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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 19 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2022

Quaternary structure independent folding of voltage-gated ion channel pore domain subunits

Every voltage-gated ion channel (VGIC) has a pore domain (PD) made from four subunits, each comprising an antiparallel transmembrane helix pair bridged by a loop. The extent to which PD subunit structure requires quaternary interactions is unclear. In this report we present crystal structures of a set of bacterial voltage-gated sodium channel (BacNa V ) 'pore only' proteins that reveal a surprising collection of non-canonical quaternary arrangements in which the PD tertiary structure is maintained. This context-independent structural robustness, supported by molecular dynamics simulations, indicates that VGIC-PD tertiary structure is independent of quaternary interactions. This fold occurs throughout the VGIC superfamily and in diverse transmembrane and soluble proteins. Strikingly, characterization of PD subunit-binding Fabs indicates that non-canonical quaternary PD conformations can occur in full-length VGICs. Together, our data demonstrate that the VGIC-PD is an autonomously folded unit. This property has implications for VGIC biogenesis, understanding functional states, de novo channel design, and VGIC structural origins.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Structural basis for severe pain caused by mutations in the S4-S5 linkers of voltage-gated sodium channel Na V 1.7

Gain-of-function mutations in voltage-gated sodium channel Na V 1.7 cause severe inherited pain syndromes, including inherited erythromelalgia (IEM). The structural basis of these disease mutations, however, remains elusive. Here, we focused on three mutations that all substitute threonine residues in the alpha-helical S4-S5 intracellular linker that connects the voltage sensor to the pore: Na V 1.7/I234T, Na V 1.7/I848T, and Na V 1.7/S241T in order of their positions in the amino acid sequence within the S4-S5 linkers. Introduction of these IEM mutations into the ancestral bacterial sodium channel Na V Ab recapitulated the pathogenic gain-of-function of these mutants by inducing a negative shift in the voltage dependence of activation and slowing the kinetics of inactivation. Remarkably, our structural analysis reveals a common mechanism of action among the three mutations, in which the mutant threonine residues create new hydrogen bonds between the S4-S5 linker and the pore-lining S5 or S6 segment in the pore module. Because the S4-S5 linkers couple voltage sensor movements to pore opening, these newly formed hydrogen bonds would stabilize the activated state substantially and thereby promote the 8 to 18 mV negative shift in the voltage dependence of activation that is characteristic of the Na V 1.7 IEM mutants. Our results provide key structural insights into how IEM mutations in the S4-S5 linkers may cause hyperexcitability of Na V 1.7 and lead to severe pain in this debilitating disease.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

CryoEM structures of the human CLC-2 voltage-gated chloride channel reveal a ball-and-chain gating mechanism

CLC-2 is a voltage-gated chloride channel that contributes to electrical excitability and ion homeostasis in many different tissues. Among the nine mammalian CLC homologs, CLC-2 is uniquely activated by hyperpolarization, rather than depolarization, of the plasma membrane. The molecular basis for the divergence in polarity of voltage gating among closely related homologs has been a long-standing mystery, in part because few CLC channel structures are available. Here, we report cryoEM structures of human CLC-2 at 2.46 – 2.76 Å, in the presence and absence of the selective inhibitor AK-42. AK-42 binds within the extracellular entryway of the Cl – -permeation pathway, occupying a pocket previously proposed through computational docking studies. In the apo structure, we observed two distinct conformations involving rotation of one of the cytoplasmic C-terminal domains (CTDs). In the absence of CTD rotation, an intracellular N-terminal 15-residue hairpin peptide nestles against the TM domain to physically occlude the Cl – -permeation pathway. This peptide is highly conserved among species variants of CLC-2 but is not present in other CLC homologs. Previous studies suggested that the N-terminal domain of CLC-2 influences channel properties via a “ball-and-chain” gating mechanism, but conflicting data cast doubt on such a mechanism, and thus the structure of the N-terminal domain and its interaction with the channel has been uncertain. Through electrophysiological studies of an N-terminal deletion mutant lacking the 15-residue hairpin peptide, we support a model in which the N-terminal hairpin of CLC-2 stabilizes a closed state of the channel by blocking the cytoplasmic Cl – -permeation pathway.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Water, Protons, and the Gating of Voltage-Gated Potassium Channels

