Stoichiometry and specific assembly of Best ion channels

Bharill, Shashank; Fu, Zhu; Palty, Raz; Isacoff, Ehud Y.

doi:10.1073/pnas.1400248111

Cited by 23 publications

(20 citation statements)

References 47 publications

(90 reference statements)

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“…6 Monomers of Best1 have four transmembrane domains as well as intracellular N-and C-termini, with the latter being at the terminus of a large, cytosolic domain. [7][8][9] Monomers of Best1 oligomerize [10][11][12][13][14] to form a highly conserved homopentameric Ca 2þ -activated ion channel. 8,9 Data from heterologous systems, 15,16 primary human RPE cell culture, 17,18 mouse models, 19,20 the structurally similar paralog bestrophin-2, 21,22 as well as recently published crystal structures 8,9 all unambiguously demonstrate that mammalian Best1 is a calcium-activated anion channel.…”

Section: Discussionmentioning

confidence: 99%

Autosomal Recessive Bestrophinopathy Is Not Associated With the Loss of Bestrophin-1 Anion Channel Function in a Patient With a NovelBEST1Mutation

Johnson

Bachman

Gilles

et al. 2015

Invest. Ophthalmol. Vis. Sci.

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Citation: Johnson AA, Bachman LA, Gilles BJ, et al. Autosomal recessive bestrophinopathy is not associated with the loss of bestrophin-1 anion channel function in a patient with a novel BEST1 mutation. Invest Ophthalmol Vis Sci. 2015;56:4619-4630. DOI:10.1167/iovs.15-16910 PURPOSE. Mutations in BEST1, encoding bestrophin-1 (Best1), cause autosomal recessive bestrophinopathy (ARB). Encoding bestrophin-1 is a pentameric anion channel localized to the basolateral plasma membrane of the RPE. Here, we characterize the effects of the mutations R141H (CGC > CAC) and I366fsX18 (c.1098_1100þ7del), identified in a patient in our practice, on Best1 trafficking, oligomerization, and channel activity. METHODS. Currents of ClÀ were assessed in transfected HEK293 cells using whole-cell patch clamp. Best1 localization was assessed by confocal microscopy in differentiated, humaninduced pluripotent stem cell-derived RPE (iPSC-RPE) cells following expression of mutants via adenovirus-mediated gene transfer. Oligomerization was evaluated by coimmunoprecipitation in iPSC-RPE and MDCK cells. À currents, this indicates that ARB in this patient is not caused by a loss of channel activity. Moreover, Best1 I366fsX18 differs from Best1 in that it lacks most of the cytosolic C-terminal domain, suggesting that the loss of this region contributes significantly to the pathogenesis of ARB in this patient. RESULTS. Compared to

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Section: Discussionmentioning

confidence: 99%

Autosomal Recessive Bestrophinopathy Is Not Associated With the Loss of Bestrophin-1 Anion Channel Function in a Patient With a NovelBEST1Mutation

Johnson

Bachman

Gilles

et al. 2015

Invest. Ophthalmol. Vis. Sci.

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“…Down-regulation of TMEM16A reduced the expression of Bestrophins, however, a reduced expression of LTCCs has also been mentioned [66]. On the other hand, Bestrophins were shown to form preferentially homomeric channels [67], similarly to TMEM16A [68], which may question the ability of the two proteins to form a single functional ion channel.…”

Section: Ca 2+ -Entry Via I Cal Is Essential For the Activation Of Imentioning

confidence: 99%

Sarcolemmal Ca 2+ -entry through L-type Ca 2+ channels controls the profile of Ca 2+ -activated Cl − current in canine ventricular myocytes

