Browsing by Subject "Voltage-gated sodium channel"

Now showing 1 - 5 of 5
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    Cardiac sodium channel palmitoylation regulates channel function and cardiac excitability with implications for arrhythmia generation
    (2016-12-09) Pei, Zifan; Cummins, Theodore R.; Oxford, Gerry S.; Hudmon, Andy; Rubart-von der Lohe, Michael; Sheets, Patrick L.
    The  cardiac  voltage-­gated  sodium  channels  (Nav1.5)  play  a  specific  and   critical  role  in  regulating  cardiac  electrical  activity  by  initiating  and  propagating   action  potentials  in  the  heart.  The  association  between  Nav1.5  dysfunctions  and   generation  of  various  types  of  cardiac  arrhythmia  disease,  including  long-­QT3   and  Brugada  syndrome,  is  well  established.  Many  types  of  post-­translational   modifications  have  been  shown  to  regulate  Nav1.5  biophysical  properties,   including  phosphorylation,  glycosylation  and  ubiquitination.  However,  our   understanding  about  how  post-­translational  lipid  modification  affects  sodium   channel  function  and  cellular  excitability,  is  still  lacking.  The  goal  of  this   dissertation  is  to  characterize  Nav1.5  palmitoylation,  one  of  the  most  common   post-­translational  lipid  modification  and  its  role  in  regulating  Nav1.5  function  and   cardiac  excitability.     In  our  studies,  three  lines  of  biochemistry  evidence  were  shown  to  confirm   Nav1.5  palmitoylation  in  both  native  expression  background  and  heterologous   expression  system.  Moreover,  palmitoylation  of  Nav1.5  can  be  bidirectionally   regulated  using  2-­Br-­palmitate  and  palmitic  acid.  Our  results  also  demonstrated   that  enhanced  palmitoylation  in  both  cardiomyocytes  and  HEK293  cells   increases  sodium  channel  availability  and  late  sodium  current  activity,  leading  to   enhanced  cardiac  excitability  and  prolonged  action  potential  duration.  In  contrast,   blocking  palmitoylation  by  2-­Br-­palmitiate  increases  closed-­state  channel inactivation  and  reduces  myocyte  excitability.  Our  computer  simulation  results   confirmed  that  the  observed  modification  in  Nav1.5  gating  properties  by  protein   palmitoylation  are  adequate  for  the  alterations  in  cardiac  excitability.  Mutations  of   potential  palmitoylation  sites  predicted  by  CSS-­Palm  bioinformatics  tool  were   introduced  into  wild-­type  Nav1.5  constructs  using  site-­directed  mutagenesis.   Further  studies  revealed  four  cysteines  (C981,  C1176,  C1178,  C1179)  as   possible  Nav1.5  palmitoylation  sites.  In  particular,  a  mutation  of  one  of  these   sites(C981)  is  associated  with  cardiac  arrhythmia  disease.  Cysteine  to   phenylalanine  mutation  at  this  site  largely  enhances  of  channel  closed-­state   inactivation  and  ablates  sensitivity  to  depalmitoylation.  Therefore,  C981  might  be   the  most  important  site  that  regulates  Nav1.5  palmitoylation.  In  summary,  this   dissertation  research  identified  novel  post-­translational  modification  on  Nav1.5   and  revealed  important  details  behind  this  process.  Our  data  provides  new   insights  on  how  post-­translational  lipid  modification  alters  cardiomyocyte   excitability  and  its  potential  role  in  arrhythmogenesis.
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    Differential Regulation of Nav1.1 and SCN1A Disease Mutant Sodium Current Properties by Fibroblast Growth Factor Homologous Factors
    (MDPI, 2025-02-15) Frazee, Ashley; Zybura, Agnes; Cummins, Theodore R.; Biology, School of Science
    Fibroblast growth factor homologous factors (FHFs) regulate the activity of several different voltage-gated sodium channels (Navs). However, more work is needed to determine how specific FHF isoforms and variants affect the properties of different Nav isoforms. In addition, it is not known if FHFs can differentially modulate the properties of Nav variants associated with disease. Here, we investigated the effects of FHF2A and FHF2B on Nav1.1 properties as well as on a familial hemiplegic migraine 3 (FHM3) causing mutation in this channel, F1774S. We found that FHF2A, but not 2B, induced prominent long-term inactivation (LTI) in the wild-type (WT) Nav1.1. Interestingly, FHF2A induced LTI in the F1774S FHM3 mutant channel to a greater extent than in the WT. Furthermore, persistent currents caused by the F1774S mutation were attenuated by the co-expression of FHF2A, leading to a possible rescue of the mutant channel phenotype. By contrast, the P1894L mutation, which is associated with epilepsy and mild intellectual disability, greatly attenuated the LTI induced by FHF2A. Overall, our data show for the first time that FHF2A might be a significant modulator of Nav1.1 that can differentially modulate the impact of Nav1.1 disease-associated mutations.