Ion channels are ubiquitous throughout all forms of life. Potassium channels are even found in viruses. Every cell must communicate with its surroundings, so all cells have them, and excitable cells, in particular, especially nerve cells, depend on the behavior of these channels. Every channel must be open at the appropriate time, and only then, so that each channel opens in response to the stimulus that tells that channel to open. One set of channels, including those in nerve cells, responds to voltage. There is a standard model for the gating of these channels that has a section of the protein moving in response to the voltage. However, there is evidence that protons are moving, rather than protein. Water is critical as part of the gating process, although it is hard to see how this works in the standard model. Here, we review the extensive evidence of the importance of the role of water and protons in gating these channels. Our principal example, but by no means the only example, will be the Kv1.2 channel. Evidence comes from the effects of D2O, from mutations in the voltage sensing domain, as well as in the linker between that domain and the gate, and at the gate itself. There is additional evidence from computations, especially quantum calculations. Structural evidence comes from X-ray studies. The hydration of ions is critical in the transfer of ions in constricted spaces, such as the gate region and the pore of a channel; we will see how the structure of the hydrated ion fits with the structure of the channel. In addition, there is macroscopic evidence from osmotic experiments and streaming current measurements. The combined evidence is discussed in the context of a model that emphasizes the role of protons and water in gating these channels.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Structural basis for severe pain caused by mutations in the voltage sensors of sodium channel NaV1.7

Voltage-gated sodium channels in peripheral nerves conduct nociceptive signals from nerve endings to the spinal cord. Mutations in voltage-gated sodium channel NaV1.7 are responsible for a number of severe inherited pain syndromes, including inherited erythromelalgia (IEM). Here, we describe the negative shifts in the voltage dependence of activation in the bacterial sodium channel NaVAb as a result of the incorporation of four different IEM mutations in the voltage sensor, which recapitulate the gain-of-function effects observed with these mutations in human NaV1.7. Crystal structures of NaVAb with these IEM mutations revealed that a mutation in the S1 segment of the voltage sensor facilitated the outward movement of S4 gating charges by widening the pathway for gating charge translocation. In contrast, mutations in the S4 segments modified hydrophobic interactions with surrounding amino acid side chains or membrane phospholipids that would enhance the outward movement of the gating charges. These results provide key structural insights into the mechanisms by which these IEM mutations in the voltage sensors can facilitate outward movements of the gating charges in the S4 segment and cause hyperexcitability and severe pain in IEM. Our work gives new insights into IEM pathogenesis at the near-atomic level and provides a molecular model for mutation-specific therapy of this debilitating disease.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Microelectrode Arrays Measure Blocking of Voltage-Gated Calcium Ion Channels on Supported Lipid Bilayers Derived from Primary Neurons

Drug studies targeting neuronal ion channels are crucial to understand neuronal function and develop therapies for neurological diseases. The traditional method to study neuronal ion-channel activities heavily relies on the whole-cell patch clamp as the industry standard. However, this technique is both technically challenging and labour-intensive, while involving the complexity of keeping cells alive with low throughput. Therefore, the shortcomings are limiting the efficiency of ion-channel-related neuroscience research and drug testing. Here, this work reports a new system of integrating neuron membranes with organic microelectrode arrays (OMEAs) for ion-channel-related drug studies. This work demonstrates that the supported lipid bilayers (SLBs) derived from both neuron-like (neuroblastoma) cells and primary neurons are integrated with OMEAs for the first time. The increased expression of voltage-gated calcium (CaV) ion channels on differentiated SH-SY5Y SLBs compared to non-differentiated ones is sensed electrically. Also, dose-response of the CaV ion-channel blocking effect on primary cortical neuronal SLBs from rats is monitored. The dose range causing ion channel blocking is comparable to literature. This system overcomes the major challenges from traditional methods (e.g., patch clamp) and showcases an easy-to-test, rapid, ultra-sensitive, cell-free, and high-throughput platform to monitor dose-dependent ion-channel blocking effects on native neuronal membranes.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2024

Dual receptor-sites reveal the structural basis for hyperactivation of sodium channels by poison-dart toxin batrachotoxin

