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Olaf Straus - One of the best experts on this subject based on the ideXlab platform.
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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: C A Muller, Nestor Gomez, Peter Ruth, Olaf StrausAbstract:Abstract Phagocytosis of shed photoreceptor outer segments by the retinal pigment epithelium (RPE) is critical for maintenance of visual function. Because changes in intracellular Ca2 + regulate phagocytosis, we studied in vitro the impact of different ion channels in addition to mice deficient for Cav1.3 L-type Ca2+ channels (Ca1.3−/−) and maxiK Ca2+-dependent K+ channels (BK−/−). The knockdown of Bestrophin-1 protein, a regulator of intracellular Ca2+ 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 Cav1.3, the predominant L-type Ca2 + channel in RPE cells, varied at different times of day. CaV1.3−/− 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 Ca2 + 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 CaV1.3 for rhythmic phagocytic activity synchronized with photoreceptor shedding.
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cav1 3 l type channels maxik ca 2 dependent k channels and Bestrophin 1 regulate rhythmic photoreceptor outer segment phagocytosis by retinal pigment epithelial cells
Cellular Signalling, 2014Co-Authors: C A Muller, Nestor Gomez, Peter Ruth, Olaf StrausAbstract: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 Cav1.3 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 Cav1.3, the predominant L-type Ca(2+) channel in RPE cells, varied at different times of day. CaV1.3(-/-) 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 CaV1.3 for rhythmic phagocytic activity synchronized with photoreceptor shedding.
Vladimir V. Matchkov - One of the best experts on this subject based on the ideXlab platform.
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Bestrophin-3 (Vitelliform Macular Dystrophy 2–Like 3 Protein) Is Essential for the cGMP-Dependent Calcium-Activated Chloride Conductance in Vascular Smooth Muscle Cells
2016Co-Authors: Vladimir V. Matchkov, Donna Briggs Boedtkjer, Finn Skou Pedersen, Christian Aalkjaer, Veronika Golubinskaya, Per Larsen, Elena V. Bouzinova, Ra Rojek, Holger NilssonAbstract:Abstract—Although the biophysical fingerprints (ion selectivity, voltage-dependence, kinetics, etc) of Ca2-activated Cl currents are well established, their molecular identity is still controversial. Several molecular candidates have been suggested; however, none of them has been fully accepted. We have recently characterized a cGMP-dependent Ca2-activated Cl current with unique characteristics in smooth muscle cells. This novel current has been shown to coexist with a “classic ” (cGMP-independent) Ca2-activated Cl current and to have characteristics distinct from those previously known for Ca2-activated Cl currents. Here, we suggest that a Bestrophin, a product of the Best gene family, is responsible for the cGMP-dependent Ca2-activated Cl current based on similarities between the membrane currents produced by heterologous expressions of Bestrophins and the cGMP-dependent Ca2-activated Cl current. This is supported by similarities in the distribution pattern of the cGMP-dependent Ca2-activated Cl current and Bestrophin-3 (the product of Best-3 gene) expression in different smooth muscle. Furthermore, downregulation of Best-3 gene expression with small interfering RNA both in cultured cells and in vascular smooth muscle cells in vivo was associated with a significant reduction of the cGMP-dependent Ca2-activated Cl current, whereas the magnitude of the classic Ca2-activated Cl current was not affected. The majority of previous suggestions that Bestrophins are a new Cl channel family were based on heterologous expression in cell culture studies. Our present results demonstrate that at least 1 family member, Bestrophin-3, is essential for a well-defined endogenous Ca2-activated Cl current in smoot
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tmem16a knockdown abrogates two different ca2 activated cl currents and contractility of smooth muscle in rat mesenteric small arteries
Pflügers Archiv: European Journal of Physiology, 2014Co-Authors: Vibeke Secher Dam, Donna Briggs Boedtkjer, Christian Aalkjaer, Jakob Nyvad, Vladimir V. MatchkovAbstract:The presence of Ca2+-activated Cl− channels (CaCCs) in vascular smooth muscle cells (SMCs) is well established. Their molecular identity is, however, elusive. Two distinct Ca2+-activated Cl− currents (ICl(Ca)) were previously characterized in SMCs. We have shown that the cGMP-dependent ICl(Ca) depends on Bestrophin expression, while the “classical” ICl(Ca) is not. Downregulation of Bestrophins did not affect arterial contraction but inhibited the rhythmic contractions, vasomotion. In this study, we have used in vivo siRNA transfection of rat mesenteric small arteries to investigate the role of a putative CaCC, TMEM16A. Isometric force, [Ca2+]i, and SMC membrane potential were measured in isolated arterial segments. ICl(Ca) and GTPγS-induced nonselective cation current were measured in isolated SMCs. Downregulation of TMEM16A resulted in inhibition of both the cGMP-dependent ICl(Ca) and the “classical” ICl(Ca) in SMCs. TMEM16A downregulation also reduced expression of Bestrophins. TMEM16A downregulation suppressed vasomotion both in vivo and in vitro. Downregulation of TMEM16A reduced agonist (noradrenaline and vasopressin) and K+-induced contractions. In accordance with the depolarizing role of CaCCs, TMEM16A downregulation suppressed agonist-induced depolarization and elevation in [Ca2+]i. Surprisingly, K+-induced depolarization was unchanged but Ca2+ entry was reduced. We suggested that this is due to reduced expression of the L-type Ca2+ channels, as observed at the mRNA level. Thus, the importance of TMEM16A for contraction is, at least in part, independent from membrane potential. This study demonstrates the significance of TMEM16A for two SMCs ICl(Ca) and vascular function and suggests an interaction between TMEM16A and L-type Ca2+ channels.
