bestrophin-1 and intracellular trafficking of the interaction partners in confluent RPE monolayers. We selected four BEST1 mutations, each representing one mutational hotspot of the disease: T6P, F80L, R218C, and F305S. Heterologously expressed L-type channels and mutant Bestrophin-1 showed reduced interaction, reduced CaV 1.3 channel activity, and changes in surface expression. Transfection of polarized RPE (porcine primary cells, iPSC-RPE) that endogenously express CaV 1.3 and wild-type Bestrophin-1, with mutant Bestrophin-1 confirmed reduction of CaV 1.3 surface expression. For the four selected BEST1 mutations, presence of mutant Bestrophin-1 led to reduced CaV 1.3 activity by modulating pore-function or decreasing surface expression. Reduced CaV 1.3 activity might open new ways to understand symptoms of Best vitelliform macular dystrophy such as reduced electro-oculogram, lipofuscin accumulation, and vision impairment.
-
Contribution of Ion Channels in Calcium Signaling Regulating Phagocytosis: MaxiK, Cav1.3 and Bestrophin-1
Retinal Degenerative Diseases, 2015Co-Authors: Olaf Strauß, Nadine Reichhart, Nestor Mas Gomez, Claudia MüllerAbstract:Mutations in the BEST1 gene lead to a variety of retinal degenerations including Best's vitelliforme macular degeneration. The BEST1 gene product, Bestrophin-1, is expressed in the retinal pigment epithelium (RPE). It is likely that mutant Bestrophin-1 impairs functions of the RPE which support photoreceptor function and will thus lead to retinal degeneration. However, the RPE function which is influenced by Bestrophin-1 is so far not identified. Previously we showed that Bestrophin-1 interacts with L-type Ca²⁺ channels of the CaV1.3 subtype and that the endogenously expressed Bestrophin-1 is required for intracellular Ca²⁺ regulation. A hallmark of Best's disease is the fast lipofuscin accumulation occurring already at young ages. Therefore, we addressed the hypothesis that Bestrophin-1 might influence phagocytosis of photoreceptor outer segments (POS) by the RPE. Here, siRNA knock-down of Bestrophin-1 expression as well as inhibition of L-type Ca²⁺ channel activity modulated the POS phagocytosis in vitro. In vivo CaV1.3 expression appeared to be diurnal regulated with a higher expression rate in the afternoon. Compared to wild-type littermates, Ca V 1.3 (-/-) mice showed a shift in the circadian POS phagocytosis with an increased activity in the afternoon. Thus we suggest that mutant Bestrophin-1 leads to an impaired regulation of the POS phagocytosis by the RPE which would explain the fast lipofuscin accumulation in Best patients.
-
The role of Bestrophin-1 in intracellular Ca(2+) signaling.
Retinal Degenerative Diseases, 2014Co-Authors: Olaf Strauß, Nadine Reichhart, Claudia A. Müller, Ernst R. Tamm, Nestor Mas GomezAbstract:Mutations in the BEST1 gene lead to a variety of retinal degenerations, among them Best’s vitelliforme macular degeneration. To clarify the mechanism of the disease, the understanding of the function of BEST1 gene product, Bestrophin-1, is mandatory. In overexpression studies Bestrophin-1 appeared to function as a Ca2+-dependent Cl channel. On the other hand, Bestrophin-1 is able to participate in intracellular Ca2+ signaling. Endogenously expressed Bestrophin-1 largely localized to the cytosolic compartment close to the basolateral membrane of the retinal pigment epithelium (RPE) as it can be shown using differential centrifugation, immunohistochemistry, and transmission electron microscopy. To elucidate a cytosolic function of Bestrophin-1, we explored the store-operated Ca2+ entry in short-time cultured porcine RPE cells. Depletion of cytosolic Ca2+stores by SERCA inhibition led to activation of Orai-1 Ca2+ channels. This resulted in an influx of extracellular Ca2+ into the cell which was reduced when Bestrophin-1 expression was knocked down using siRNA techniques. Quantification of Ca2+ which can be released from cytosolic Ca2+ stores revealed that after reduction of Bestrophin-1 expression less Ca2+ is stored in ER Ca2+ stores. Thus, Bestrophin-1 functions as an intracellular Cl channel which helps to accumulate and to release Ca2+ from stores by conducting the counterion for Ca2+.