The poison dart toxin batrachotoxin is exceptional for its high potency and toxicity, and for its multifaceted modification of the function of voltage-gated sodium channels. By using cryogenic electron microscopy, we identify two homologous, but nonidentical receptor sites that simultaneously bind two molecules of toxin, one at the interface between Domains I and IV, and the other at the interface between Domains III and IV of the cardiac sodium channel. Together, these two bound toxin molecules stabilize α/π helical conformation in the S6 segments that gate the pore, and one of the bound BTX-B molecules interacts with the crucial Lys1421 residue that is essential for sodium conductance and selectivity via an apparent water-bridged hydrogen bond. Overall, our structure provides insight into batrachotoxin’s potency, efficacy, and multifaceted functional effects on voltage-gated sodium channels via a dual receptor site mechanism.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Design and structural basis of selective 1,4-dihydropyridine inhibitors of the calcium-activated potassium channel K Ca 3.1

The 1,4-dihydropyridines, drugs with well-established bioavailability and toxicity profiles, have proven efficacy in treating human hypertension, peripheral vascular disorders, and coronary artery disease. Every 1,4-dihydropyridine in clinical use blocks L-type voltage-gated calcium channels. We now report our development, using selective optimization of a side activity (SOSA), of a class of 1,4-dihydropyridines that selectively and potently inhibit the intermediate-conductance calcium-activated K + channel K Ca 3.1, a validated therapeutic target for diseases affecting many organ systems. One of these 1,4-dihydropyridines, DHP-103, blocked K Ca 3.1 with an IC 50 of 6 nM and exhibited exquisite selectivity over calcium channels and a panel of >100 additional molecular targets. Using high-resolution structure determination by cryogenic electron microscopy together with mutagenesis and electrophysiology, we delineated the drug binding pocket for DHP-103 within the water-filled central cavity of the K Ca 3.1 channel pore, where bound drug directly impedes ion permeation. DHP-103 inhibited gain-of-function mutant K Ca 3.1 channels that cause hereditary xerocytosis, suggesting its potential use as a therapeutic for this hemolytic anemia. In a rat model of acute ischemic stroke, the second leading cause of death worldwide, DHP-103 administered 12 h postischemic insult in proof-of-concept studies reduced infarct volume, improved balance beam performance (measure of proprioception) and decreased numbers of activated microglia in infarcted areas. K Ca 3.1-selective 1,4-dihydropyridines hold promise for the many diseases for which K Ca 3.1 has been experimentally confirmed as a therapeutic target.

Ong, Seow Theng [Lee Kong Chian School of Medicine↗
DOE OSTI2025

Electrosome assembly: Structural insights from high voltage-activated calcium channel (CaV)–chaperone interactions

Ion channels are multicomponent complexes (termed here as“electrosomes”) that conduct the bioelectrical signals required for life. It has been appreciated for decades that assembly is critical for proper channel function, but knowledge of the factors that undergird this important process has been lacking. Although there are now exemplar structures of representatives of most major ion channel classes, there has been no direct structural information to inform how these complicated, multipart complexes are put together or whether they interact with chaperone proteins that aid in their assembly. Recent structural characterization of a complex of the endoplasmic membrane protein complex (EMC) chaperone and a voltage-gated calcium channel (CaV) assembly intermediate comprising the pore-forming CaVα1 and cytoplasmic CaVβ subunits offers the first structural view into the assembly of a member of the largest ion channel class, the voltagegated ion channel (VGIC) superfamily. The structure shows how the EMC remodels the CaVα1/CaVβ complex through a set of rigid body movements for handoff to the extracellular CaVα2δ subunit to complete channel assembly in a process that involves intersubunit coordination of a divalent cation and ordering of CaVα1 elements. These findings set a new framework for deciphering the structural underpinnings of ion channel biogenesis that has implications for understanding channel function, how drugs and disease mutations act, and for investigating how other membrane proteins may engage the ubiquitous EMC chaperone.

