Cholesterol Alters the Phase Separation in Model Membranes Containing hBest1.

Human retinal pigment epithelial (RPE) cells express the transmembrane Ca2+-dependent Cl- channel bestrophin-1 (hBest1) of the plasma membrane. Mutations in the hBest1 protein are associated with the development of distinct pathological conditions known as bestrophinopathies. The interactions between hBest1 and plasma membrane lipids (cholesterol (Chol), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and sphingomyelin (SM)) determine its lateral organization and surface dynamics, i.e., their miscibility or phase separation. Using the surface pressure/mean molecular area (π/A) isotherms, hysteresis and compressibility moduli (Cs-1) of hBest1/POPC/Chol and hBest1/SM/Chol composite Langmuir monolayers, we established that the films are in an LE (liquid-expanded) or LE-LC (liquid-condensed) state, the components are well-mixed and the Ca2+ ions have a condensing effect on the surface molecular organization. Cholesterol causes a decrease in the elasticity of both films and a decrease in the ΔGmixπ values (reduction of phase separation) of hBest1/POPC/Chol films. For the hBest1/SM/Chol monolayers, the negative values of ΔGmixπ are retained and equalized with the values of ΔGmixπ in the hBest1/POPC/Chol films. Shifts in phase separation/miscibility by cholesterol can lead to changes in the structure and localization of hBest1 in the lipid rafts and its channel functions.

Self-organization and surface properties of hBest1 in models of biological membranes.

The transmembrane Ca2+ - activated Cl- channel - human bestrophin-1 (hBest1) is expressed in retinal pigment epithelium and mutations of BEST1 gene cause ocular degenerative diseases colectivelly referred to as "bestrophinopathies". A large number of genetical, biochemical, biophysical and molecular biological studies have been performed to understand the relationship between structure and function of the hBest1 protein and its pathophysiological significance. Here, we review the current understanding of hBest1 surface organization, interactions with membrane lipids in model membranes, and its association with microdomains of cellular membranes. These highlights are significant for modulation of channel activity in cells.

The Clinical Features and Genetic Spectrum of a Large Cohort of Chinese Patients With Vitelliform Macular Dystrophies.

PURPOSE: To provide the clinical and genetic characteristics of a large cohort of Chinese patients with vitelliform macular dystrophies. DESIGN: Cross-sectional study. METHODS: One hundred and thirty-four unrelated Chinese patients diagnosed with Best vitelliform macular dystrophy (BVMD), autosomal recessive bestrophinopathy (ARB), or adult vitelliform macular dystrophy (AVMD) were enrolled. Detailed ophthalmic examinations and genetic testing on vitelliform macular dystrophy-related genes were performed. Genotype and phenotype association were analyzed among different diagnostic groups. RESULTS: In total, 87 BVMD, 30 AVMD, and 17 ARB patients were enrolled in this study. Genetic analysis identified 37 BEST1 mutations in 53 patients with BVMD and ARB. Of these, 5 variants (c.254A>C, c.291C>G, c.722C>G, c.848_850del, c.1740-2A>C) were novel. The variant c.898G>A was a hotspot mutation, which was identified in 13 patients with BVMD and 1 patient with ARB. There were significant differences of ocular biometric parameters among patients with homozygous or compound heterozygous mutations, heterozygous mutations, and those without mutations of BEST1. Homozygous or compound heterozygous patients had shortest axial length (AL), shallowest anterior chamber depth (ACD), and highest intraocular pressure (IOP); patients without mutations had longest AL, deepest ACD, and lowest IOP; and heterozygous patients were in between. Moreover, 7 patients harboring heterozygous mutations in BEST1 and 3 patients without BEST1 mutations showed similar clinical appearance to ARB in our cohort. CONCLUSIONS: This is the largest sample size study of Chinese vitelliform macular dystrophy patients. Our results indicated that assessment of angle-closure risk is a necessary consideration for all types of BEST1-related vitelliform macular dystrophies. The study expanded both the clinical and genetic findings of 3 common types of vitelliform macular dystrophies in a Chinese population.