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Bestrophin IS IMPORTANT FOR THE RHYTHMIC BUT NOT THE TONIC CONTRACTION IN RAT MESENTERIC SMALL ARTERIES
Cardiovascular Research, 2011Co-Authors: Torbjoern Broegger, Jens Christian Brings Jacobsen, Vibeke Secher Dam, Donna Briggs Boedtkjer, Henrik Kold-petersen, Finn Skou Pedersen, Christian Aalkjaer, Vladimir V. MatchkovAbstract:Aims We have previously characterized a cGMP-dependent Ca2+-activated Cl– current in vascular smooth muscle cells (SMCs) and have shown its dependence on Bestrophin-3 expression. We hypothesize that this current is important for synchronization of SMCs in the vascular wall. In the present study, we aimed to test this hypothesis by transfecting rat mesenteric small arteries in vivo with siRNA specifically targeting Bestrophin-3. Methods and results The arteries were tested 3 days after transfection in vitro for isometric force development and for intracellular Ca2+ in SMCs. Bestrophin-3 expression was significantly reduced compared with arteries transfected with mutated siRNA. mRNA levels for Bestrophin-1 and -2 were also significantly reduced by Bestrophin-3 down-regulation. This is suggested to be secondary to specific Bestrophin-3 down-regulation since siRNAs targeting different exons of the Bestrophin-3 gene had identical effects on Bestrophin-1 and -2 expression. The transfection affected neither the maximal contractile response nor the sensitivity to norepinephrine and arginine-vasopressin. The amplitude of agonist-induced vasomotion was significantly reduced in arteries down-regulated for Bestrophins compared with controls, and asynchronous Ca2+ waves appeared in the SMCs. The average frequency of vasomotion was not different. 8Br-cGMP restored vasomotion in arteries where the endothelium was removed, but oscillation amplitude was still significantly less in Bestrophin-down-regulated arteries. Thus, vasomotion properties were consistent with those previously characterized for rat mesenteric small arteries. Data from our mathematical model are consistent with the experimental results. Conclusion This study demonstrates the importance of Bestrophins for synchronization of SMCs and strongly supports our hypothesis for generation of vasomotion.
