Document text
Principal Investigator: MIGUEL HOLMGREN
Organization: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
Fiscal Year: 2023
Award: $1,616,783
Funding agency: National Institute of Neurological Disorders and Stroke
Calcium ions increase the open probability of the large conductance potassium (BK) channels by binding to high affinity sites within the large cytosolic Ca2+ sensing structure called the gating ring. This Ca2+ sensing structure is formed by eight Regulator of K+ Conductance (RCK) domains, two from each subunit of the channel, termed them RCK1 and RCK2. Other divalent species, like Ba2+ ions, are also able to activate BK channels by interacting with the Ca2+ sensor. Unlike Ca2+ that activate BK channel through all RCK domains, Ba2+ does it selectively through the RCK2 domain. In addition to activation, Ba2+ can also block K+ permeation by binding to the selectivity filter of BK channels. We have solved Cryo-EM structures of BK channels with various concentrations of Ba2+. Structurally, blockade arises from one Ba2+ occupying the traditional K+ site S3 in the selectivity filter of the channel. In addition, electronic densities attributed to K+ ions are detected at K+ sites S2 and S4. In the gating ring, eight Ba2+ ions are bound to the corresponding high affinity Ca2+ binding sites. Thereby, the lack of functional significance of the RCK1 on the activation by Ba2+ indicates that the RCK1 is not in the activated configuration. Finally, these Ba2+ structures reveal an intricate series of concerted changes of the RCK 1 and RCK2 from the same subunit, suggesting coordinated intra-subunit dynamics.
The Na+/K+-ATPase is a membrane embedded enzyme responsible for the exchange of 3 intracellular Na+ for 2 extracellular K+, fueled by the hydrolysis of 1 molecule of ATP. By performing this transport process, the Na+/K+-ATPase maintains the electrochemical gradients of these ions between the internal and external environments of animal cells, which are used for action potential signaling and for cotransport of substrates such as calcium, nutrients, and neurotransmitters. Reduction of Na+/K+-ATPase activity thus causes disease symptoms attributable to impairment of action potentials, disruption of other cellular functions, and cell death. Pathogenic variants in the ATP1A3 Na+/K+-ATPase gene, which encodes an isoform of the catalytic subunit that is highly expressed in neural and heart tissues, cause disabling neurological diseases with varying cardiac involvement. When ATP1A3 residues that participate in ion binding are involved, the resulting phenotypes often fall within the spectrum of rapid-onset dystonia-parkinsonism (RDP) to alternating hemiplegia of childhood (AHC). AHC and RDP are broadly characterized by asymmetric movement disorders plus other features such as plegia, hypotonia, intellectual disability, epilepsy, and cardiac arrhythmia in patients toward the more severe AHC end of the spectrum. The core enzyme is an obligatory heterodimer of an and subunit, in which the subunit contains the ATP binding domain as well as all three ion binding sites. Normal export of Na+ through the Albers-Post enzymatic cycle starts with the binding of 3 intracellular Na+ to the ATP-bound E1 form of the enzyme, which has high Na+ affinity (Na3-E1-ATP). These Na+ occupy 3 sites, numbered I-III in the crystal structure. Binding of all 3 Na+ triggers occlusion, enzyme phosphorylation with release of ADP, and change to a conformation that has an extracellular ion pathway and high affinity for K+ (P-E2-Na3). Extracellular Na+ release occurs sequentially with deocclusion followed by stepwise unbinding of each of the 3 Na+ at different rates. After releasing Na+, the phosphorylated E2 enzyme binds 2 K+ at sites I and II and imports them in a series of steps involving dephosphorylation and ATP binding. We studied mutations of three ATP1A3 residues thought to exclusively coordinate Na+ but not K+: Y768, T771, and D923. Missense variants of the first two (Y768C, Y768H, T771I, T771N) cause the severe phenotype, AHC, but their functional effects have not previously been characterized in ATP1A3. Missense variants of D923 (D923N, D923Y) cause phenotypes ranging across the AHC/RDP spectrum and reduce ion transport, with a reduction in the overall apparent affinity for Na+ observed for D923N. From Na+-bound crystal structures, the side chains of D923, Y768 and T771 specifically participate in the coordination of one Na+ at site III. Unlike sites I and II which are shared by K+ during the transport cycle, site III is uniquely used by one Na+. We also studied a new mutation, P775L that causes a cation leak through the ATP1A3 leading to spasticity and intellectual disability, symptoms that do not fulfil criteria for ATP1A3 related syndromes.
The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) constituted a serious threat to global human health and underscored the need of detailed research into the SARS-CoV-2 virus. This virus which had high sequence similarity to SARS-CoV and bat-borne coronaviruses encodes four structural proteins, sixteen non-structural proteins and nine accessory proteins for viral replication and release. Structural proteins, including spike (S) protein, membrane (M) protein, envelope (E) protein, and nucleocapsid (N) protein, are incorporated into the structurally complete viral particles. Non-structural proteins act as various enzymes and transcription factors for viral replication and pathogenicity. Accessory proteins perform a diverse range of functions, involved in viral release, stability, and pathogenesis. Among these accessory proteins in SARS-CoV-2, ORF3a which is located between S and E proteins on the 3 terminal genome is the largest. It is also a membrane protein, present exclusively in SARS-like coronavirus but no other viruses. SARS-CoV-2 ORF3a had a high sequence identity (73%) with its SARS-CoV homolog, particularly in functional domains, such as the 3 transmembrane (TM) segments, suggesting functional similarity among the homologous ORF3a proteins.
