Functional Expression Of A Novel SCN5A Mutation K317N Identified In A Chinese Brugada Syndrome Family

Introduction:Since its introduction as a clinical entity in 1992, the Brugada syndrome hasprogressed from being a rare disease to one that is second only to automobile accidents as a cause of death among young adults in some countries. Electrocardiographically characterized by a distinct ST-segment elevation and complete or incomplete right bundle branch block in the right precordial leads, the syndrome is associated with a high risk for sudden cardiac death in young and otherwise healthy adults. One of the genes linked to this syndrome is SCN5A, the gene encoding for the cardiac sodium channel. Mutations in SCN5A cause a functional reduction in the availability of cardiac sodium current in Brugada syndrome. ST-segment elevation in the Brugada syndrome is thought to be due to a rebalancing of the currents active at the end of phase 1,leading to an accentuation of the action potential notch in RV epicardium .A transinet outward current Ito mediated spike and dome morphology,or notch,in ventricular epicardium,but not endocardium ,creates a transmural voltage gradient responsible for the inscription of the electrocardiographic J wave in humans. Several dozen SCN5A mutations have been linked to the syndrome over the past years and shown to result in either 1failure of the sodium channel to express; 2 reduced current due to a shift in the voltage and time dependence of sodium channel current activation , inactivation,or reactivation ; or 3 reduced contribution of Iwa during the early phases of the action potential resulting from accelerated inactivation of the sodium channel. We have reported a case of a novel SCN5A mutation associated with Brugada syndrome in a Chinese family. SSCP and DNA sequence analysis of SCN5A in this patient revealed a missense mutation (K317N) in the P-Loop region of domain I. This mutational change was not present in 100 healthy Chinese controls.Objective :We have found a new mutation K317N of SCN5A in a Chinese Brugada syndrome family. However, the molecular and cellular electrophysiological mechanism of such mutation causing Brugada syndrome keeps unknown.For the purpose of exploring the function role of K317N of SCN5A, In the first part we intended to site-directed mutagenize and construct human cardiac sodium a unit cDNA (hHl) mutated recombinant pRc/CMV-hHl. And in the second part, sodium channel current (Ins) and kinetics were investigated using patch clamp whole-cell recording in human embryonic kidney 293 cell into which the hHl or mhHl was transfected successfully. Methods :The first,We mutagenized SCN5A K317N mutation through a single step PCR with a forward primer (30bp) located 5' to a Sse8387 I site and a reverse mega-primer (94bp) incorporated with K317N mutation and Agel restricted site using plasmid pRc/CMV-hHl as PCR template. The purified PCR product and plasmid pRc/CMV-hHl were digested with Agel and sse8387I respectively, then ligated into a full length pRc/CMV-hHl. The ligated product was transformed intoE.coli JM109 competent cell. Positive clones were identified using restricted enzyme Agel and sse8387I and performed DNA sequencing of the amplified region to identify mutant K317N site.The second, Mammalian cell transfection and expression: A 293 cell line that stably expressed Na channel & i-subunits was established by using Lipofectamine?2000. Positive colonies were then selected by G418 resistance.Then, Standard liposome methods were used to transiently transfect HEK293 cells with Na+ channel a-subunits hHl or mhHl.Electrophysiological recording: Macroscopic Na+ currents were recorded using the patch-clamp technique in the whole cell mode. Patch pipettes were pulled from borosilicate capillary glass, lightly fire-polished to resistance 2. 5M1Q -3. 5 MD when filled with pipette solution, and connected to the head stage of a patch-clamp amplifier (CEZ-2300,NihonKohden Japan). Cells were transferred on glass coverslips to the recording chamber on the stage of an inverted microscope .Unless otherwise noted,recordings were made at 25 °C. Solutions. External Tyrode bathing solution consisted of (in mM) 137 NaCl, 5.4 KC1, 1 MgCl2, 2 CaCl2, 10 glucose, and 10 HEPES, pH 7.3. Internal pipette solution contained (inmM) 120 CsF, 10 CsCl, 10 EGTA, and 10 HEPES, pH 7.3. Data acquisition and analysis. Generation of voltage commands,data acquisition, data analysis, and curve fitting was accomplished with pClamp8.0 software (Axon Instruments). Result:Recombinant pRc/CMV-hHl (K317N) has been constructed successfully and confirmed by DNA sequencing as with K317N mutation and without PCR incorporated nucleotide error.A 293 cell line that stably expressed Na+channel P rsubunits was established and the positive colonies were selected by G418 resistance.And the cells expressingthe construst appeared green under UV epifluorescence. After transient transfection with WT a-subunits, we recorded large Na+ currents from the stable 3 i-cell line. No detectable currents were observed in the absence of a-subunit transfection.However, after transient transfection with K317N a-subunits,we did not record any Na+ currents from the stable P i-cell line. Conclusion :We have successfully site-directed mutagenized and constructed the recombinant expression plasmid pRc/CMV-hHl (K317N) carrying human cardiac sodium a unit cDNA (hHl) with SCN5A mutation K317N identified in a Chinese Brugada syndrome family, and provided molecular material for further functional genomics research of SCN5A .The improved PCR-based megaprimer method for site-directed mutagenesis is rapid,simple and highly efficient.This method is suitable for routine application in moleculur cloning.The sodium channel kinetics characteristic of WT hHl is the same as the normal cardiac muscle cell. The missense mutation (K317N) in the P-Loop region of domain I maybe cause the failure of the sodium channel to express.