Functional Roles Of Small Gtpases SAR1A And SAR1B In The Trafficking Of The Cardiac Sodium Channel Na_v1.5

The human SCN5 A gene is located on chromosome 3p21,and encodes the cardiac voltage-gated sodium channel α-subunit Na_v1.5.Na_v1.5 plays an essential role in the initiation and propagation of the cardiac action potential.Mutations in SCN5A/Na_v1.5 cause cardiac arrhythmias and sudden death,including long QT syndrome(LQTS),Brugada syndrome(Br S),idiopathic ventricular tachycardia(VT)and fibrillation(VF),sudden infant death,sick sinus syndrome(SSS)and atrial fibrillation(AF).Little is known about the folding,maturation,translocation from the ER,and targeting of Na_v1.5 to cell surface.However,numerous loss-of-function mutations in SCN5 A disrupt the trafficking of Na_v1.5,leading to decreased Na_v1.5 expression on cell surface and reduced sodium current densities,and cause Br S,idiopathic VT/VF,and SSS.However,the mechanisms by which Na_v1.5 missense mutations cause impaired Na_v1.5 trafficking to the cell surface are mostly unknown.Therefore,understanding the molecular basis of Na_v1.5 trafficking may yield critical molecular insight into the pathogenesis of cardiac arrhythmias,and may suggest novel therapeutic strategies for prevention or treatment of cardiac arrhythmias.Protein trafficking is regulated by small GTPases.Typically,these proteins ha_ve sequence homology and share several conserved domains,including consensus amino acid sequences responsible for interaction with guanosine-5’-diphosphate(GDP)and guanosine-5’-triphosphate(GTP),and a region for interacting with downstream effectors.SAR1 belongs to the family of small GTPases,and regulates the formation or assembly of the ER-derived coat protein complex II(COPII)vesicles involved in the ER export of proteins.There are two paralogs of the SAR1 genes,SAR1A and SAR1B.Bioinformatic analysis showed that SAR1A and SAR1B share 89.9% identify at the amino acid level.However,the role of SAR1A or SAR1B GTPases in ER export of Na_v1.5 is unknown and indeed the critical regulators of ER export of Na_v1.5 remain poorly defined.MOG1 was initially identified as a suppressor that was able to rescue the temperature sensitive defect of S.cerevisiae Ran,a protein involved in nucleocytoplasmic transport,microtubule and nuclear assembly.Our Lab previously reported that MOG1 plays a role in ER export of Na_v1.5.Specifically,MOG1 interacted with an intracellular loop II of Na_v1.5,and facilitated Na_v1.5 cell surface expression to increase the sodium current density.Our Lab further showed that knockdown of MOG1 expression caused retention of Na_v1.5 in the ER and reduced targeting of Na_v1.5 to cell surface,in particular,to the ca_veolae structure on cell surface.However,the mechanisms by which knockdown of MOG1 expression caused retention of Na_v1.5 in the ER is remain unclear.In this study,we investigated the role of the SAR1 GTPases in trafficking of Na_v1.5.Overexpression of dominant-negative mutant SAR1A(T39N or H79G)or SAR1B(T39N or H79G)significantly reduces the expression level of Na_v1.5 on cell surface in HEK293 cells.These data suggest that SAR1A or SAR1B is involved in regulation of the level of cell surface Na_v1.5.Furthermore,overexpression of dominant-negative mutant SAR1A or SAR1B significantly decreases the peak sodium current density(INa)in HEK/Na_v1.5 cells.The findings that dominant-negative mutants of SAR1A and SAR1B significantly reduce the densities of INa in HEK293 cells were confirmed in primary neonatal cardiomyocytes.Together,these data suggest that dominant-negative mutants of SAR1A and SAR1B significantly reduce the densities of cardiac INa in HEK/Na_v1.5 cells and neonatal rat cardiomyocytes,demonstrating a regulatory role of SAR1A and SAR1B in cardiac INa.Simultaneous knockdown of SAR1A and SAR1B expression by siRNAs significantly reduces the INa density in HEK/Na_v1.5 cells,whereas single knockdown of either SAR1A or SAR1B has minimal effect.Computer modeling showed that proteins SAR1 and Ran shared similar structures with each other.RAN was reported to interact with MOG1,a small protein involved in regulation of the ER exit of Na_v1.5.These results suggest that MOG1 could interact with SAR1 in a similar way as MOG1 interaction with Ran.Our GST-pull down analysis showed that Flag-MOG1 overexpressed in HEK293 cells was pulled down by either GST-SAR1A or GST-SAR1B,but not by GST,suggesting that SAR1A or SAR1B interacts with MOG1.Furthermore,co-immunoprecipitation(Co-IP)assays showed that the anti-Flag antibody recognizing Flag-MOG1 successfully precipitated both GFP-SAR1A and SAR1B.Reciprocal Co-IP assays showed that the anti-GFP antibody recognizing SAR1A or SAR1B precipitated Flag-MOG1.Together,these data suggest that SAR1A or SAR1B interacts with MOG1.Interestingly,knockdown of SAR1A and SAR1B expression abolished the MOG1-mediated increases in both cell surface trafficking of Na_v1.5 and the density of INa.These data suggest that the increased trafficking of Na_v1.5 to plasma membranes and enhanced INa density by MOG1 is dependent on SAR1A/B.For the first time,we demonstrated that SAR1A and SAR1B are the critical regulators of trafficking of Na_v1.5 in HEK/Na_v1.5 cells and neonatal rat cardiomyocytes.Moreover,SAR1A and SAR1B interact with MOG1,and are required for MOG1-mediated cell surface expression and function of Na_v1.5.These findings provide important insights into the molecular mechanism of Na_v1.5 trafficking and further characterization of Na_v1.5 trafficking by dissecting the SAR1A/SAR1B-MOG1-Na_v1.5 interaction complex during ER exit,which may be used to enhance the efficacy of MOG1 gene therapy for BrS,SIDS and SSS.