Biology subjects

Long, S. B.

Publications and source records attributed to Long, S. B..

5 recordsLinked to original sources

bioRxiv2025

Structure of the human TWIK-2 potassium channel and its inhibition by pimozide

The potassium channel TWIK-2 is crucial for ATP-induced activation of the NLRP3 inflammasome in macrophages. The channel is a member of the two-pore domain potassium (K2P) channel superfamily and an emerging therapeutic target to mitigate severe inflammatory injury involving NLRP3 activation. We report the cryo-EM structure of human TWIK-2. In comparison to other K2P channels, the structure reveals a unique up conformation of Tyr111 in the selectivity filter and a SF1-P1 pocket behind the filter that could serve as a binding site for channel modulators. Density for acyl chains is present in fenestrations within the transmembrane region that connect the central cavity of the pore to the lipid membrane. Limited pharmacological tools are available for TWIK-2 despite its importance as a drug target. We show that the small molecule pimozide inhibits TWIK-2 and determine a structure of the channel with pimozide. Pimozide displaces the acyl chains and binds below the selectivity filter to block ion conduction. The drug may access its binding site via the membrane, suggesting that other hydrophobic small molecules could have utility for inhibiting TWIK-2. The work defines the structure of TWIK-2 and provides a structural foundation for development of specific inhibitors with potential utility as anti-inflammatory drugs. Significance StatementThe TWIK-2 potassium channel is a member of the two-pore domain potassium (K2P) channel superfamily and a potential therapeutic target to control severe inflammatory injury involving the NLRP3 inflammasome. We report the cryo-EM structure of the human TWIK-2 channel at 2.85 [A] resolution, revealing differences in comparison to other K2P channels. We identify that pimozide, an FDA-approved drug for Tourette syndrome, inhibits TWIK-2. A cryo-EM structure of TWIK-2 in complex with pimozide identifies its binding location and mechanism of inhibition. The work provides a structural foundation for development of specific TWIK-2 inhibitors that have potential therapeutic utility for inflammatory diseases involving NLRP3 activation.

biochemistry↗

bioRxiv2024

The pentameric chloride channel BEST1 is activated by extracellular GABA

Bestrophin 1 (BEST1) is chloride channel expressed in the eye, central nervous system (CNS), and other tissues in the body. A link between BEST1 and the principal inhibitory neurotransmitter {gamma}-aminobutyric acid (GABA) has been proposed. The most appreciated receptors for extracellular GABA are the GABAB G-protein coupled receptors and the pentameric GABAA chloride channels, both of which have fundamental roles in the CNS. Here, we demonstrate that BEST1 is directly activated by GABA. Through functional studies and atomic-resolution structures of human and chicken BEST1, we identify a GABA binding site on the channels extracellular side and determine the mechanism by which GABA binding induces opening of the channels central gate. This same gate is activated by intracellular [Ca2+], indicating that BEST1 is controlled by ligands from both sides of the membrane. The studies demonstrate that BEST1, which shares no structural homology with GABAA, is a GABA-activated chloride channel. The physiological implications of this finding remain to be studied. Significance Statement{gamma}-aminobutyric acid (GABA) is the principal inhibitory neurotransmitter in the central nervous system. Extracellular GABA is primarily sensed by GABAB G-protein coupled receptors, and GABAA pentameric chloride channels. We show that the chloride channel bestrophin-1 (BEST1) is also activated by extracellular GABA. The application of GABA, but not glycine or other endogenous molecules we tested, markedly potentiates chloride currents through the channel. Structural studies combined with electrophysiology and other functional studies reveal the mechanism of GABA activation. GABA binding within the outer entryway of the channel allosterically controls its gate, the neck. Molecular interactions with GABA resemble those in GABAA receptors, despite a lack of homology between the channels. The physiological significance of GABA-activation in BEST1 remains to be studied.

biochemistry↗

bioRxiv2020

Cryo-EM structure of the calcium release-activated calcium channel Orai in an open conformation

The calcium release-activated calcium channel Orai regulates Ca2+ entry into non-excitable cells and is required for proper immune function. The channel typically opens following the release of Ca2+ from the endoplasmic reticulum. Certain pathologic mutations render the channel constitutively open. Here, using one such mutation (H206A), we present a cryo-EM structure of Orai from Drosophila melanogaster in an open conformation at 3.3 [A] resolution. Comparison with previous closed structures reveals that opening occurs through the outward movements of M1 helices that dilate the central pore. Repositionings of a ring of phenylalanine residues (F171) expose previously shielded glycine residues (G170) to the channel pore, despite the absence of significant rotational movement of the associated pore-lining helices. This phenylalanine ring and two rings of flanking hydrophobic amino acids act as a hydrophobic gate to control ion permeation. Extracellular M1-M2 turrets, not evident from previous Orai structures, form an electronegative pore entrance. Single sentence summaryA structure of the Ca2+ channel Orai in an open conformation provides insight into the opening mechanism of the channel and its role in regulating selective Ca2+ entry into immune and other non-excitable cells.

biochemistry↗

bioRxiv2020

Structure and reconstitution of a MCU-EMRE mitochondrial Ca2+ uniporter complex

The proteins MCU and EMRE form the minimal functional unit of the mitochondrial calcium uniporter complex in metazoans, a highly selective and tightly controlled Ca2+ channel of the inner mitochondrial membrane that regulates cellular metabolism. Here we present functional reconstitution of an MCU-EMRE complex from the red flour beetle, Tribolium castaneum, and a cryo-EM structure of the complex at 3.5 [A] resolution. Robust Ca2+ uptake is observed into proteoliposomes containing the purified complex and is dependent on EMRE. The structure reveals a tetrameric channel with a single ion pore. EMRE is located at the periphery of the transmembrane domain and associates primarily with the first transmembrane helix of MCU. Coiled coil and juxtamembrane domains within the matrix portion of the complex adopt markedly different conformations than in a structure of a human MCU-EMRE complex, suggesting that the structures represent different conformations of these functionally similar metazoan channels.

biochemistry↗

bioRxiv2018

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

Bestrophin (BEST1-4 in humans) channels are ligand gated chloride (Cl-) channels that are activated by calcium (Ca2+). Mutations in BEST1 cause retinal degenerative diseases. Partly because these 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 (cryo-EM) structures of chicken BEST1, determined at 3.1 [A] resolution or better, that represent the principal gating states of the channel. 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 within the protein core. The neck serves as both the activation and the inactivation gate. The binding of Ca2+ to a cytosolic domain instigates pore opening and the structures reveal that, unlike voltage-gated Na+ and K+ channels, similar molecular rearrangements are responsible for inactivation and deactivation. A single aperture within the 95 [A]-long opened pore separates the cytosol from the extracellular milieu and controls anion permeability. The studies define the basis for Ca2+-activated Cl- channel function and reveal a new molecular paradigm for gating in ligand-gated ion channels.

biochemistry↗