Structural and functional characterization of the bestrophin-2 anion channel.

The bestrophin family of calcium (Ca2+)-activated chloride (Cl-) channels, which mediate the influx and efflux of monovalent anions in response to the levels of intracellular Ca2+, comprises four members in mammals (bestrophin 1-4). Here we report cryo-EM structures of bovine bestrophin-2 (bBest2) bound and unbound by Ca2+ at 2.4- and 2.2-Å resolution, respectively. The bBest2 structure highlights four previously underappreciated pore-lining residues specifically conserved in Best2 but not in Best1, illustrating the differences between these paralogs. Structure-inspired electrophysiological analysis reveals that, although the channel is sensitive to Ca2+, it has substantial Ca2+-independent activity for Cl-, reflecting the opening at the cytoplasmic restriction of the ion conducting pathway even when Ca2+ is absent. Moreover, the ion selectivity of bBest2 is controlled by multiple residues, including those involved in gating.

Miscibility of hBest1 and sphingomyelin in surface films - A prerequisite for interaction with membrane domains.

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.

Pathogenicity of new BEST1 variants identified in Italian patients with best vitelliform macular dystrophy assessed by computational structural biology.

BACKGROUND: Best vitelliform macular dystrophy (BVMD) is an autosomal dominant macular degeneration. The typical central yellowish yolk-like lesion usually appears in childhood and gradually worsens. Most cases are caused by variants in the BEST1 gene which encodes bestrophin-1, an integral membrane protein found primarily in the retinal pigment epithelium. METHODS: Here we describe the spectrum of BEST1 variants identified in a cohort of 57 Italian patients analyzed by Sanger sequencing. In 13 cases, the study also included segregation analysis in affected and unaffected relatives. We used molecular mechanics to calculate two quantitative parameters related to calcium-activated chloride channel (CaCC composed of 5 BEST1 subunits) stability and calcium-dependent activation and related them to the potential pathogenicity of individual missense variants detected in the probands. RESULTS: Thirty-six out of 57 probands (63% positivity) and 16 out of 18 relatives proved positive to genetic testing. Family study confirmed the variable penetrance and expressivity of the disease. Six of the 27 genetic variants discovered were novel: p.(Val9Gly), p.(Ser108Arg), p.(Asn179Asp), p.(Trp182Arg), p.(Glu292Gln) and p.(Asn296Lys). All BEST1 variants were assessed in silico for potential pathogenicity. Our computational structural biology approach based on 3D model structure of the CaCC showed that individual amino acid replacements may affect channel shape, stability, activation, gating, selectivity and throughput, and possibly also other features, depending on where the individual mutated amino acid residues are located in the tertiary structure of BEST1. Statistically significant correlations between mean logMAR best-corrected visual acuity (BCVA), age and modulus of computed BEST1 dimerization energies, which reflect variations in the in CaCC stability due to amino acid changes, permitted us to assess the pathogenicity of individual BEST1 variants. CONCLUSIONS: Using this computational approach, we designed a method for estimating BCVA progression in patients with BEST1 variants.

Dual Ca2+-dependent gates in human Bestrophin1 underlie disease-causing mechanisms of gain-of-function mutations.

Mutations of human BEST1, encoding a Ca2+-activated Cl- channel (hBest1), cause macular degenerative disorders. Best1 homolog structures reveal an evolutionarily conserved channel architecture highlighted by two landmark restrictions (named the "neck" and "aperture", respectively) in the ion conducting pathway, suggesting a unique dual-switch gating mechanism, which, however, has not been characterized well. Using patch clamp and crystallography, we demonstrate that both the neck and aperture in hBest1 are Ca2+-dependent gates essential for preventing channel leakage resulting from Ca2+-independent, spontaneous gate opening. Importantly, three patient-derived mutations (D203A, I205T and Y236C) lead to Ca2+-independent leakage and elevated Ca2+-dependent anion currents due to enhanced opening of the gates. Moreover, we identify a network of residues critically involved in gate operation. Together, our results suggest an indispensable role of the neck and aperture of hBest1 for channel gating, and uncover disease-causing mechanisms of hBest1 gain-of-function mutations.