Horváth

Váczi

Hegyi

et al. 2016

Journal of Molecular and Cellular Cardiology

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Highlights• Ca 2+ -entry via I Ca,L is essential for the activation of I Cl(Ca) • I Cl(Ca) can be activated even in the absence of CICR• TMEM16A and Bestrophin-3 are expressed on human left ventricular muscle• TMEM16A and Bestrophin-3 co-localize with each other and with Ca v 1.2 channels (I Cl(Ca) ) mediated by TMEM16A and/or Bestrophin-3 may contribute to cardiac arrhythmias. The true profile of I Cl(Ca) during an actual ventricular action potential (AP), however, is poorly understood. We aimed to study the profile of I Cl(Ca) systematically under physiological conditions (normal Ca 2+ cycling and AP voltage-clamp) as well as in conditions designed to change [Ca 2+ ] i . The expression of TMEM16A and/or Bestrophin-3 in canine and human left ventricular myocytes was examined. The possible spatial distribution of these proteins and their co-localization with Ca v 1.2 was also studied. The profile of I Cl(Ca), identified as a 9-anthracene carboxylic acid-sensitive current under AP voltageclamp conditions, contained an early fast outward and a late inward component, overlapping early and terminal repolarizations, respectively. Both components were moderately reduced by ryanodine, while fully abolished by BAPTA, but not EGTA. [Ca 2+ ] i was monitored using Fura-2-AM. Setting [Ca 2+ ] i to the systolic level measured in the bulk cytoplasm (1.1 µM) decreased I Cl(Ca), while application of Bay K8644, isoproterenol, and faster stimulation rates increased the amplitude of I Cl(Ca) . Ca 2+ -entry through L-type Ca 2+ channels was essential for activation of I Cl(Ca) . TMEM16A and Bestrophin-3 showed strong co-localization with one another and also with Ca v 1.2 channels, when assessed using immunolabeling and confocal microscopy in both canine myocytes and human ventricular myocardium. Activation of I Cl(Ca) in canine ventricular cells requires Ca 2+ -entry through neighboring L-type Ca 2+ channels and is only augmented by SR Ca 2+ -release. Substantial activation of I Cl(Ca) requires high Ca 2+ in the dyadic clefts which can be effectively buffered by BAPTA, but not EGTA.

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“…Properties including subunit topology and stoichiometry are unresolved. One recent study using the single-molecule photobleaching technique led the authors to conclude that bestrophins are tetramers 25 , while other experiments suggest pentameric stoichiometry 5 .…”

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confidence: 99%

Structure and insights into the function of a Ca2+-activated Cl− channel

Dickson

Pedi

Long

2014

Nature

184

299

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Bestrophin calcium-activated chloride channels (CaCCs) regulate the flow of chloride and other monovalent anions across cellular membranes in response to intracellular calcium (Ca2+) levels. Mutations in bestrophin-1 (Best1) cause certain eye diseases. Here we present X-ray structures of chicken Best1-Fab complexes, at 2.85 Å resolution, with permeant anions and Ca2+. Representing the first structure of a CaCC, the eukaryotic Best1 channel, which recapitulates CaCC function in liposomes, is formed from a pentameric assembly of subunits. Ca2+ binds to the channel's large cytosolic region. A single ion pore, approximately 95 Å in length, is located along the central axis and contains at least fifteen binding sites for anions. A hydrophobic neck within the pore likely forms the gate. Phenylalanine residues within it may coordinate permeating anions via anion-π interactions. Conformational changes observed near the “Ca2+ clasp” hint at the mechanism of Ca2+-dependent gating. Disease-causing mutations are prevalent within the gating apparatus.

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Stoichiometry and specific assembly of Best ion channels

Cited by 23 publications

References 47 publications

Autosomal Recessive Bestrophinopathy Is Not Associated With the Loss of Bestrophin-1 Anion Channel Function in a Patient With a NovelBEST1Mutation

Autosomal Recessive Bestrophinopathy Is Not Associated With the Loss of Bestrophin-1 Anion Channel Function in a Patient With a NovelBEST1Mutation

Sarcolemmal Ca 2+ -entry through L-type Ca 2+ channels controls the profile of Ca 2+ -activated Cl − current in canine ventricular myocytes

Structure and insights into the function of a Ca2+-activated Cl− channel

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