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Bestrophin 3 vitelliform macular dystrophy 2 like 3 protein is essential for the cgmp dependent calcium activated chloride conductance in vascular smooth muscle cells
Circulation Research, 2008Co-Authors: Vladimir V. Matchkov, Donna Briggs Boedtkjer, Finn Skou Pedersen, Christian Aalkjaer, Veronika Golubinskaya, Per Larsen, Elena V. Bouzinova, Aleksandra Rojek, Holger NilssonAbstract:Although the biophysical fingerprints (ion selectivity, voltage-dependence, kinetics, etc) of Ca 2+ -activated Cl − currents are well established, their molecular identity is still controversial. Several molecular candidates have been suggested; however, none of them has been fully accepted. We have recently characterized a cGMP-dependent Ca 2+ -activated Cl − current with unique characteristics in smooth muscle cells. This novel current has been shown to coexist with a “classic” (cGMP-independent) Ca 2+ -activated Cl − current and to have characteristics distinct from those previously known for Ca 2+ -activated Cl − currents. Here, we suggest that a Bestrophin, a product of the Best gene family, is responsible for the cGMP-dependent Ca 2+ -activated Cl − current based on similarities between the membrane currents produced by heterologous expressions of Bestrophins and the cGMP-dependent Ca 2+ -activated Cl − current. This is supported by similarities in the distribution pattern of the cGMP-dependent Ca 2+ -activated Cl − current and Bestrophin-3 (the product of Best-3 gene) expression in different smooth muscle. Furthermore, downregulation of Best-3 gene expression with small interfering RNA both in cultured cells and in vascular smooth muscle cells in vivo was associated with a significant reduction of the cGMP-dependent Ca 2+ -activated Cl − current, whereas the magnitude of the classic Ca 2+ -activated Cl − current was not affected. The majority of previous suggestions that Bestrophins are a new Cl − channel family were based on heterologous expression in cell culture studies. Our present results demonstrate that at least 1 family member, Bestrophin-3, is essential for a well-defined endogenous Ca 2+ -activated Cl − current in smooth muscles in the intact vascular wall.
Holger Nilsson - One of the best experts on this subject based on the ideXlab platform.
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Table_1_Bestrophin-3 Expression in a Subpopulation of Astrocytes in the Neonatal Brain After Hypoxic-Ischemic Injury.pdf
2019Co-Authors: Veronika Golubinskaya, Helena Gustafsson, Carina Mallard, Regina Vontell, Veena Supramaniam, Josephine Wyatt-ashmead, Holger NilssonAbstract:Bestrophin-3, a potential candidate for a calcium-activated chloride channel, recently was suggested to have cell-protective functions. We studied the expression and alternative splicing of Bestrophin-3 in neonatal mouse brain and after hypoxic-ischemic (HI) injury and in human neonatal brain samples. HI brain injury was induced in 9-day old mice by unilateral permanent common carotid artery occlusion in combination with exposure to 10% oxygen for 50 min. Endoplasmic reticulum stress was induced by thapsigargin treatment in primary culture of mouse brain astrocytes. We also investigated expression of Bestrophin-3 protein in a sample of human neonatal brain tissue. Bestrophin-3 protein expression was detected with immunohistochemical methods and western blot; mRNA expression and splicing were analyzed by RT-PCR. HI induced a brain tissue infarct and a pronounced increase in the endoplasmic reticulum-associated marker CHOP. Three days after HI a population of astrocytes co-expressed Bestrophin-3 and nestin in a penumbra-like area of the injured hemisphere. However, total levels of Bestrophin-3 protein in mouse cortex were reduced after injury. Mouse astrocytes in primary culture also expressed Bestrophin-3 protein, the amount of which was reduced by endoplasmic reticulum stress. Bestrophin-3 protein was detected in astrocytes in the hippocampal region of the human neonatal brain which had patchy white matter gliosis and neuronal loss in the Sommer’s sector of the Ammon’s horn (CA1). Analysis of Bestrophin-3 mRNA in mouse brain with and without injury showed the presence of two truncated spliced variants, but no full-length mRNA. Total amount of Bestrophin-3 mRNA increased after HI, but showed only minor injury-related change. However, the splice variants of Bestrophin-3 mRNA were differentially regulated after HI depending on the presence of tissue injury. Our results show that Bestrophin-3 is expressed in neonatal mouse brain after injury and in the human neonatal brain with pathology. In mouse brain Bestrophin-3 protein is upregulated in a specific astrocyte population after injury and is co-expressed with nestin. Splice variants of Bestrophin-3 mRNA respond differently to HI, which might indicate their different roles in tissue injury.
