Dissertation
Dissertation > Medicine, health > Neurology and psychiatry > Neurology > Brain diseases > Epilepsy

The Pathogenesis of Epilepsy of SCN1A Mutation

Author LiuChao
Tutor LiaoWeiPing
School Guangzhou Medical College
Course Internal Medicine
Keywords Epilesy Mutation Voltage-gated sodium channel Patch clamp Nav1.1 SCN1A SMEI Dravet syndrome Channelopathy
CLC R742.1
Type PhD thesis
Year 2011
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BackgroundEpilepsy is a group of central nervous system dysfunction syndrome with the alteration of neuronal excitability, a high degree of synchronization of abnormal brain neurons discharging, and characterized as episodic, transient, repetitive and stereotyped. Epileptic discharge is easily formed in the case of increasing membrane excitability of neurons, which is the basis of the pathophysiology of epilepsy. Research has uncovered a growing number of single-gene mutations that cause epilepsy. The majority of these genes encode ion channels or receptors, including voltage-gated sodium, potassium, calcium, chloride channels and acetylcholine receptors andγ-aminobutyric acid (GABA) receptor. The voltage-gated sodium channels are in part responsible for controlling cell electrical excitability, and its abnormal structure and function can cause changes in membrane excitability, which play an important role in the pathogenesis of epilepsy.The mammalian voltage-gated sodium channel consists ofαsubunit and twoβsubunits. Theαsubunits are encoded by nine genes of the same family (SCN1A-SCN11A), SCN1A encode one isoform, termed Nav1.1. Theαsubunit comprises four homologous domains termed I–IV. Within each domain, there are six transmembrane segments called S1–S6. The Nav1.1 is predominantly expressed in inhibitory interneurons. Four accessoryβ1,β2,β3, orβ4 subunits are encoded by SCN1B-SCN4B respectively. In the adult CNS, theαsubunits are associated withβ1 andβ2. SCN1A mutations mainly can cause the three epilepsy syndrome, generalized epilepsy with febrile seizures plus (GEFS+), severe myoclonic epilepsy of infancy (SMEI), and partial epilepsy with febrile seizures plus (PEFS+). Mutations in SCN1B lead to GEFS+. We found a novel missense mutation c.4868A>C in SCN1A genetic screening of patients with SMEI. The mutation lead to the 1623 amino acid glutamate into alanine (E1623A), it was located at S3 segments of the fourth domain where highly conserved. Up to now, there are 10 mutations located in S3 of Nav1.1 in patients with SMEI, but the effect of these mutations on the function of Nav1.1 has not been reported. The mechanism of Nav1.1 E1623A leading to epilepsy is worthy of further study.PurposeTo explore the pathogenesis of epilepsy caused by the mutation of SCN1A c.4868 A>C. by comparing the electrophysiological changes using whole cell patch clamp in HEK 293T cells with expression of Nav1.1 E1623A andβ1 andβ2 subunits.Methods1. Sequencing the pCMV-SCN1A plasmid and identificating the expression of Nav1.1pCMV-SCN1A plasmid was identified first by endonuclease, and then the SCN1A coding region was confirmed by sequencing. Total protein of HEK 293T cells transfected with pCMV-SCN1A plasmid was extracted after 48h, and the expression of Nav1.1 was detected by using western blot.2. Constructing the pDsred-IRES-SCN1B plasmidRed fluorescent DNA fragment was amplified from pCMV-Dsred plasmid using PCR methods with disigned restriction enzyme cutting site, and it was cloned to TA vector. pDsred-TA plasmid which was confirmed by sequencing was digested with endonuclease, and Dsred DNA fragment was gotten. pCD8 marks was removed from pCD8-IRES-SCN1B plasmid by endonuclease and IRES-SCN1B vector was gotten. pDsred-IRES-SCN1B plasmid was constructed by ligating Dsred DNA fragment with IRES-SCN1B vector with DNA ligase. SCN1B coding region in pDsred-IRES- SCN1B plasmid was confirmed by sequencing. 