Structural and functional characterization of the bestrophin-2 anion channel

Owji, Aaron P.; Zhao, Qingqing; Ji, Changyi; Kittredge, Alec; Hopiavuori, Austin; Fu, Ziao; Ward, Nancy E.; Clarke, Oliver; Shen, Yin; Zhang, Yu; Hendrickson, Wayne A.; Yang, Tingting

doi:10.1038/s41594-020-0402-z

Cited by 28 publications

(51 citation statements)

References 61 publications

(90 reference statements)

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“…Similar dual hydrophobic gating mechanisms are characteristic of ion channels with a lyotropic selectivity sequence 37 , including calcium-activated Clchannels 48,49 and members of the Cysloop receptor family 50 . The hydrophobicity of residues that make up the two gates are conserved in other members of the SLC1A family, including the neutral amino acid exchangers ASCT1/2, which also exhibit dual transporter/channel function 51,52 .…”

Section: Discussionmentioning

confidence: 79%

Glutamate transporters have a chloride channel with two hydrophobic gates

Chen

Pant

et al. 2021

Nature

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Glutamate is the most abundant excitatory neurotransmitter in the central nervous system, therefore its precise control is vital for maintaining normal brain function and preventing excitotoxicity 1 . Removal of extracellular glutamate is achieved by plasma membrane-bound transporters, which couple glutamate transport to sodium, potassium and pH gradients using an elevator mechanism [2][3][4][5] . Glutamate transporters also conduct chloride ions via a channel-like process that is thermodynamically uncoupled from transport [6][7][8] . However, the molecular mechanisms that allow these dual-function transporters to carry out two seemingly contradictory roles are unknown. Here we report the cryo-electron microscopy structure of a glutamate transporter homologue in an open-channel state, revealing an aqueous cavity that is formed during the transport cycle. Using functional studies and molecular dynamics simulations, we show that this cavity is an aqueous-accessible chloride permeation pathway gated by two hydrophobic regions, and is conserved across mammalian and archaeal glutamate transporters. Our findings provide insight into the mechanism by which glutamate transporters support their dual function and add a crucial piece of information to aid mapping of the complete transport cycle shared by the SLC1A transporter family.

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Section: Discussionmentioning

confidence: 79%

Glutamate transporters have a chloride channel with two hydrophobic gates

Chen

Pant

et al. 2021

Nature

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“…For single-particle Cryo-EM APols provide an attractive proposition as they eliminate the background noise, often present due to free micelles in detergent-protein samples, and potentially enable proteins to retain co-purifying lipids that may lock them in more stable and or native conformations. APol A8-35 and PMAL-C8 are the most commonly used for determining high-resolution structures with the highest achieved being the bovine-bestrophin-2 anion channel at 2.17 Å [ 90 ]. The aptness of APols for Cryo-EM stimulated the development of novel CyclApols which consolidate the properties of SMA and A8–35.…”

Section: Amphipolsmentioning

confidence: 99%

Methods for the solubilisation of membrane proteins: the micelle-aneous world of membrane protein solubilisation

Ratkeviciute

Cooper

Knowles

2021

Biochemical Society Transactions

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The solubilisation of membrane proteins (MPs) necessitates the overlap of two contradictory events; the extraction of MPs from their native lipid membranes and their subsequent stabilisation in aqueous environments. Whilst the current myriad of membrane mimetic systems provide a range of modus operandi, there are no golden rules for selecting the optimal pipeline for solubilisation of a specific MP hence a miscellaneous approach must be employed balancing both solubilisation efficiency and protein stability. In recent years, numerous diverse lipid membrane mimetic systems have been developed, expanding the pool of available solubilisation strategies. This review provides an overview of recent developments in the membrane mimetic field, with particular emphasis placed upon detergents, polymer-based nanodiscs and amphipols, highlighting the latest reagents to enter the toolbox of MP research.

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“…Both KpBest and cBest1 have been used as models to dissect molecular mechanisms of mammalian bestrophins through functional and structural studies [ 75–79 ]. Recently, the first structure of a mammalian bestrophin channel was solved by single-particle cryogenic electron microscopy (cryoEM) using bovine ( Bos taurus ) Best2 (bBest2, Figure 1 , right) [ 80 , 81 ]. KpBest, cBest1, and bBest2 are the only bestrophin structures to date, laying the basis for structure–function studies.…”

Section: Overall Architecturementioning

confidence: 99%

“…An ion passing through the channel from the extracellular side of the membrane will first encounter the “neck,” a hydrophobic gate located at the level of the intracellular leaflet of the plasma membrane. This channel feature is composed of three highly conserved hydrophobic residues residing on the second transmembrane alpha-helix (helix S2b, also termed transmembrane domain 2 (TMD2) in earlier studies) with the conserved hydrophobic residues pointing in toward the central axis of the channel [ 74 , 81 , 82 , 3 , 4 , 72 ]. In cBest1 and bBest2, the neck is composed of I76, F80, and F84 ( Figure 2a , left), with their counterparts in KpBest being I62, I66, and F70, respectively.…”

Section: The Neck: a Hydrophobic Gate Within The Membranementioning

confidence: 99%

Structure and Function of the Bestrophin family of calcium-activated chloride channels

et al. 2021

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Bestrophins are a family of calcium-activated chloride channels (CaCCs) with relevance to human physiology and a myriad of eye diseases termed “bestrophinopathies”. Since the identification of bestrophins as CaCCs nearly two decades ago, extensive studies from electrophysiological and structural biology perspectives have sought to define their key channel features including calcium sensing, gating, inactivation, and anion selectivity. The initial X-ray crystallography studies on the prokaryotic homolog of Best1, Klebsiella pneumoniae (KpBest), and the Best1 homolog from Gallus gallus (chicken Best1, cBest1), laid the foundational groundwork for establishing the architecture of Best1. Recent progress utilizing single-particle cryogenic electron microscopy has further elucidated the molecular mechanism of gating in cBest1 and, separately, the structure of Best2 from Bos taurus (bovine Best2, bBest2). Meanwhile, whole-cell patch clamp, planar lipid bilayer, and other electrophysiologic analyses using these models as well as the human Best1 (hBest1) have provided ample evidence describing the functional properties of the bestrophin channels. This review seeks to consolidate these structural and functional results to paint a broad picture of the underlying mechanisms comprising the bestrophin family’s structure-function relationship.

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Structural and functional characterization of the bestrophin-2 anion channel

Cited by 28 publications

References 61 publications

Glutamate transporters have a chloride channel with two hydrophobic gates

Glutamate transporters have a chloride channel with two hydrophobic gates

Methods for the solubilisation of membrane proteins: the micelle-aneous world of membrane protein solubilisation

Structure and Function of the Bestrophin family of calcium-activated chloride channels

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