-
CaV1.3 L-type channels, maxiK Ca2 +-dependent K+ channels and Bestrophin-1 regulate rhythmic photoreceptor outer segment phagocytosis by retinal pigment epithelial cells
Cellular Signalling, 2014Co-Authors: Claudia A. Müller, Nestor Gomez, Peter Ruth, Olaf StraußAbstract:Abstract Phagocytosis of shed photoreceptor outer segments by the retinal pigment epithelium (RPE) is critical for maintenance of visual function. Because changes in intracellular Ca 2 + regulate phagocytosis, we studied in vitro the impact of different ion channels in addition to mice deficient for Ca v 1.3 L-type Ca 2+ channels (Ca1.3 −/− ) and maxiK Ca 2+ -dependent K + channels (BK −/− ). The knockdown of Bestrophin-1 protein, a regulator of intracellular Ca 2+ homeostasis, affected phagocytosis in porcine RPE cultures. Blockage of voltage-gated L-type channels by (+)BayK8644 inhibitor reduced phagocytosis in vitro, in contrast L-type activation by (−)BayK8644 had no impact. The expression rate of Ca v 1.3, the predominant L-type Ca 2 + channel in RPE cells, varied at different times of day. Ca V 1.3 −/− RPE lacked peak phagocytic activity following morning photoreceptor shedding in wild-type RPE and retained a higher number of phagosomes at a later time of day. The BK-channel blocker paxilline lowered phagocytosis in RPE cultures in a concentration-dependent manner. BK −/− RPE in vivo retained phagocytic capability but this activity, which is normally well synchronized with circadian photoreceptor shedding, shifted out of phase. Retinae of older BK −/− mice showed shortened photoreceptor outer segments and diminished rhodopsin content. Store-operated Ca 2 + channels Orai-1 did not affect phagocytosis in cultured RPE. TRPV channel inhibition by ruthenium-red reduced phagocytosis, whereas activation at high concentrations of 2-APB increased phagocytosis. Our data demonstrate essential roles for Bestrophin-1, BK, TRPV and L-type channels in regulating retinal phagocytosis. These data indicate further the importance of BK and Ca V 1.3 for rhythmic phagocytic activity synchronized with photoreceptor shedding.
-
Interaction of Bestrophin-1 and Ca 2+ Channel b-Subunits: Identification of New Binding Domains on the Bestrophin-1 C-Terminus
2013Co-Authors: Vladimir M. Milenkovic, Sarka Krejcova, Nadine Reichhart, Andrea Wagner, Olaf StraußAbstract:Bestrophin-1 modulates currents through voltage-dependent L-type Ca 2+ channels by physically interacting with the b-subunits of Ca 2+ channels. The main function of b-subunits is to regulate the number of pore-forming CaV-subunits in the cell membrane and modulate Ca 2+ channel currents. To understand the influence of full-length Bestrophin-1 on b-subunit function, we studied binding and localization of Bestrophin-1 and Ca 2+ channel subunits, together with modulation of CaV1.3 Ca 2+ channels currents. In heterologeous expression, Bestrophin-1 showed co-immunoprecipitation with either, b3-, or b4-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 b-subunits. A Bestrophin-1 that lacks these proline-rich motifs (DCT-PxxP Bestrophin-1) showed reduced efficiency to co-immunoprecipitate with b3 and b4subunits. In the presence of DCT-PxxP Bestrophin-1, b4-subunits and CaV1.3 subunits partly lost membrane localization. Currents from CaV1.3 subunits were modified in the presence of b4-subunit and wild-type Bestrophin-1: accelerated timedependent activation and reduced current density. With DCTPxxP 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 Ca 2+ channels proteins in the cell membrane. In summary, we described new proline-rich motifs on Bestrophin-1 C-terminus
Kirilka Mladenova - One of the best experts on this subject based on the ideXlab platform.