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    Distinctive Properties and Powerful Neuromodulation of Nav1.6 Sodium Channels Regulates Neuronal Excitability
    (MDPI, 2021-06-25) Zybura, Agnes; Hudmon, Andy; Cummins, Theodore R.; Biology, School of Science
    Voltage-gated sodium channels (Navs) are critical determinants of cellular excitability. These ion channels exist as large heteromultimeric structures and their activity is tightly controlled. In neurons, the isoform Nav1.6 is highly enriched at the axon initial segment and nodes, making it critical for the initiation and propagation of neuronal impulses. Changes in Nav1.6 expression and function profoundly impact the input-output properties of neurons in normal and pathological conditions. While mutations in Nav1.6 may cause channel dysfunction, aberrant changes may also be the result of complex modes of regulation, including various protein-protein interactions and post-translational modifications, which can alter membrane excitability and neuronal firing properties. Despite decades of research, the complexities of Nav1.6 modulation in health and disease are still being determined. While some modulatory mechanisms have similar effects on other Nav isoforms, others are isoform-specific. Additionally, considerable progress has been made toward understanding how individual protein interactions and/or modifications affect Nav1.6 function. However, there is still more to be learned about how these different modes of modulation interact. Here, we examine the role of Nav1.6 in neuronal function and provide a thorough review of this channel’s complex regulatory mechanisms and how they may contribute to neuromodulation.
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    Identification and Characterization of Novel Proteins from Arizona Bark Scorpion Venom That Inhibit Nav1.8, a Voltage-Gated Sodium Channel Regulator of Pain Signaling
    (MDPI, 2021-07-18) El-Aziz, Tarek Mohamed Abd; Xiao, Yucheng; Kline, Jake; Gridley, Harold; Heaston, Alyse; Linse, Klaus D.; Ward, Micaiah J.; Rokyta, Darin R.; Stockand, James D.; Cummins, Theodore R.; Fornelli, Luca; Rowe, Ashlee H.; Biology, School of Science
    The voltage-gated sodium channel Nav1.8 is linked to neuropathic and inflammatory pain, highlighting the potential to serve as a drug target. However, the biophysical mechanisms that regulate Nav1.8 activation and inactivation gating are not completely understood. Progress has been hindered by a lack of biochemical tools for examining Nav1.8 gating mechanisms. Arizona bark scorpion (Centruroides sculpturatus) venom proteins inhibit Nav1.8 and block pain in grasshopper mice (Onychomys torridus). These proteins provide tools for examining Nav1.8 structure-activity relationships. To identify proteins that inhibit Nav1.8 activity, venom samples were fractioned using liquid chromatography (reversed-phase and ion exchange). A recombinant Nav1.8 clone expressed in ND7/23 cells was used to identify subfractions that inhibited Nav1.8 Na+ current. Mass-spectrometry-based bottom-up proteomic analyses identified unique peptides from inhibitory subfractions. A search of the peptides against the AZ bark scorpion venom gland transcriptome revealed four novel proteins between 40 and 60% conserved with venom proteins from scorpions in four genera (Centruroides, Parabuthus, Androctonus, and Tityus). Ranging from 63 to 82 amino acids, each primary structure includes eight cysteines and a "CXCE" motif, where X = an aromatic residue (tryptophan, tyrosine, or phenylalanine). Electrophysiology data demonstrated that the inhibitory effects of bioactive subfractions can be removed by hyperpolarizing the channels, suggesting that proteins may function as gating modifiers as opposed to pore blockers.
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    Structural and Functional Characterization of a Novel Scorpion Toxin that Inhibits NaV1.8 via Interactions With the DI Voltage Sensor and DII Pore Module
    (Frontiers Media, 2022-05-19) George, Kiran; Lopez-Mateos, Diego; El-Aziz, Tarek Mohamed Abd; Xiao, Yucheng; Kline, Jake; Bao, Hong; Raza, Syed; Stockand, James D.; Cummins, Theodore R.; Fornelli, Luca; Rowe, Matthew P.; Yarov-Yarovoy, Vladimir; Rowe, Ashlee H.; Biology, School of Science
    Voltage-gated sodium channel NaV1.8 regulates transmission of pain signals to the brain. While NaV1.8 has the potential to serve as a drug target, the molecular mechanisms that shape NaV1.8 gating are not completely understood, particularly mechanisms that couple activation to inactivation. Interactions between toxin producing animals and their predators provide a novel approach for investigating NaV structure-function relationships. Arizona bark scorpions produce Na+ channel toxins that initiate pain signaling. However, in predatory grasshopper mice, toxins inhibit NaV1.8 currents and block pain signals. A screen of synthetic peptide toxins predicted from bark scorpion venom showed that peptide NaTx36 inhibited Na+ current recorded from a recombinant grasshopper mouse NaV1.8 channel (OtNaV1.8). Toxin NaTx36 hyperpolarized OtNaV1.8 activation, steady-state fast inactivation, and slow inactivation. Mutagenesis revealed that the first gating charge in the domain I (DI) S4 voltage sensor and an acidic amino acid (E) in the DII SS2 – S6 pore loop are critical for the inhibitory effects of NaTx36. Computational modeling showed that a DI S1 – S2 asparagine (N) stabilizes the NaTx36 – OtNaV1.8 complex while residues in the DI S3 – S4 linker and S4 voltage sensor form electrostatic interactions that allow a toxin glutamine (Q) to contact the first S4 gating charge. Surprisingly, the models predicted that NaTx36 contacts amino acids in the DII S5 – SS1 pore loop instead of the SS2 – S6 loop; the DII SS2 – S6 loop motif (QVSE) alters the conformation of the DII S5 – SS1 pore loop, enhancing allosteric interactions between toxin and the DII S5 – SS1 pore loop. Few toxins have been identified that modify NaV1.8 gating. Moreover, few toxins have been described that modify sodium channel gating via the DI S4 voltage sensor. Thus, NaTx36 and OtNaV1.8 provide tools for investigating the structure-activity relationship between channel activation and inactivation gating, and the connection to alternative pain phenotypes.