Biochemistry & Molecular Biology↗
DOE OSTI2024

Chemical Imaging for In Situ Detection and Discrimination of Aquatic Toxins Targeting Voltage Gated Sodium Channels

Biologically derived neurotoxins from cyanobacteria and algae impact environmental resources in addition to being considered a potential biological threat to human and animal health. Activity based assays are essential to detecting and responding to toxic neurotoxin events either naturally occurring or deliberate. Two toxins of interest include saxitoxin and brevetoxin. These toxins bind to and alter the function of voltage-gated sodium channels (NaV channels) which are essential for generating cell membrane action potential. We report the development and refinement of a System for Analysis at Liquid Vacuum Interface (SALVI) to assess the functional activity of saxitoxin and brevetoxin. This approach utilizes a vacuum-compatible microfluidic reactor that permits analysis at the liquid vacuum interface of human derived cells with a neurotoxin of interest in a biologically relevant environment.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2026

Molecular pathways for learning in the single-cell Stentor coeruleus

The single-cell Stentor coeruleus contracts in response to mechanical taps but habituates and learns to ignore the taps after repeated stimulation. Here, we explored the molecular changes that occur during the formation of this cellular memory in order to improve our understanding of non-synaptic learning. We impaired cellular protein synthesis with cycloheximide and puromycin and found that, contrary to the effects of such treatments on metazoa, these drugs accelerate habituation and prolong memory retention in Stentor . Exploratory proteomic and transcriptomic analyses identified candidate proteins and genes that changed over the course of habituation and response recovery, pointing toward the regulation of Stentor learning by calcium signaling and protein phosphorylation. Building on these results, we found that using RNA interference to knock down the calcium-binding, EF-hand domain-containing protein SteCoe_6763 accelerated habituation. Furthermore, increased extracellular calcium improved Stentor learning, while treatment with kinase and phosphatase inhibitors impaired learning. In particular, KN-93, a drug known to inhibit calcium/calmodulin-dependent kinase II and voltage-gated calcium channels, decreased both the rate and extent of habituation in Stentor , similar to its effects on learning in metazoa. We also discovered that habituation memory can be maintained in progeny following cell division. Taken together, these results suggest that response recovery in Stentor requires new protein synthesis and that memory formation involves the modification of delocalized mechanoreceptors by phosphorylation and calcium signaling. This is consistent with our previous model of Stentor learning, in which habituation occurs through the inactivation of cell-surface receptors.

CaMKII↗
DOE OSTI2025

Prediction of Specificity of α-Conotoxins to Subtypes of Human Nicotinic Acetylcholine Receptors with Semi-supervised Machine Learning

Conotoxins are a family of highly toxic neurotoxins composed of cysteine-rich peptides produced by marine cone snails. The most lethal cone snail species to humans is Conus geographus, with fatality rates of up to ∼65% from a single sting, which is caused mostly by the activity of α-conotoxins against human nicotinic acetylcholine receptors (nAChRs). While sequence-based machine learning (ML) classifiers have been trained to identify targets of conotoxins binding voltage-gated ion channels, no ML model has been built to predict the subtype-specific nAChR targets of α-conotoxins. Here, we trained an ML model in a semi-supervised manner to predict the specificity of α-conotoxin binding toward different human nAChR subtypes to overcome the challenge of limited data in subtype-specific nAChR targets of α-conotoxins and the issue that one α-conotoxin can bind multiple nAChR subtypes with high selectivity. We considered additional features of sequences of α-conotoxins in training our ML model, including the secondary structure propensities and electrostatic properties, which resulted in better prediction capability for the ML model. Notably, we identify that most α-conotoxins bind to α3β2, α1γδ, and α7 subtypes of human nAChRs. Our findings from this study provide a framework for predicting targets of various kinds of toxins.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Coupling sensor to enzyme in the voltage sensing phosphatase

Voltage-sensing phosphatases (VSPs) dephosphorylate phosphoinositide (PIP) signaling lipids in response to membrane depolarization. VSPs possess an S4-containing voltage sensor domain (VSD), resembling that of voltage-gated cation channels, and a lipid phosphatase domain (PD). The mechanism by which voltage turns on enzyme activity is unclear. Structural analysis and modeling suggest several sites of VSD-PD interaction that could couple voltage sensing to catalysis. Voltage clamp fluorometry reveals voltage-driven rearrangements in three sites implicated earlier in enzyme activation—the VSD-PD linker, gating loop and R loop—as well as the N-terminal domain, which has not yet been explored. N-terminus mutations perturb both rearrangements in the other segments and enzyme activity. Our results provide a model for a dynamic assembly by which S4 controls the catalytic site.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2024

