Indiana University-Purdue University Indianapolis (IUPUI) / 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.

Indiana University-Purdue University Indianapolis (IUPUI) / The voltage-gated sodium channels (VGSCs) are critical determinants of neuronal excitability. They set the threshold for action potential generation. The VGSC isoform Nav1.6 participates in various physiological processes and contributes to distinct pathological conditions, but how Nav1.6 function is differentially regulated in different cell types and subcellular locations is not clearly understood. Some VGSC isoforms are subject to S-palmitoylation and exhibit altered functional properties in different S-palmitoylation states. This dissertation investigates the role of S-palmitoylation in Nav1.6 regulation and explores the consequences of S-palmitoylation in modulating neuronal excitability in physiological and pathological conditions. The aims of this dissertation were to 1) provide biochemical and electrophysiological evidence of Nav1.6 regulation by S-palmitoylation and identify specific S-palmitoylation sites in Nav1.6 that are important for excitability modulation, 2) determine the biophysical consequences of epilepsy-associated mutations in Nav1.6 and employ computational models for excitability prediction and 3) test the modulating effects of S-palmitoylation on aberrant Nav1.6 activity incurred by epilepsy mutations. To address these aims, an acyl-biotin exchange assay was used for Spalmitoylation detection and whole-cell electrophysiology was used for channel characterization and excitability examination. The results demonstrate that 1) Nav1.6 is biochemically modified and functionally regulated by S-palmitoylation in an isoform- and site-specific manner and altered S-palmitoylation status of the channel results in substantial changes of neuronal excitability, 2) epilepsy associated Nav1.6 mutations affect different aspects of channel function, but may all converge to gain-of-function alterations with enhanced resurgent currents and increased neuronal excitability and 3) S-palmitoylation can target specific Nav1.6 properties and could possibly be used to rescue abnormal channel function in diseased conditions. Overall, this dissertation reveals S-palmitoylation as a new mechanism for Nav1.6 regulation. This knowledge is critical for understanding the potential role of S-palmitoylation in isoform-specific regulation for VGSCs and providing potential targets for the modulation of excitability disorders. / 2022-05-06

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<p dir="ltr">Migraine is a condition that has affected many for generations and yet remains poorly understood. Mutations to the Na_v1.1 voltage gated sodium channels have been implicated in various diseases such as Familial Hemiplegic Migraine 3 (FHM3), epilepsy, and autism spectrum disorder (ASD). Various proteins have been found to modify the function of these channels. Fibroblast growth factor homologous factors (FHFs) have been found to regulate the activity of some voltage-gated sodium channels (Na_vs). More work is needed to determine which FHFs affect which Na_vs. Here I looked at FHF2A and FHF2B in Nav1.1 as well as an FHM3-causing mutation to this channel, F1774S. I found that FHF2A, but not 2B, induced long-term inactivation (LTI) in the wild-type (WT) Nav1.1 and that FHF2A induced LTI in the F1774S mutant channel to a greater extent. Several changes in channel function caused by the mutation were attenuated with the addition of FHF2A, including persistent currents, leading to a possible rescue in the mutant phenotype. By contrast, the P1894L mutation, which has been found to cause ASD, greatly attenuated LTI and other impacts of FHF2A on Nav1.1. The inflammatory cytokine IL-6 was also investigated as a possible modulator of the Na_v1.1 channel. There does not appear to be any direct interaction between this cytokine and the channel. Overall, my data shows for the first time that FHF2A, but FHF2B or IL-6, might be a significant modulator of Nav1.1 and can differentially modulate disease mutations.</p>

Voltage-gated sodium channels are composed of pore-forming α-subunits, and modulatory and multifunctional associated β subunits. While much of the field of cardiac electrophysiology and pathology has focused on treating and preventing cardiac arrhythmias by targeting the α subunit, there is also evidence that targeting the β subunits, particularly SCN1B, the gene that encodes β1 and an alternatively spliced variant β1B, has therapeutic potential. The first attempt at targeting the β1 subunit was with the generation of and treatment with an SCN1B Ig domain mimetic peptide βadp1. Here we describe further investigation into the function and mode-of-action of both βadp1 and novel peptides derived from the original βadp1 sequence. We find that in a heterologous expression system βadp1 initially disrupts β1-mediated trans-homophilic adhesion, but after approximately 30 hours eventually increases adhesion. Novel mimetic dimers increase β1 adhesion up to 48 hours post-treatment. Furthermore, it appears that βadp1 may increase β1 adhesion by upregulating the intramembrane proteolysis of β1, a process which has important downstream implications and effects on translation. Despite these exciting findings, we were unable to translate them into a primary culture of cardiac cells with endogenous expression of β1 because we found that both neonatal rat cardiomyocytes and isolated adult mouse cardiomyocytes do not express β1 at detectable levels, whereas they do appear to express β1B. In summary, we show exciting findings on the function and mode-of-action of SCN1B mimetic peptides and their therapeutic potential in targeting the β1 subunit, but further work is needed to determine the translatability of our findings to in vivo models and eventually to humans. / Doctor of Philosophy / Voltage-gated sodium channels have two main parts: the pore-forming α-subunits and the modulatory β subunits. Most research in heart function and issues has focused on fixing problems with the α subunit. However, there's evidence that working on the β subunits, specifically the SCN1B gene that makes β1 and another version called β1B, could be helpful. Previously, researchers used a peptide that is designed exactly like a part of β1, called βadp1, to target the β1 subunit. In our study, we explore more about how βadp1 works and test new peptides based on βadp1. We found that βadp1 initially disrupts trans-homophilic adhesion, where 2 β1 subunits interact with each other across the space between 2 cells, but after about 30 hours, it actually increases adhesion. New mimetic dimers also boost adhesion up to 48 hours later. It seems like βadp1 might enhance adhesion by triggering a process called intramembrane proteolysis of β1, which has important effects on translation. Despite these exciting findings, we couldn't confirm the presence of this protein in heart cells because we discovered that certain heart cells don't have enough β1, although they do have β1B. In conclusion, our study shows promising results about how SCN1B mimetic peptides work and their potential for treating arrhythmia. However, more research is needed to see if these findings apply to real-life situations and eventually to help people with cardiac conduction abnormalities.

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