Whole-exome sequencing in a Japanese pedigree implicates a rare non-synonymous single-nucleotide variant in BEST3 as a candidate for mandibular prognathism.

Mandibular prognathism is a phenotype of facial deformity seen in populations around the world, but with higher incidence among East Asian populations. Five genome-wide nonparametric linkage analyses and a genome-wide association study to identify susceptibility loci of the phenotype have shown inconsistent results. To explore variants related to mandibular prognathism, we undertook whole-exome sequencing in a Japanese pedigree. The pedigree was ascertained as mandibular prognathism. The pedigree comprised 15 individuals from 4 generations. Four affected individuals across 2 generations and 5 unaffected individuals were chosen for whole-exome sequencing. Five non-synonymous single-nucleotide variants (SNVs) of UBASH3B, OR6M1, OR8D4, OR8B4, and BEST3 genes were detected in all 4 affected individuals, but in none of the 5 unaffected individuals. A non-synonymous SNV of the BEST3 gene, Chr12(GRCh37):g.70048878G>T, NM_032735.2:c.1816C>A, p.(L606I), was identified as rare missense variant. BEST3 is located on chromosome 12q15 and encodes bestrophin 3 from the bestrophin family of anion channels. The 4 other non-synonymous SNVs of UBASH3B, OR6M1, OR8D4, and OR8B4 were not considered plausible candidates for mandibular prognathism. Our whole-exome sequencing implicates a rare non-synonymous SNV of BEST3 as a candidate for mandibular prognathism in the Japanese pedigree.

Molecular mechanisms of gating in the calcium-activated chloride channel bestrophin.

Bestrophin (BEST1-4) ligand-gated chloride (Cl-) channels are activated by calcium (Ca2+). Mutation of BEST1 causes retinal disease. Partly because bestrophin channels have no sequence or structural similarity to other ion channels, the molecular mechanisms underlying gating are unknown. Here, we present a series of cryo-electron microscopy structures of chicken BEST1, determined at 3.1 Å resolution or better, that represent the channel's principal gating states. Unlike other channels, opening of the pore is due to the repositioning of tethered pore-lining helices within a surrounding protein shell that dramatically widens a neck of the pore through a concertina of amino acid rearrangements. The neck serves as both the activation and the inactivation gate. Ca2+ binding instigates opening of the neck through allosteric means whereas inactivation peptide binding induces closing. An aperture within the otherwise wide pore controls anion permeability. The studies define a new molecular paradigm for gating among ligand-gated ion channels.

Effects of Ca2+, Glu and GABA on hBest1 and composite hBest1/POPC surface films.

Bestrophinopathies are ocular diseases caused by mutations in the human bestrophin-1 (hBest1) - transmembrane Ca2+-activated chloride channel protein, mainly expressed in the retinal pigment epithelium (RPE) cells. hBest1 is also an important transporter for neurotransmitters such as glutamate (Glu) and γ-aminobutyric acid (GABA) in the nervous system. Recently, a new biological role of hBest1, related to its possible involvement in the pathology of brain diseases (Alzheimer's, Parkinson's disease) has been proposed. Here, we report the effects of Ca2+, Glu and GABA on hBest1 and composite hBest1/POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) Langmuir and Langmuir-Blodgett monolayers based on surface dynamics (π/A isotherms, hysteresis and compressibility), morphology (Brewster angle microscopy, BAM) and visualization of protein molecular organization (Atomic force microscopy, AFM). Ca2+ ions and neurotransmitters Glu and GABA affect hBest1 topology at the air/water interface altering its surface activity, size, orientation and organization. In contrast, no significant changes were detected on π/A isotherms and hysteresis of the composite hBest1/POPC films but their effects on structure, aggregation state and orientation hBest1 established by BAM and AFM differentiate. We found that the binary films of hBest1 and POPC are phase separated at the air/water interface, suggesting stronger lipid-lipid and protein-protein interactions than lipid-protein interactions that can significantly alter the molecular organization and activity of hBest1 in cell membranes. Our data shed light on structure, surface behavior and organization of hBest1 that define relationship structure-functional activity of hBest1 as transport channel.