-
Miscibility of hBest1 and sphingomyelin in surface films – A prerequisite for interaction with membrane domains
Colloids and Surfaces B: Biointerfaces, 2020Co-Authors: Nikola Mladenov, Kirilka Mladenova, Svetla Petrova, Veselina Moskova-doumanova, Tanya Topouzova-hristova, Desislava Bozhinova, Pavel Videv, Ralitsa Veleva, Aneliya Kostadinova, Galya StanevaAbstract:Abstract Human Bestrophin-1 (hBest1) is a transmembrane Ca2+- dependent anion channel, associated with the transport of Cl−, HCO3- ions, γ-aminobutiric acid (GABA), glutamate (Glu), and regulation of retinal homeostasis. Its mutant forms cause retinal degenerative diseases, defined as Bestrophinopathies. Using both physicochemical - surface pressure/mean molecular area (π/A) isotherms, hysteresis, compressibility moduli of hBest1/sphingomyelin (SM) monolayers, Brewster angle microscopy (BAM) studies, and biological approaches - detergent membrane fractionation, Laurdan (6-dodecanoyl-N,N-dimethyl-2-naphthylamine) and immunofluorescence staining of stably transfected MDCK-hBest1 and MDCK II cells, we report: 1) Ca2+, Glu and GABA interact with binary hBest1/SM monolayers at 35 °C, resulting in changes in hBest1 surface conformation, structure, self-organization and surface dynamics. The process of mixing in hBest1/SM monolayers is spontaneous and the effect of protein on binary films was defined as “fluidizing”, hindering the phase-transition of monolayer from liquid-expanded to intermediate (LE-M) state; 2) in stably transfected MDCK-hBest1 cells, Bestrophin-1 was distributed between detergent resistant (DRM) and detergent-soluble membranes (DSM) - up to 30 % and 70 %, respectively; in alive cells, hBest1 was visualized in both liquid-ordered (Lo) and liquid-disordered (Ld) fractions, quantifying protein association up to 35 % and 65 % with Lo and Ld. Our results indicate that the spontaneous miscibility of hBest1 and SM is a prerequisite to diverse protein interactions with membrane domains, different structural conformations and biological functions.
-
Effects of Ca2+ ions on Bestrophin-1 surface films
Colloids and Surfaces B: Biointerfaces, 2017Co-Authors: Kirilka Mladenova, Svetla Petrova, Veselina Moskova-doumanova, Tanya Topouzova-hristova, Christina Chakarova, Tonya D. Andreeva, Yuri Kalvachev, Konstantin Balashev, Shomi S. Bhattacharya, Zdravko LalchevAbstract:Abstract Human Bestrophin-1 (hBest1) is a transmembrane calcium-activated chloride channel protein – member of the Bestrophin family of anion channels, predominantly expressed in the membrane of retinal pigment epithelium (RPE) cells. Mutations in the protein cause ocular diseases, named Bestrophinopathies. Here, we present the first Fourier transform infrared (FTIR) study of the secondary structure elements of hBest1, π/A isotherms and hysteresis, Brewster angle microscopy (BAM) and atomic force microscopy (AFM) visualization of the aggregation state of protein molecules dispersed as Langmuir and Langmuir-Blodgett films. The secondary structure of hBest1 consists predominantly of 3 10 -helices (27.2%), α-helixes (16.3%), β-turns and loops (32.2%). AFM images of hBest1 suggest approximate lateral dimensions of 100 × 160 A and 75 A height. Binding of calcium ions (Ca 2+ ) induces conformational changes in the protein secondary structure leading to assembly of protein molecules and changes in molecular and macro-organization of hBest1 in monolayers. These data provide basic information needed in pursuit of molecular mechanisms underlying retinal and other pathologies linked to this protein.