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Image_1_Bestrophin-3 Expression in a Subpopulation of Astrocytes in the Neonatal Brain After Hypoxic-Ischemic Injury.pdf
2019Co-Authors: Veronika Golubinskaya, Helena Gustafsson, Carina Mallard, Regina Vontell, Veena Supramaniam, Josephine Wyatt-ashmead, Holger NilssonAbstract:Bestrophin-3, a potential candidate for a calcium-activated chloride channel, recently was suggested to have cell-protective functions. We studied the expression and alternative splicing of Bestrophin-3 in neonatal mouse brain and after hypoxic-ischemic (HI) injury and in human neonatal brain samples. HI brain injury was induced in 9-day old mice by unilateral permanent common carotid artery occlusion in combination with exposure to 10% oxygen for 50 min. Endoplasmic reticulum stress was induced by thapsigargin treatment in primary culture of mouse brain astrocytes. We also investigated expression of Bestrophin-3 protein in a sample of human neonatal brain tissue. Bestrophin-3 protein expression was detected with immunohistochemical methods and western blot; mRNA expression and splicing were analyzed by RT-PCR. HI induced a brain tissue infarct and a pronounced increase in the endoplasmic reticulum-associated marker CHOP. Three days after HI a population of astrocytes co-expressed Bestrophin-3 and nestin in a penumbra-like area of the injured hemisphere. However, total levels of Bestrophin-3 protein in mouse cortex were reduced after injury. Mouse astrocytes in primary culture also expressed Bestrophin-3 protein, the amount of which was reduced by endoplasmic reticulum stress. Bestrophin-3 protein was detected in astrocytes in the hippocampal region of the human neonatal brain which had patchy white matter gliosis and neuronal loss in the Sommer’s sector of the Ammon’s horn (CA1). Analysis of Bestrophin-3 mRNA in mouse brain with and without injury showed the presence of two truncated spliced variants, but no full-length mRNA. Total amount of Bestrophin-3 mRNA increased after HI, but showed only minor injury-related change. However, the splice variants of Bestrophin-3 mRNA were differentially regulated after HI depending on the presence of tissue injury. Our results show that Bestrophin-3 is expressed in neonatal mouse brain after injury and in the human neonatal brain with pathology. In mouse brain Bestrophin-3 protein is upregulated in a specific astrocyte population after injury and is co-expressed with nestin. Splice variants of Bestrophin-3 mRNA respond differently to HI, which might indicate their different roles in tissue injury.
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Bestrophin-3 Expression in a Subpopulation of Astrocytes in the Neonatal Brain After Hypoxic-Ischemic Injury
Frontiers Media S.A., 2019Co-Authors: Veronika Golubinskaya, Helena Gustafsson, Carina Mallard, Regina Vontell, Veena Supramaniam, Josephine Wyatt-ashmead, Holger NilssonAbstract:Bestrophin-3, a potential candidate for a calcium-activated chloride channel, recently was suggested to have cell-protective functions. We studied the expression and alternative splicing of Bestrophin-3 in neonatal mouse brain and after hypoxic-ischemic (HI) injury and in human neonatal brain samples. HI brain injury was induced in 9-day old mice by unilateral permanent common carotid artery occlusion in combination with exposure to 10% oxygen for 50 min. Endoplasmic reticulum stress was induced by thapsigargin treatment in primary culture of mouse brain astrocytes. We also investigated expression of Bestrophin-3 protein in a sample of human neonatal brain tissue. Bestrophin-3 protein expression was detected with immunohistochemical methods and western blot; mRNA expression and splicing were analyzed by RT-PCR. HI induced a brain tissue infarct and a pronounced increase in the endoplasmic reticulum-associated marker CHOP. Three days after HI a population of astrocytes co-expressed Bestrophin-3 and nestin in a penumbra-like area of the injured hemisphere. However, total levels of Bestrophin-3 protein in mouse cortex were reduced after injury. Mouse astrocytes in primary culture also expressed Bestrophin-3 protein, the amount of which was reduced by endoplasmic reticulum stress. Bestrophin-3 protein was detected in astrocytes in the hippocampal region of the human neonatal brain which had patchy white matter gliosis and neuronal loss in the Sommer’s sector of the Ammon’s horn (CA1). Analysis of Bestrophin-3 mRNA in mouse brain with and without injury showed the presence of two truncated spliced variants, but no full-length mRNA. Total amount of Bestrophin-3 mRNA increased after HI, but showed only minor injury-related change. However, the splice variants of Bestrophin-3 mRNA were differentially regulated after HI depending on the presence of tissue injury. Our results show that Bestrophin-3 is expressed in neonatal mouse brain after injury and in the human neonatal brain with pathology. In mouse brain Bestrophin-3 protein is upregulated in a specific astrocyte population after injury and is co-expressed with nestin. Splice variants of Bestrophin-3 mRNA respond differently to HI, which might indicate their different roles in tissue injury