Direct interaction with 14-3-3γ promotes surface expression of Best1 channel in astrocyte.

BACKGROUND: Bestrophin-1 (Best1) is a calcium-activated anion channel (CAAC) that is expressed broadly in mammalian tissues including the brain. We have previously reported that Best1 is expressed in hippocampal astrocytes at the distal peri-synaptic regions, called microdomains, right next to synaptic junctions, and that it disappears from the microdomains in Alzheimer's disease mouse model. Although Best1 appears to be dynamically regulated, the mechanism of its regulation and modulation is poorly understood. It has been reported that a regulatory protein, 14-3-3 affects the surface expression of numerous membrane proteins in mammalian cells. METHODS: The protein-protein interaction between Best1 and 14-3-3γ was confirmed by yeast-two hybrid assay and BiFC method. The effect of 14-3-3γ on Best1-mediated current was measured by whole-cell patch clamp technique. RESULTS: We identified 14-3-3γ as novel binding partner of Best1 in astrocytes: among 7 isoforms of 14-3-3 protein, only 14-3-3γ was found to bind specifically. We determined a binding domain on the C-terminus of Best1 which is critical for an interaction with 14-3-3γ. We also revealed that interaction between Best1 and 14-3-3γ was mediated by phosphorylation of S358 in the C-terminus of Best1. We confirmed that surface expression of Best1 and Best1-mediated whole-cell current were significantly decreased after a gene-silencingof 14-3-3γ without a significant change in total Best1 expression in cultured astrocytes. Furthermore, we discovered that 14-3-3γ-shRNA reduced Best1-mediated glutamate release from hippocampal astrocyte by recording a PAR1 receptor-induced NMDA receptor-mediated current from CA1 pyramidal neurons in hippocampal slices injected with adenovirus carrying 14-3-3γ-shRNA. Finally, through a structural modeling, we found critical amino acid residues containing S358 of Best1 exhibiting binding affinities to 14-3-3γ. CONCLUSIONS: 14-3-3γ promotes surface expression of Best1 channel in astrocytes through direct interaction.

Patient-specific mutations impair BESTROPHIN1's essential role in mediating Ca2+-dependent Cl- currents in human RPE.

Mutations in the human BEST1 gene lead to retinal degenerative diseases displaying progressive vision loss and even blindness. BESTROPHIN1, encoded by BEST1, is predominantly expressed in retinal pigment epithelium (RPE), but its physiological role has been a mystery for the last two decades. Using a patient-specific iPSC-based disease model and interdisciplinary approaches, we comprehensively analyzed two distinct BEST1 patient mutations, and discovered mechanistic correlations between patient clinical phenotypes, electrophysiology in their RPEs, and the structure and function of BESTROPHIN1 mutant channels. Our results revealed that the disease-causing mechanism of BEST1 mutations is centered on the indispensable role of BESTROPHIN1 in mediating the long speculated Ca2+-dependent Cl- current in RPE, and demonstrate that the pathological potential of BEST1 mutations can be evaluated and predicted with our iPSC-based 'disease-in-a-dish' approach. Moreover, we demonstrated that patient RPE is rescuable with viral gene supplementation, providing a proof-of-concept for curing BEST1-associated diseases.

Two heterozygous mutations identified in one Chinese patient with bilateral macular coloboma.