We are focusing our study to four aspects of the biology of ORF3a: 1) its oligoramization state in mammalian cells, 2) its functional properties as an ion channel, 3) its localization within the infected cells, and 4) its proteomic vicinity.
Terms: <2019 novel corona virus><2019 novel coronavirus><2019-nCoV><ATP1A1><ATP1A1 gene><ATP1A3><ATP1A3 gene><Action Potentials><Affinity><Amino Acids><Animals><Arrhythmia><BK channels><Basal Transcription Factor><Basal transcription factor genes><Behavioral><Big K channels><Binding><Binding Sites><Biology><Biophysics><Birth><Body Tissues><COVID crisis><COVID epidemic><COVID pandemic><COVID-19 crisis><COVID-19 epidemic><COVID-19 genome><COVID-19 global health crisis><COVID-19 global pandemic><COVID-19 health crisis><COVID-19 pandemic><COVID-19 public health crisis><COVID-19 virus><COVID-19 virus genome><COVID19 crisis><COVID19 epidemic><COVID19 genome><COVID19 global health crisis><COVID19 global pandemic><COVID19 health crisis><COVID19 pandemic><COVID19 public health crisis><COVID19 virus><COVID19 virus genome><Calcium><Calcium ion><Cardiac><Cardiac Arrhythmia><Catalytic Core><Catalytic Domain><Catalytic Region><Catalytic Site><Catalytic Subunit><Cations><Cell Body><Cell Communication and Signaling><Cell Death><Cell Function><Cell Process><Cell Signaling><Cell membrane><Cell physiology><Cells><Cellular Function><Cellular Physiology><Cellular Process><Cerebellar Ataxia><Cerebellar Incoordination><Childhood><CoV-2><CoV2><Cognitive Disturbance><Cognitive Impairment><Cognitive decline><Cognitive function abnormal><Combining Site><Coronaviridae><Coronavirus><Cryo-electron Microscopy><Cryoelectron Microscopy><Cytoplasmic Membrane><Decreased Muscle Tone><Dephosphorylation><Disease><Disorder><Disturbance in cognition><Dyskinesia Syndromes><Dystonia><Dystonia 12><E protein><Electron Cryomicroscopy><Electronics><Electrophysiology><Electrophysiology (science)><Encephalopathies><Environment><Enzyme Gene><Enzymes><Epilepsy><Epileptic Seizures><Epileptics><Eye Movements><Fever><GeneHomolog><General Transcription Factor Gene><General Transcription Factors><Genes><Genetic Alteration><Genetic Change><Genetic defect><Genome><Goals><Health><Heart><Heart Arrhythmias><Homolog><Homologous Gene><Homologue><Human><Hydrolysis><Hypomyotonia><Hypotonia><Impaired cognition><Impairment><Integral Membrane Protein><Intellectual disability><Intellectual functioning disability><Intellectual limitation><Intracellular Communication and Signaling><Intrinsic Membrane Protein><Ion Channel><Ion Transport><Ionic Channels><Ions><Isoforms><K ATPase><K element><KI mice><Knock-in Mouse><Mammalian Cell><MaxiK channels><Membrane><Membrane Channels><Membrane Potentials><Membrane Protein Gene><Membrane Proteins><Membrane Structure and Function><Membrane-Associated Proteins><Mitochondria><Modern Man><Molecular><Molecular Configuration><Molecular Conformation><Molecular Interaction><Molecular Stereochemistry><Molecular Transport><Monitor><Movement><Movement Disorder Syndromes><Movement Disorders><Muscle Dystonia><Muscle Hypotony><Muscle Tone Poor><Muscle hypotonia><Muscular Hypotonia><Mutation><Na element><Nerve Transmitter Substances><Nervous System Diseases><Neurologic><Neurologic Disorders><Neurologic outcome><Neurological><Neurological Disorders><Neurological outcome><Neurophysiology / Electrophysiology><Neurotransmitters><Non-structural Protein><Nonstructural Protein><Nucleocapsid><Nutrient><ORFs><Open Reading Frames><Optic Atrophy><Organelles><Palsy><Paralysed><Parturition><Pathogenesis><Pathogenicity><Pathway interactions><Patients><Pes Cavus><Phenotype><Phosphorylation><Plasma Membrane><Plegia><Potassium><Probability><Property><Protein Coding Region><Protein Conformation><Protein Dephosphorylation><Protein Dynamics><Protein Isoforms><Protein Phosphorylation><Proteins><Proteomics><Pump><Pyrexia><Rapid onset Dystonia Parkinsonism><Reactive Site><Recurrence><Recurrent><Research><Resistance><Resting Potentials><SARS><SARS Virus><SARS corona virus><SARS corona virus 2><SARS coronavirus><SARS coronavirus disease><SARS-Associated Coronavirus><SARS-CO-V2><SARS-COVID-2><SARS-CoV><SARS-CoV disease><SARS-CoV-1><SARS-CoV-2><SARS-CoV-2 epidemic><SARS-CoV-2 