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Bestrophin-3 (Vitelliform Macular Dystrophy 2–Like 3 Protein) Is Essential for the cGMP-Dependent Calcium-Activated Chloride Conductance in Vascular Smooth Muscle Cells
2016Co-Authors: Vladimir V. Matchkov, Donna Briggs Boedtkjer, Finn Skou Pedersen, Christian Aalkjaer, Veronika Golubinskaya, Per Larsen, Elena V. Bouzinova, Ra Rojek, Holger NilssonAbstract:Abstract—Although the biophysical fingerprints (ion selectivity, voltage-dependence, kinetics, etc) of Ca2-activated Cl currents are well established, their molecular identity is still controversial. Several molecular candidates have been suggested; however, none of them has been fully accepted. We have recently characterized a cGMP-dependent Ca2-activated Cl current with unique characteristics in smooth muscle cells. This novel current has been shown to coexist with a “classic ” (cGMP-independent) Ca2-activated Cl current and to have characteristics distinct from those previously known for Ca2-activated Cl currents. Here, we suggest that a Bestrophin, a product of the Best gene family, is responsible for the cGMP-dependent Ca2-activated Cl current based on similarities between the membrane currents produced by heterologous expressions of Bestrophins and the cGMP-dependent Ca2-activated Cl current. This is supported by similarities in the distribution pattern of the cGMP-dependent Ca2-activated Cl current and Bestrophin-3 (the product of Best-3 gene) expression in different smooth muscle. Furthermore, downregulation of Best-3 gene expression with small interfering RNA both in cultured cells and in vascular smooth muscle cells in vivo was associated with a significant reduction of the cGMP-dependent Ca2-activated Cl current, whereas the magnitude of the classic Ca2-activated Cl current was not affected. The majority of previous suggestions that Bestrophins are a new Cl channel family were based on heterologous expression in cell culture studies. Our present results demonstrate that at least 1 family member, Bestrophin-3, is essential for a well-defined endogenous Ca2-activated Cl current in smoot
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Bestrophin 3 expression in mouse glomeruli 694 1
The FASEB Journal, 2014Co-Authors: Veronika Golubinskaya, Johannes Elvin, Kerstin Ebefors, Helena Gustafsson, Carina Mallard, Jenny Nystrom, Holger NilssonAbstract:The Bestrophin isoform, Bestrophin-3 (Best3), acts as a calcium-activated chloride channel in cardiomyocytes and in vascular smooth muscle. It may have cell-protective functions in other cell types...
Criss H. Hartzell - One of the best experts on this subject based on the ideXlab platform.
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Cellular/Molecular The Anion-Selective Pore of the Bestrophins, a Family of Chloride Channels Associated with Retinal Degeneration
2014Co-Authors: Li-ting Chien, Yuanyuan Cui, Criss H. HartzellAbstract:Mutations in human Bestrophin-1 (VMD2) are genetically linked to a juvenile form of macular degeneration and autosomal dominant vitreoretinochoroidopathy. Recently, it has been proposed that Bestrophins are Cl � channels and that the putative second transmembrane domain participates in forming the Bestrophin pore. However, the structural determinants of Cl � ion permeation through the channel pore are not known. Here we systematically replaced every amino acid in mouse Bestrophin-2 (mBest2) between positions 69 and 104 with cysteine. We then measured the effects on the relative permeability and conductance of the channel to Cl � and SCN � (thiocyanate) and determined the accessibility of the cysteine-substituted amino acids to extracellularly applied, membrane-impermeant sulfhydryl reagents. Unlike K � channels, the amino acids forming the mBest2 selectivity filter are not discretely localized but are distributed over �20 amino acids within the transmembrane domain. Cysteine-substituted amino acids in the selectivity filter are easily accessible to extracellularly applied sulfhydryl reagents and select for anionic sulfhydryl reagents over cationic ones. Understanding the structure of the anion conduction pathway of Bestrophins provides insights into how mutations produce channel dysfunction and may provide important information for development of therapeutic strategies for treating macular degeneration. Key words: ion channel; anion channel; chloride channel; macular degeneration; Bestrophin; patch clam
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Neurobiology of Disease The Best Disease-Linked Cl � Channel hBest1 Regulates Ca V1 (L-type) Ca 2 � Channels via src-Homology-Binding Domains