Congenital macular coloboma is characterized by defined punched out atrophic lesions of the macula. The present study aimed to investigate the genetic alterations of one Chinese sporadic patient with bilateral large macular coloboma. Complete ophthalmic examinations, including best­corrected visual acuity, slit­lamp examination, fundus examination, fundus photograph and fundus fluorescein angiography imaging, Pentacam, and optical coherence tomography were performed on the patient. Genomic DNA was extracted from leukocytes in a peripheral blood sample collected from the patient, the patient's unaffected family members and from 200 unrelated control subjects from the same population. Next­generation sequencing of the known genes involved in ocular disease was performed. The functional effects of the mutation were analyzed using Polymorphism Phenotyping (PolyPhen) and Sorting Intolerant From Tolerant (SIFT). One heterozygous bestrophin 1 (BEST1) mutation c.1037C>A (p.Pro346His, p.P346H) in exon 9 and one heterozygous regulating synaptic membrane exocytosis 1 (RIMS1) mutation c.3481A>G (p.Arg1161Gly, p.R1161G) in exon 23 were identified in the patient being investigated, but not in the unaffected family members or unrelated control subjects. Polyphen and SIFT predicted that the amino acid substitution p.P346H in the BEST1 protein is damaging. In addition, Polyphen predicted that the amino acid substitution p.R1161G in the RIM1 protein is damaging. The results of the current study have increased the mutation spectrums of BEST1 and RIMS1, and are valuable for improving the current genetic counseling process and developing novel therapeutic interventions for patients with macular coloboma.

A BEST example of channel structure annotation by molecular simulation.

An increasing number of ion channel structures are being determined. This generates a need for computational tools to enable functional annotation of channel structures. However, several studies of ion channel and model pores have indicated that the physical dimensions of a pore are not always a reliable indicator of its conductive status. This is due to the unusual behavior of water within nano-confined spaces, resulting in a phenomenon referred to as "hydrophobic gating". We have recently demonstrated how simulating the behavior of water within an ion channel pore can be used to predict its conductive status. In this addendum to our study, we apply this method to compare the recently solved structure of a mutant of the bestrophin chloride channel BEST1 with that of the wild-type channel. Our results support the hypothesis of a hydrophobic gate within the narrow neck of BEST1. This provides further validation that this simulation approach provides the basis for an accurate and computationally efficient tool for the functional annotation of ion channel structures.

Distinct regions that control ion selectivity and calcium-dependent activation in the bestrophin ion channel.

Cytoplasmic calcium (Ca2+) activates the bestrophin anion channel, allowing chloride ions to flow down their electrochemical gradient. Mutations in bestrophin 1 (BEST1) cause macular degenerative disorders. Previously, we determined an X-ray structure of chicken BEST1 that revealed the architecture of the channel. Here, we present electrophysiological studies of purified wild-type and mutant BEST1 channels and an X-ray structure of a Ca2+-independent mutant. From these experiments, we identify regions of BEST1 responsible for Ca2+ activation and ion selectivity. A "Ca2+ clasp" within the channel's intracellular region acts as a sensor of cytoplasmic Ca2+. Alanine substitutions within a hydrophobic "neck" of the pore, which widen it, cause the channel to be constitutively active, irrespective of Ca2+. We conclude that the primary function of the neck is as a "gate" that controls chloride permeation in a Ca2+-dependent manner. In contrast to what others have proposed, we find that the neck is not a major contributor to the channel's ion selectivity. We find that mutation of a cytosolic "aperture" of the pore does not perturb the Ca2+ dependence of the channel or its preference for anions over cations, but its mutation dramatically alters relative permeabilities among anions. The data suggest that the aperture functions as a size-selective filter that permits the passage of small entities such as partially dehydrated chloride ions while excluding larger molecules such as amino acids. Thus, unlike ion channels that have a single "selectivity filter," in bestrophin, distinct regions of the pore govern anion-vs.-cation selectivity and the relative permeabilities among anions.