genome><SARS-CoV-2 global health crisis><SARS-CoV-2 global pandemic><SARS-CoV-2 pandemic><SARS-CoV2><SARS-CoV2 epidemic><SARS-CoV2 genome><SARS-CoV2 pandemic><SARS-Related Coronavirus><SARS-associated corona virus 2><SARS-associated coronavirus 2><SARS-coronavirus-2><SARS-coronavirus-2 epidemic><SARS-coronavirus-2 pandemic><SARS-related corona virus 2><SARS-related coronavirus 2><SARSCoV2><Seizure Disorder><Sensorineural Deafness><Sensorineural Hearing Loss><Sensory Hearing Loss><Series><Severe Acute Respiratory Coronavirus><Severe Acute Respiratory Coronavirus 2><Severe Acute Respiratory Distress Syndrome CoV 2><Severe Acute Respiratory Distress Syndrome Corona Virus 2><Severe Acute Respiratory Distress Syndrome Coronavirus 2><Severe Acute Respiratory Syndrome><Severe Acute Respiratory Syndrome CoV 2><Severe Acute Respiratory Syndrome CoV 2 epidemic><Severe Acute Respiratory Syndrome CoV 2 pandemic><Severe Acute Respiratory Syndrome CoV disease><Severe Acute Respiratory Syndrome Virus><Severe Acute Respiratory Syndrome corona virus><Severe Acute Respiratory Syndrome coronavirus><Severe Acute Respiratory Syndrome coronavirus disease><Severe Acute Respiratory Syndrome-associated coronavirus 2><Severe Acute Respiratory Syndrome-related coronavirus 2><Severe acute respiratory syndrome associated corona virus 2><Severe acute respiratory syndrome coronavirus 2><Severe acute respiratory syndrome coronavirus 2 epidemic><Severe acute respiratory syndrome coronavirus 2 pandemic><Severe acute respiratory syndrome related corona virus 2><Side><Signal Transduction><Signal Transduction Systems><Signaling><Site><Site-Directed Mutagenesis><Site-Specific Mutagenesis><Sodium><Structural Protein><Structure><Subcellular Process><Surface Proteins><Symptoms><Syndrome><TM Domain><Talipes cavus><Targeted DNA Modification><Targeted Modification><Techniques><Tissues><Transcription Factor Proto-Oncogene><Transcription factor genes><Transmembrane Domain><Transmembrane Potentials><Transmembrane Protein><Transmembrane Protein Gene><Transmembrane Region><Transport Process><Variant><Variation><Viral><Viral Gene Products><Viral Gene Proteins><Viral Proteins><Virus><Virus Replication><Work><Wuhan coronavirus><alternating hemiplegia><aminoacid><bat-borne><batborne><biological signal transduction><biophysical foundation><biophysical principles><biophysical sciences><body movement><cognitive dysfunction><cognitive loss><conformation><conformational><conformational state><conformationally><conformations><corona virus><corona virus disease 2019 epidemic><corona virus disease 2019 pandemic><coronavirus disease 2019 crisis><coronavirus disease 2019 epidemic><coronavirus disease 2019 genome><coronavirus disease 2019 global health crisis><coronavirus disease 2019 global pandemic><coronavirus disease 2019 health crisis><coronavirus disease 2019 pandemic><coronavirus disease 2019 public health crisis><coronavirus disease 2019 virus><coronavirus disease 2019 virus genome><coronavirus disease crisis><coronavirus disease epidemic><coronavirus disease pandemic><coronavirus disease-19 global pandemic><coronavirus disease-19 pandemic><coronavirus disease-19 virus><cryo-EM><cryoEM><cryogenic electron microscopy><electronic><electronic device><electrophysiological><epilepsia><epileptogenic><experiment><experimental research><experimental study><experiments><extracellular><falls><febrile><febris><genome mutation><hCoV19><intellectual and developmental disability><knockin mice><large-conductance calcium-activated potassium channels><limited intellectual functioning><maxi-K channels><membrane structure><mitochondrial><nCoV2><necrocytosis><nervous system disorder><neural><neurological disease><paralysis><paralytic><particle><pathway><pediatric><plasmalemma><potassium ATPase><recruit><resistant><sensor><sensorineural hearing impairment><severe acute respiratory syndrome coronavirus 2 genome><severe acute respiratory syndrome coronavirus 2 global health crisis><severe acute respiratory syndrome coronavirus 2 global pandemic><severe acute respiratory syndrome-CoV><slowpoke protein><spasticity><transcription factor><viral multiplication><viral replication><virus multiplication><virus protein><voltage>