-
Effects of Ca2+ ions on Bestrophin-1 surface films B Biointerfaces
Colloids and Surfaces, 2017Co-Authors: Kirilka Mladenova, Svetla Petrova, Veselina Moskova-doumanova, Tanya Topouzova-hristova, Christina Chakarova, Tonya D. Andreeva, Yuri Kalvachev, Konstantin Balashev, Shomi S. Bhattacharya, Zdravko LalchevAbstract:Human Bestrophin-1 (hBest1) is a transmembrane calcium-activated chloride channel protein – member of the Bestrophin family of anion channels, predominantly expressed in the membrane of retinal pigment epithelium (RPE) cells. Mutations in the protein cause ocular diseases, named Bestrophinopathies. Here, we present the first Fourier transform infrared (FTIR) study of the secondary structure elements of hBest1, π/A isotherms and hysteresis, Brewster angle microscopy (BAM) and atomic force microscopy (AFM) visualization of the aggregation state of protein molecules dispersed as Langmuir and Langmuir-Blodgett films. The secondary structure of hBest1 consists predominantly of 310-helices (27.2%), α-helixes (16.3%), β-turns and loops (32.2%). AFM images of hBest1 suggest approximate lateral dimensions of 100×160A and 75A height. Binding of calcium ions (Ca²⁺) induces conformational changes in the protein secondary structure leading to assembly of protein molecules and changes in molecular and macro-organization of hBest1 in monolayers. These data provide basic information needed in pursuit of molecular mechanisms underlying retinal and other pathologies linked to this protein.
-
transepithelial resistance in human Bestrophin 1 stably transfected madin darby canine kidney cells
Biotechnology & Biotechnological Equipment, 2015Co-Authors: Kirilka Mladenova, Svetla Petrova, Stoyanka Stoitsova, Irena Tabashka, Christina Chakarova, Zdravko Lalchev, Veselina Moskovadoumanova, Tanya Topouzovahristova, Jordan DoumanovAbstract:Bestrophin-1 (Best1) is a transmembrane protein, found in the basolateral plasma membrane of retinal pigmented epithelial cells. The exact structure and functions of Best1 protein are still unclear. The protein is thought to be a regulator of ion channels, or an ion channel itself: it was shown to be permeable for chloride, thiocyanate, bicarbonate, glutamate and γ-aminobutyric acid (GABA). Mutations in the gene for Best1 are leading to best vitelliform macular dystrophy (BVMD) and are found in several other types of maculopathy. In order to obtain additional information about Best1 protein, we determined cell polarization of a stably transfected Madin-Darby canine kidney cell line II (MDCK II) cell line, expressing human Best1. We measured the transepithelial resistance of transfected and non-transfected MDCK cells by voltmeter EVOM, over 10 days at 24 hour intervals. The first few days (first-fourth day) both cell lines showed the same or similar values of transmembrane resistance. As expected, on the fifth day the non-transfected cells showed maximum value of epithelial resistance, corresponding to the forming of monolayer. The transfected cells showed maximum value of transepithelial resistance on the ninth day of their cultivation. Phalloidin staining of actin demonstrated the difference in actin arrangements between transfected and non-transfected cells due to Best1. As a consequence of actin rearrangement, Best1 strongly affects the transepithelial resistance of polarizing stably transfected MDCK cells. Our results suggest that Best1 protein has an effect on transepithelial resistance and actin rearrangements of polarized stably transfected MDCK cells.
-
ARTICLE; MEDICAL BIOTECHNOLOGY Transepithelial resistance in human Bestrophin-1 stably transfected MadinDarby canine kidney cells
2015Co-Authors: Kirilka Mladenova, Svetla Petrova, Veselina Moskova-doumanova, Tanya Topouzova-hristova, Stoyanka Stoitsova, Irena Tabashka, Christina Chakarova, Zdravko Lalchev, Jordan DoumanovAbstract:Kirilka Mladenova, Svetla Petrova, Veselina Moskova-Doumanova, Tanya Topouzova-Hristova, StoyankaStoitsova, Irena Tabashka, Christina Chakarova, Zdravko Lalchev & Jordan Doumanov (2014): Transepithelial resistance inhuman Bestrophin-1 stably transfected Madin–Darby canine kidney cells, Biotechnology & Biotechnological Equipment, DOI:10.1080/13102818.2014.988078
Vladimir M. Milenkovic - One of the best experts on this subject based on the ideXlab platform.