3. Sequencing the pGFP-IRES-SCN2B plasmidpGFP-IRES-SCN2B plasmid was identified first by endonuclease, and then the SCN2B coding region was confirmed by sequencing.4. Constructing the pCMV-SCN1A-E1623A plasmidpCMV-SCN1A-E123A plasmid was constructed with site-directed mutagenesis kit and confirmed by sequencing.5. Patch clamp detectionpCMV-SCN1A plasmid or pCMV-SCN1A-E1623A plasmid, pDsred-IRES- SCN1B plasmid and pGFP-IRES-SCN2B plasmid were co-transfected in HEK 293T cells, the red and green fluorescence were observed under fluorescent microscope after 48h. Only cell with the red and green fluorescence can be used for next patch clamp detection. During patch clamp detection, the standard stimulus was given in the whole-cell voltage clamp mode. According to current data, the current density curves of sodium current, voltage dependent activation curve, steady-state inactivation curve and recovery curve after inactivation were established, and the current density of sodium current, half activation voltage, half inactivation voltage and resurrection time constant were calculated and compared.6. Statistical analysisData were shown as mean±standard deviation ( X±S). SPSS l6.0 software package was used for the statistical methods. Statistical comparisons were done with the two-sample t-test. P <0.05 was statistically significant difference.Results1. Sequencing the pCMV-SCN1A plasmid and identificating the expression of Nav1.1No mutation was found in coding sequence of SCN1A in pCMV-SCN1A plasmid. Western blot results showed that: the level of Nav1.1 has been markedly expressed in HEK 293T cells transfected with pCMV-SCN1A plasmids 48h later.2. Constructing the pDsred-IRES-SCN1B plasmidDsred PCR products were successfully ligated to the IRES-SCN1B vector. pDsred-IRES-SCN1B plasmid was sequenced and no mutation was found in its coding region.3. Sequencing the pGFP-IRES-SCN2B plasmidNo mutation was found in coding sequence of SCN2B in pGFP-IRES-SCN2B plasmid.4. Constructing the pCMV-SCN1A-E1623A plasmidThe mutation of 1623 glutamic acid codon (GAG) to alanine codon (GCG) in SCN1A was confirmed by sequencing, while other mutations in SCN1A in the plasmid were not found.5. Patch clamp detectionAt -10mv, the level of sodium channel current was maximal in wild type Nav1.1 and E1623A mutant. Peak current density of wild type Nav1.1 is about -232.1±19.1pA/pF (n=10), half activation potential (V1/2) of activation curve was -24.8±0.4mv (n=10), slope factor K was 5.2±0.3; half inactivation potential (V1/2) of steady-state inactivation curve was -53.0±0.5mv (n=10), slope factor K was -8.6±0.4; Recovery time constantτfast andτslow were 1.4±0.1ms (n=9) and 39.9±6.0ms .Compared with that of wild type Nav1.1, peak current density of E1623A mutant was about -100.7±16.2pA/pF (n=7) (p <0.05); half activation potential (V1/2) of activation curve was -24.6±0.5mv (n=7), slope factor K is 8.4±0.4 (p<0.05); steady-state inactivation curve shifted in the negative direction, half inactivation potential (V1/2) was -60.9±0.5mv (n=7) (p <0.05), slope factor K was -11.5±0.5 (p <0.05); Recovery time constantτfast andτslow were 2.1±0.2ms (n=6) ( p <0.05) and 42.5±6.9ms.Conclusions1. Nav1.1 E1623A displayed a decreased current density, a slower activation, a negative shift in the steady-state inactivation and a significantly delayed recovery from inactivation. These data indicates that Nav1.1 E1623A results in partial loss of function. 2. The E1623A missense mutation may decrease the availability of the Nav1.1 channel in inhibitory interneurons, resulting in the partial dysfunction of network inhibition. The resulting dysfunction of network inhibition can hyperactivate excitatory neurons leading to epilepsy.

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