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.