-
Bestrophin 1 is indispensable for volume regulation in human retinal pigment epithelium cells
Proceedings of the National Academy of Sciences of the United States of America, 2015Co-Authors: Andrea Milenkovic, Vladimir M. Milenkovic, Franziska Horling, Caroline Brandl, Thomas Jendryke, Lalida Sirianant, Potchanart Wanitchakool, Stephanie Zimmermann, Charlotte M Reiff, Heinrich SchreweAbstract: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.
-
Interaction of Bestrophin-1 and Ca 2+ Channel b-Subunits: Identification of New Binding Domains on the Bestrophin-1 C-Terminus
2013Co-Authors: Vladimir M. Milenkovic, Sarka Krejcova, Nadine Reichhart, Andrea Wagner, Olaf StraußAbstract:Bestrophin-1 modulates currents through voltage-dependent L-type Ca 2+ channels by physically interacting with the b-subunits of Ca 2+ channels. The main function of b-subunits is to regulate the number of pore-forming CaV-subunits in the cell membrane and modulate Ca 2+ channel currents. To understand the influence of full-length Bestrophin-1 on b-subunit function, we studied binding and localization of Bestrophin-1 and Ca 2+ channel subunits, together with modulation of CaV1.3 Ca 2+ channels currents. In heterologeous expression, Bestrophin-1 showed co-immunoprecipitation with either, b3-, or b4-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 b-subunits. A Bestrophin-1 that lacks these proline-rich motifs (DCT-PxxP Bestrophin-1) showed reduced efficiency to co-immunoprecipitate with b3 and b4subunits. In the presence of DCT-PxxP Bestrophin-1, b4-subunits and CaV1.3 subunits partly lost membrane localization. Currents from CaV1.3 subunits were modified in the presence of b4-subunit and wild-type Bestrophin-1: accelerated timedependent activation and reduced current density. With DCTPxxP 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 Ca 2+ channels proteins in the cell membrane. In summary, we described new proline-rich motifs on Bestrophin-1 C-terminus
-
A Potential Cytosolic Function of Bestrophin-1
Retinal Degenerative Diseases, 2011Co-Authors: Olaf Strauß, Claudia A. Müller, Rudgar Neussert, Vladimir M. MilenkovicAbstract:Mutations in the BEST1 gene lead to a variety of retinal degenerations. In the eye, the BEST1 gene product Bestrophin-1 is expressed in the retinal pigment epithelium (RPE). It is likely that retinal degenerations are caused by a change of the RPE function. Recent publications indicated that endogenously expressed Bestrophin-1 function as a cytosolic Cl channel in Ca2+ stores of the endoplasmic reticulum influences intracellular Ca2+ signaling. Possible cytosolic function of BEST1 could help to understand discrepancies between the Bestrophin-1 function in overexpression systems and in animal models as well as to develop new ways to understand BEST1-associated retinal degenerations.
-
Disease-associated missense mutations in Bestrophin-1 affect cellular trafficking and anion conductance.
Journal of cell science, 2011Co-Authors: Vladimir M. Milenkovic, Bernhard H. F. Weber, Elena Röhrl, Olaf StraußAbstract: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.
-
Interaction of Bestrophin-1 and Ca2+ channel β-subunits: identification of new binding domains on the Bestrophin-1 C-terminus.
PLoS ONE, 2011Co-Authors: Vladimir M. Milenkovic, Sarka Krejcova, Nadine Reichhart, Andrea Wagner, Olaf StraußAbstract:Bestrophin-1 modulates currents through voltage-dependent L-type Ca(2+) channels by physically interacting with the β-subunits of Ca(2+) channels. The main function of β-subunits is to regulate the number of pore-forming Ca(V)-subunits in the cell membrane and modulate Ca(2+) channel currents. To understand the influence of full-length Bestrophin-1 on β-subunit function, we studied binding and localization of Bestrophin-1 and Ca(2+) channel subunits, together with modulation of Ca(V)1.3 Ca(2+) 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 Ca(V)1.3 subunits partly lost membrane localization. Currents from Ca(V)1.3 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 Ca(2+) 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 Ca(V) Ca(2+)-channel subunits.