2014Co-Authors: Qinghuan Xiao, Guiying Cui, Amy Lee, Criss H. HartzellAbstract:Mutations in the Bestrophin-1 (Best1) gene are linked to several kinds of macular degeneration in both humans and dogs. Although Bestrophins have been shown clearly to be Cl � ion channels, it is controversial whether Cl � channel dysfunction can explain the diseases. It has been suggested that Bestrophins are multifunctional proteins: they may regulate voltage-gated Ca 2 � channels in addition to functioning as Cl � channels. Here, we show that human Best1 gene (hBest1) differentially modulates Ca V1.3 (L-type) voltage-gated Ca 2� channels through association with the Ca V � subunit. In transfected human embryonic kidney 293 cells, hBest1 inhibited Ca V1.3. Inhibition of Ca V1.3 was not observed in the absence of the � subunit. Also, the hBest1 C terminus binds to Ca V � subunits, suggesting that the effect of hBest1 was mediated by the Ca V � subunit. The region of hBest1 responsible for the effect was localized to a region (amino acids 330–370) in the cytoplasmic C terminus that contains a predicted src-homology-binding domain that is not present in other Bestrophin subtypes. Mutation of Pro 330 and Pro 334 abolished the effects of hBest1 on Ca V1.3. The effect was specific to hBest1; it was not observed with mouse Best1 (mBest1), mBest2, or mBest3. Wild-type hBest1 and the disease-causing mutants R92S, G299R, and D312N inhibited Ca V currents the same amount, whereas the A146K and G222E mutants were less effective. We propose that hBest1 regulates Ca V channels by interacting with the Ca V � subunit and altering channel availability. Our findings reveal a novel function of Bestrophin in regulation of Ca V channels and suggest a possible mechanism for the role of hBest1 in macular degeneration. Key words: macular degeneration; retinal pigment epithelium; ion channel; calcium; src-homology; chlorid
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Bestrophin-2 Is Involved in the Generation of Intraocular Pressure
2014Co-Authors: Benjamin Bakall, Brett J Stanton, Lihua Y Marmorstein, Youwen Zhang, Criss H. Hartzell, Precious Mclaughlin, Alan D. MarmorsteinAbstract:PURPOSE. The Bestrophin family of proteins has been demonstrated to generate or regulate Ca 2 �-activated Cl � conductances. Mutations in Bestrophin-1 (Best1) cause several blinding eye diseases, but little is known about other Bestrophin family members. This study involved disruption of the Best2 gene in mice. METHODS. The mouse Best2 gene was disrupted by replacing exons 1, 2, and part of exon 3 with a Lac Z. The expression profile of Bestrophin-2 (Best2) was examined using RT-PCR, X-gal staining, and immunohistochemistry. Intraocular pressure (IOP) was measured by anterior chamber cannulation. RESULTS. RT-PCR of mouse tissues revealed Best2 mRNA in eye, colon, nasal epithelia, trachea, brain, lung, and kidney. X-gal staining, confirmed expression in colon epithelia and in the eye, in the nonpigmented epithelia (NPE). Best2 was not expresse
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Two Bestrophins cloned from Xenopus laevis oocytes express Ca-activated Cl currents
2014Co-Authors: Raymond W. Wei, Wesley Mann, Criss H. HartzellAbstract:Ca 2 �-activated Cl � channels play important diverse roles from fast block to polyspermy to olfactory transduction, but their molecular identity has not been firmly established. By searching sequence databases with the M2 pore domain of ligand-gated anion channels, we identified potential Ca 2 �-activated Cl � channels, which included members of the Bestrophin family. We cloned two Bestrophins from Xenopus oocytes, which express high levels of Ca 2 �-activated Cl � channels. The Xenopus Bestrophins were expressed in a variety of tissues. We predict that Bestrophin has six transmembrane domains with the conserved RFP domain playing an integral part in ionic selectivity. When Xenopus Bestrophins were heterologously expressed in human embryonic kidney-293 cells, large Ca 2 �-activated Cl � currents were observed
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molecular physiology of Bestrophins multifunctional membrane proteins linked to best disease and other retinopathies
Physical Review, 2008Co-Authors: Criss H. Hartzell, Qinghuan Xiao, Li-ting ChienAbstract:This article reviews the current state of knowledge about the Bestrophins, a newly identified family of proteins that can function both as Cl− channels and as regulators of voltage-gated Ca2+ chann...
Olaf Strauß - One of the best experts on this subject based on the ideXlab platform.
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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.
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©2005 FASEB
2013Co-Authors: Rita Rosenthal, Benjamin Bakall, Tyson R. Kinnick, Sönke Wimmers, Claes Wadelius, Neal Peachey, Alan Marmorstein, Olaf StraußAbstract:Expression of Bestrophin-1, the product of the VMD2 gene, modulates voltage-dependent Ca 2+ channels in retinal pigment epithelial cell
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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
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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.
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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.