Molecular Basis Of Na_v1.5-MOG1 Interaction And Its Key Role In Cardiac Arrhythmias

Cardiac arrhythmias cause more than 1 000 000 sudden death each year worldwide.Genetic basis is one of the main pathogenic factors,such as SCN5A mutation.It is well known that loss-of-function mutations in SCN5A gene cause Brugada syndrome（Br S）and gain-of-function mutations cause Long QT syndrome（LQTS）.However,the molecular mechanism needs to be further studied.The cardiac sodium channel is a multiprotein complex consisting of anα-subunit of Na_v1.5 encoded by the SCN5A gene and other associated proteins.Using yeast two-hybrid analysis,our lab previously reported MOG1,a Ran-GTP binding protein,binds to Na_v1.5 and facilitates its trafficking to cell surface,thereby increasing cardiac sodium current(I_Na)density.To date,～200 and～300 SCN5A mutations were found in patients with Br S and LQTS,respectively.However,evidences for these mutations causing the disease,and the pathogenic molecular mechanisms are limited.Thus,the aim of the present study is:（1）to characterize the molecular basis for MOG1-Na_v1.5 interaction and its function on Na_v1.5,and（2）to identify whether the SCN5A mutations located at MOG1 binding sites are disease-causing mutations and reveal molecular mechanism for cardiac arrythmia caused by SCN5A mutations.To characterize the molecular basis for MOG1-Na_v1.5 interaction,truncations or point mutations were firstly created in Na_v1.5 expression vector.Then,combined glutathione S-transferases（GST）pull-down assay,Na_v1.5 surface protein isolation,and whole cell patch clamp for recording I_Na were performed to identify the MOG1 binding region（site）on Na_v1.5 and whether the binding region（site）is required for MOG1 function on increased Na_v1.5 surface protein expression and I_Nadensities.By detection of the interaction between MOG1 and all cytoplasmic regions of Na_v1.5,the results showed that the binding between MOG1 and Na_v1.5 Loop I was obviously the strongest.To narrow down the MOG1 binding regions on Na_v1.5 Loop I,truncation analysis was performed and the results showed that MOG1 interacted with S₄₇₆～H₅₈₅of Na_v1.5 Loop I,which was critical for MOG1 function on increased Na_v1.5 surface expression and I_Nadensities.Moreover,F₅₃₀～R₅₃₅of Na_v1.5was essential for Na_v1.5 interaction with MOG1 by further truncation analysis.Point mutation analysis for F₅₃₀～R₅₃₅showed that MOG1 effectively interacted with residues F₅₃₀,F₅₃₂,R₅₃₃,R₅₃₄of Na_v1.5,which were required for MOG1 function on increased Na_v1.5 surface expression and I_Nadensities.In addition,the residue（s）of MOG1 mediated MOG1-Na_v1.5binding were also determined by creating point mutations in MOG1 expression vector.Results showed that the Na_v1.5 Loop I binding sites on MOG1 as residues D₂₄,E₃₆,D₄₄,E₅₃,and E₁₀₁,which were critical for MOG1 function on increased I_Na densities.Next,the molecular mechanisms for cardiac arrhythmia caused by SCN5A mutations located at Na_v1.5-MOG1 binding region were investigated.Firstly,whether the mutations affect the Na_v1.5-MOG1 interaction were detected by GST pull-down assay.Secondly,whole cell patch clamp assays were performed to determine whether the mutations affect I_Na densities,gating kinetics,or late I_Na.Results showed the SCN5A mutation p.F532C identified in Br S patient markedly disrupted MOG1-Na_v1.5 binding and impaired MOG1function on increased I_Na densities,Interestingly,this mutation markedly reduced I_Nadensities in rat neonatal cardiomyocytes.However,the SCN5A mutation p.R535Q identified in LQTS patients neither affect MOG1 binding,nor late I_Na,which is associated with LQTS.Moreover,the mutation markedly reduced I_Na densities and positively shifted the activation potential.The SCN5A mutation p.F530V identified in LQTS patients neither affect late I_Na,MOG1-Na_v1.5 binding,I_Na densities nor gating kinetics of Na_v1.5.In the present study,three main findings are as follows:（1）full-understanding the mechanism of how MOG1 regulates Na_v1.5,which requires D₂₄,E₃₆,D₄₄,E₅₃ and E₁₀₁ of MOG1 and F₅₃₀,F₅₃₂,R₅₃₃,R₅₃₄ of Na_v1.5 for their interaction and MOG1 mediated the increased I_Na densities.（2）Revealing the potential pathogenic mechanism of SCN5A mutation p.F532C found in Br S patient:the mutation disrupted MOG1-Na_v1.5 binding and decreased I_Na densities in cardiomyocytes thereby may causing Br S.（3）The SCN5A mutation p.R535Q identified in LQTS patient leads to loss-of-function of Na_v1.5,speculating that this mutation may cause Br S or atrial fibrillation（AF）.The SCN5A mutation p.F530V found in LQTS patient may be a benign single nucleotide polymorphism（SNP）,but not the cause of LQTS.Together,the study contributes to the genetic diagnosis of Br S and LQTS,and provides a theoretical basis for treatment of Br S and sudden death in further.