Zdravko Lalchev - One of the best experts on this subject based on the ideXlab platform.
-
Effects of Ca2+ ions on Bestrophin-1 surface films
Colloids and Surfaces B: Biointerfaces, 2017Co-Authors: Kirilka Mladenova, Svetla Petrova, Veselina Moskova-doumanova, Tanya Topouzova-hristova, Christina Chakarova, Tonya D. Andreeva, Yuri Kalvachev, Konstantin Balashev, Shomi S. Bhattacharya, Zdravko LalchevAbstract:Abstract Human Bestrophin-1 (hBest1) is a transmembrane calcium-activated chloride channel protein – member of the Bestrophin family of anion channels, predominantly expressed in the membrane of retinal pigment epithelium (RPE) cells. Mutations in the protein cause ocular diseases, named Bestrophinopathies. Here, we present the first Fourier transform infrared (FTIR) study of the secondary structure elements of hBest1, π/A isotherms and hysteresis, Brewster angle microscopy (BAM) and atomic force microscopy (AFM) visualization of the aggregation state of protein molecules dispersed as Langmuir and Langmuir-Blodgett films. The secondary structure of hBest1 consists predominantly of 3 10 -helices (27.2%), α-helixes (16.3%), β-turns and loops (32.2%). AFM images of hBest1 suggest approximate lateral dimensions of 100 × 160 A and 75 A height. Binding of calcium ions (Ca 2+ ) induces conformational changes in the protein secondary structure leading to assembly of protein molecules and changes in molecular and macro-organization of hBest1 in monolayers. These data provide basic information needed in pursuit of molecular mechanisms underlying retinal and other pathologies linked to this protein.
-
Effects of Ca2+ ions on Bestrophin-1 surface films B Biointerfaces
Colloids and Surfaces, 2017Co-Authors: Kirilka Mladenova, Svetla Petrova, Veselina Moskova-doumanova, Tanya Topouzova-hristova, Christina Chakarova, Tonya D. Andreeva, Yuri Kalvachev, Konstantin Balashev, Shomi S. Bhattacharya, Zdravko LalchevAbstract:Human Bestrophin-1 (hBest1) is a transmembrane calcium-activated chloride channel protein – member of the Bestrophin family of anion channels, predominantly expressed in the membrane of retinal pigment epithelium (RPE) cells. Mutations in the protein cause ocular diseases, named Bestrophinopathies. Here, we present the first Fourier transform infrared (FTIR) study of the secondary structure elements of hBest1, π/A isotherms and hysteresis, Brewster angle microscopy (BAM) and atomic force microscopy (AFM) visualization of the aggregation state of protein molecules dispersed as Langmuir and Langmuir-Blodgett films. The secondary structure of hBest1 consists predominantly of 310-helices (27.2%), α-helixes (16.3%), β-turns and loops (32.2%). AFM images of hBest1 suggest approximate lateral dimensions of 100×160A and 75A height. Binding of calcium ions (Ca²⁺) induces conformational changes in the protein secondary structure leading to assembly of protein molecules and changes in molecular and macro-organization of hBest1 in monolayers. These data provide basic information needed in pursuit of molecular mechanisms underlying retinal and other pathologies linked to this protein.