An artificial intelligence accelerated virtual screening platform for drug discovery

Abstract Structure-based virtual screening is a key tool in early drug discovery, with growing interest in the screening of multi-billion chemical compound libraries. However, the success of virtual screening crucially depends on the accuracy of the binding pose and binding affinity predicted by computational docking. Here we develop a highly accurate structure-based virtual screen method, RosettaVS, for predicting docking poses and binding affinities. Our approach outperforms other state-of-the-art methods on a wide range of benchmarks, partially due to our ability to model receptor flexibility. We incorporate this into a new open-source artificial intelligence accelerated virtual screening platform for drug discovery. Using this platform, we screen multi-billion compound libraries against two unrelated targets, a ubiquitin ligase target KLHDC2 and the human voltage-gated sodium channel Na V 1.7. For both targets, we discover hit compounds, including seven hits (14% hit rate) to KLHDC2 and four hits (44% hit rate) to Na V 1.7, all with single digit micromolar binding affinities. Screening in both cases is completed in less than seven days. Finally, a high resolution X-ray crystallographic structure validates the predicted docking pose for the KLHDC2 ligand complex, demonstrating the effectiveness of our method in lead discovery.

Science & Technology - Other Topics↗
DOE OSTI2025

Structural basis for saxitoxin congener binding and neutralization by anuran saxiphilins

Dinoflagellates and cyanobacteria produce saxitoxin (STX) and ~50 congeners that disrupt bioelectrical signals by blocking voltage-gated sodium channels (NaVs). Consuming seafood carrying these toxins causes paralytic shellfish poisoning (PSP). Although NaVs and anuran STX binding proteins (saxiphilins, Sxphs) use convergent STX binding modes, the structural basis for STX congener recognition is unknown. Here, we show that American bullfrog (Rana catesbeiana) RcSxph and High Himalaya frog (Nanorana parkeri) NpSxph sequester STX congeners using a ‘lock and key’ mode shared with STX. Importantly, functional studies demonstrate that Sxph ‘toxin sponges’ reverse NaV block by multiple STX congeners and detect these toxins in a radioligand binding assay (RBA) used for environmental testing. Together, our study establishes how Sxphs sequester select neurotoxins and uncover STX congener-specific interactions distinct from NaVs. These findings expand understanding of toxin sponge action and provide a foundation for strategies to monitor and mitigate the harmful effects of STX congeners.

Zakrzewska, Sandra↗
DOE OSTI2025

CACNA1D is a circadian gene and causes familial advanced sleep phase

Familial advanced sleep phase (FASP) is a heritable human sleep trait characterized by early sleep onset and offset times. We have identified five variants in five different families in the human voltage-gated calcium channel subunit alpha1 D (CACNA1D) that cosegregate with FASP. The variants inCACNA1Dlead to altered channel dynamics in vitro. A mouse model of the E427K variant has a normal circadian period under constant darkness but displays altered phase shifts in response to light in the subjective night at circadian time (CT) 16 and CT22. Overall, these experiments establishCACNA1Das an FASP gene with altered entrainment, highlighting the ability of human genetics to uncover novel aspects of human circadian regulation.

Science & Technology - Other Topics↗
DOE OSTI2022

Structure of the Human BK Ion Channel in Lipid Environment

Voltage-gated and ligand-modulated ion channels play critical roles in excitable cells. To understand the interplay among voltage sensing, ligand binding, and channel opening, the structures of ion channels in various functional states and in lipid membrane environments need to be determined. Here, the random spherically constrained (RSC) single-particle cryo-EM method was employed to study human large conductance voltage- and calcium-activated potassium (hBK or hSlo1) channels reconstituted into liposomes. The hBK structure was determined at 3.5 Å resolution in the absence of Ca 2+ . Instead of the common fourfold symmetry observed in ligand-modulated ion channels, a twofold symmetry was observed in hBK in liposomes. Compared with the structure of isolated hSlo1 Ca 2+ sensing gating rings, two opposing subunits in hBK unfurled, resulting in a wider opening towards the transmembrane region of hBK. In the pore gate domain, two opposing subunits also moved downwards relative to the two other subunits.

59 BASIC BIOLOGICAL SCIENCES↗