Mechanistic Insights Into The Interaction Of The MOG1 Protein With Cardiac Sodium Channel Na_V1.5 Clarify The Molecular Basis Of Brugada Syndrome

The sodium ion channel is a protein embedded in the cell membrane that allows sodium ions(Na~+)to pass through.Sodium ion channels play an important role in the action potential,distributed in muscle cells,neurons,glial cells.According to activating mode,the sodium ion channel can be divided into voltage gating channel(activated by voltage)and ligand gating channel(activated by ligand combination).Cardiac sodium channels belong to voltage-gated channels and play an important role in the generation and conduction of action potentials in cardiac myocytes.The heart sodium channel is a protein complex consisting of?subunit,?subunits,and other regulating proteins.?subunit is the center of the heart sodium ion channel that is folded by four homologous domains.Each domain consists of six transmembrane segments(S1-S6),and the fourth fragment(S4)is sensitive to voltage.?subunit can form the channel that conduct Na~+independently,?subunits and other regulatory proteins regulate the voltage gating properties and distribution on the cell membrane of the heart sodium channel.Na _V1.5 is the?subunit of the cardiac sodium channel.Abnormal expression or function of Na _V1.5 cause a variety of arrhythmias including Brugada syndrome(Br S),sick sinus syndrome(SSS),cardiac conduction disease(CCD),dilated cardiomyopathy(DCM),and sudden infant death syndrome(SIDS).MOG1 is a small,evolutionarily conserved protein(20 k Da)that can intereact with Ran GTPase and regulate the transport of m RNAs and proteins between the nucleus and cytoplasm.However,in 2008,our laboratory reported that Ran-binding protein MOG1interacts with Na _V1.5,plays an important role in promoting Na _V1.5 intracellular trafficking from endoplasmic reticulum to plasma membranes.Knockdown of MOG1expression,Na _V1.5 was retained in the endoplasmic reticulum and failed to be transported to the cell membrane.Overexpression of MOG1 promotes the trafficking of Na _V1.5 from the endoplasmic reticulum to the cell membrane,thus promoting its expression on the membrane.In addition,MOG1 does not affect the kinetic properties of Na _V1.5.Therefore,overexpression of MOG1 is a potential treatment option for arrhythmia and sudden death diseases caused by decreased sodium current density.However,the molecular basis for the MOG1/Na _V1.5 interaction and the binding site of MOG1 protein are still unclear.In 2011,MOG1 was identified as a Brugada syndrome susceptibility gene and two mutations(p.E83D and p.E61X)are associated with Brugada syndrome.However,the molecular mechanism by which MOG1 mutations cause Brugada syndrome remains unknown.This paper reveals the key amino acids on the MOG1 protein that bind to Na _V1.5and the molecular mechanisms of the MOG1 mutations that cause of Brugada syndrome.We assessed the effects of defined MOG1 deletions and alanine-scanning substitutions on MOG1's interaction with Na _V1.5.Large deletion analysis mapped the MOG1 domain required for the interaction with Na _V1.5 to the region spanning amino acids 146–174,and a refined deletion analysis further narrowed this domain to amino acids 146–155.Site-directed mutagenesis further revealed that Asp-148,Arg-150,and Ser-151 residues in a loop are essential for binding to Na _V1.5.GST pull down and electrophysiological analyses disclosed that the substitutions D148Q,R150Q,and S151Q disrupt MOG1's interaction with Na _V1.5 and significantly reduce its trafficking to the cell surface.Examination of MOG1's 3D structure revealed that Glu-83 and the loop containing Asp-148,Arg-150,and Ser-151 are spatially proximal,suggesting that these residues may form a critical binding site for Na _V1.5.GST pull down and sodium current density analysis showed that Glu-83 plays an important role in the binding of MOG1 and Na _V1.5.The region of the MOG1 protein that binds to Na _V1.5 includes two parts.The loop from Asp-148 to Ser-151 and Glu-83 form a forceps-like structure,and bind to Na _V1.5.Overall,the loop at Asp-148 to Ser-151 and Glu-83 form the two separate sides of a clamp,respectively,which may make MOG1 bind to Na _V1.5 by clamping down on a specific structure of Na _V1.5.E83D mutation failed to bind to Na _V1.5,which caused Na _V1.5 trafficking defects and decreased Na _V1.5 on the membrane,resulting in Brugada syndrome.The other Brugada syndrome mutation,MOG1 E61X,encodes 60 amino acids at the N-terminus of MOG1.However,these 60 amino acids do not contain the binding sites of MOG1 we identified.Therefore,E61X does not contain the domains that MOG1 binds to Na _V1.5,which will lead to a defect in Na _V1.5 trafficking and a decrease in sodium current density and cause Brugada syndrome.In conclusion,our findings identify the structural elements in MOG1 that are crucial for its interaction with Na _V1.5 and improve our understanding of how the MOG1mutations cause Brugada syndrome.These results provide important foundation for development of drugs and therapeutic techniques to treat Brugada syndrome,lethal arrhythmias and sudden death.