-
transepithelial resistance in human Bestrophin 1 stably transfected madin darby canine kidney cells
Biotechnology & Biotechnological Equipment, 2015Co-Authors: Kirilka Mladenova, Svetla Petrova, Stoyanka Stoitsova, Irena Tabashka, Christina Chakarova, Zdravko Lalchev, Veselina Moskovadoumanova, Tanya Topouzovahristova, Jordan DoumanovAbstract:Bestrophin-1 (Best1) is a transmembrane protein, found in the basolateral plasma membrane of retinal pigmented epithelial cells. The exact structure and functions of Best1 protein are still unclear. The protein is thought to be a regulator of ion channels, or an ion channel itself: it was shown to be permeable for chloride, thiocyanate, bicarbonate, glutamate and γ-aminobutyric acid (GABA). Mutations in the gene for Best1 are leading to best vitelliform macular dystrophy (BVMD) and are found in several other types of maculopathy. In order to obtain additional information about Best1 protein, we determined cell polarization of a stably transfected Madin-Darby canine kidney cell line II (MDCK II) cell line, expressing human Best1. We measured the transepithelial resistance of transfected and non-transfected MDCK cells by voltmeter EVOM, over 10 days at 24 hour intervals. The first few days (first-fourth day) both cell lines showed the same or similar values of transmembrane resistance. As expected, on the fifth day the non-transfected cells showed maximum value of epithelial resistance, corresponding to the forming of monolayer. The transfected cells showed maximum value of transepithelial resistance on the ninth day of their cultivation. Phalloidin staining of actin demonstrated the difference in actin arrangements between transfected and non-transfected cells due to Best1. As a consequence of actin rearrangement, Best1 strongly affects the transepithelial resistance of polarizing stably transfected MDCK cells. Our results suggest that Best1 protein has an effect on transepithelial resistance and actin rearrangements of polarized stably transfected MDCK cells.
-
ARTICLE; MEDICAL BIOTECHNOLOGY Transepithelial resistance in human Bestrophin-1 stably transfected MadinDarby canine kidney cells
2015Co-Authors: Kirilka Mladenova, Svetla Petrova, Veselina Moskova-doumanova, Tanya Topouzova-hristova, Stoyanka Stoitsova, Irena Tabashka, Christina Chakarova, Zdravko Lalchev, Jordan DoumanovAbstract:Kirilka Mladenova, Svetla Petrova, Veselina Moskova-Doumanova, Tanya Topouzova-Hristova, StoyankaStoitsova, Irena Tabashka, Christina Chakarova, Zdravko Lalchev & Jordan Doumanov (2014): Transepithelial resistance inhuman Bestrophin-1 stably transfected Madin–Darby canine kidney cells, Biotechnology & Biotechnological Equipment, DOI:10.1080/13102818.2014.988078
-
Transepithelial resistance in human Bestrophin-1 stably transfected Madin–Darby canine kidney cells
Biotechnology & Biotechnological Equipment, 2014Co-Authors: Kirilka Mladenova, Svetla Petrova, Veselina Moskova-doumanova, Tanya Topouzova-hristova, Stoyanka Stoitsova, Irena Tabashka, Christina Chakarova, Zdravko Lalchev, Jordan DoumanovAbstract:Bestrophin-1 (Best1) is a transmembrane protein, found in the basolateral plasma membrane of retinal pigmented epithelial cells. The exact structure and functions of Best1 protein are still unclear. The protein is thought to be a regulator of ion channels, or an ion channel itself: it was shown to be permeable for chloride, thiocyanate, bicarbonate, glutamate and γ-aminobutyric acid (GABA). Mutations in the gene for Best1 are leading to best vitelliform macular dystrophy (BVMD) and are found in several other types of maculopathy. In order to obtain additional information about Best1 protein, we determined cell polarization of a stably transfected Madin-Darby canine kidney cell line II (MDCK II) cell line, expressing human Best1. We measured the transepithelial resistance of transfected and non-transfected MDCK cells by voltmeter EVOM, over 10 days at 24 hour intervals. The first few days (first-fourth day) both cell lines showed the same or similar values of transmembrane resistance. As expected, on the fifth day the non-transfected cells showed maximum value of epithelial resistance, corresponding to the forming of monolayer. The transfected cells showed maximum value of transepithelial resistance on the ninth day of their cultivation. Phalloidin staining of actin demonstrated the difference in actin arrangements between transfected and non-transfected cells due to Best1. As a consequence of actin rearrangement, Best1 strongly affects the transepithelial resistance of polarizing stably transfected MDCK cells. Our results suggest that Best1 protein has an effect on transepithelial resistance and actin rearrangements of polarized stably transfected MDCK cells.