Cloning And Distribution Of The Nav1.5 Channels In Human Brain

ObjectiveVoltage-gated sodium channels (VGSC), which play an essential role in the generation and propagation of action potentials, consist of a large pore-forming a-subunit (≈260kDa, Navl) associated with smaller auxiliaryβ-subunits,β1 (36kDa),β2 (33kDa),β3 andβ4. According to their binding affinity to the specific inhibitor TTX, voltage-gated sodium channels are classified into two types. One type is sensitive to TTX, including Nav1.1, Nav1.2, Nav1.3, Nav1.4, Nav1.6 and Nav1.7, and the other type is resistant or insensitive to TTX, including Navl.5, Nav1.8 and Nav1.9. Among those channels, Nav1.1, Nav1.2 and Navl.3 were first cloned from the brain and functionally analyzed, as they were originally considered as brain sodium channel typesⅠ,ⅡandⅢ. As mentioned above, these channels are sensitive to TTX, interestingly, Na+currents with TTX resistance have also been observed in neurons, but the full-length genes corresponding to these currents in central nervous system (CNS) remain unknown. Although PCR amplified moderate levels of the Navl.5 mRNA from human cerebral cortex, the full-length Nav1.5 cDNA of the brain has not been identified and its specific distribution among different part of the brain was unknown either, let alone its expression patterns with age development. Navl.5 channel was first cloned from the heart and has been well studied, but the structural and functional differences between cardiac Navl.5 channels and cerebral Navl.5 channels still remain unknown, so one of the aims of this study was to clone the full-length Navl.5 cDNA from the brain in order to find the differences between them. Interestingly, our results show that Nav1.5 channels in human and rat brains are encoded by new variants of Nav1.5/SCN5A gene and this gene is more widely distributed and expressed than previously thought.Materials and methods1. MaterialsThe investigation was approved by the Ethic Committee and the Committee of Animal Experimentation of China Medical University and conformed to the principles outlined in the declaration of Helsinki. Human brain tissue was obtained from the human frontal lobe which was dissected for curing basilar meningioma at the first hospital affiliated to China Medical University. And the experiment was undertaken with the understanding and written consent of the patient. Healthy male Wistar rats at different developmental stages (P1-P120) were provided by the Animal Experimentation Center of China Medical University. The rats were anaesthetized by inhalation of a mixture of 50% O2 and 50% CO2 and killed by cerebral dislocation, with all tissues used carefully excised.2. Methods(1) RNA isolation and RT-PCRTotal RNA was extracted from all tissues using RNA out kit (TaKaRa, Japan) according to the manufacture's instructions. The first-strand cDNA was synthesized using the RT-PCR kit (TaKaRa, Japan) with oligo-(dt) and random primers. The PCR primers for the amplification of Navl.5 and Navβ1 were designed from highly conserved sequences among species but not among isoforms and the PCR was carried out according to its instructions. All PCRs for detecting the relative amount of Nav1.5 were repeated at least three times.(2) Cloning of Nav1.5 cDNAFor the isolation of a variant of Nav1.5 from human brain, we used 10 primer pairs to amplify ten fragments of the full length cDNA by the PCR method described above. All of the ten fragments partially over-lapped each other allowing subsequent assembly using common restriction enzyme sites. The PCR products (300-1300bp) were separated on 1-2% agarose gel, and fragments of the expected size were extracted by using gel extraction kit (Qiagen, USA) and then were subcloned into the PGEM-T.easy vector (Promega, USA) and sequenced by using 3700 DNA sequencer(USA) after plasmid extraction(Qiagen, USA). Multiple independent recombinants of each construct were sequenced completely to identify clones without polymerase errors.(3) Statistical analysisPCR products were analyzed by gel electrophoreses (1%-2% agarose).The signal of each band was determined using Quantity One 4.5 software (USA). For normalization of the amount of cDNA, we usedβ-actin and GAPDH as an internal standard. So the gene expression levels were presented as amplicon densities toβ-actin or GAPDH and data for gene expression values were presented as means±S.E.M. Student's t-tests were used as appropriate to evaluate the statistical significance of differences between two group means, and analysis of variance used for multiple groups. Values of P＜0.05 were considered to indicate statistical significance.Results1. It is exon6A rather than exon6 of Nav1.5/SCN5A that encodes Nav1.5 channels in the brainIn order to investigate whether exon6 and/or exon6A encode(s) Nav1.5 channels in the brain, special primer pairs for amplification of exon6A and exon6 were designed. The PCR products were separated by electrophoresis onl.5% agarose gel after 35 cycles. The expected fragment of exon6 was not found from the brain cDNAs and that of exon6A was not found in non-brain tissues, such as heart, lung and testis. Sequence analysis further confirmed that it was exon6A rather than exon6 that encoded Navl.5 channels in the brain. Both exon6A and exon6 had 92 base pairs, which encoded 30 amino acid residues. But 7 amino acid residues were ifferent between them. There was only one different nucleotide between the exon6A of human Navl.5/SCN5A and that of the rat or human neuroblastoma cell Nav1.5/SCN5A, but they encode identical amino acid residues.2. Cloning of Nav1.5 channel a-subunit from the brainTo obtain the full-length cDNA encoding Navl.5 in the human cerebral cortex, ten primer pairs (table 1) were designed based on the published human hHl (accession no: M77235) sequence, which predicted fragment sizes ranging between 300bp to 1300bp. Then ten different sub-regions of the full-length cDNA were separately amplified using PCR method.In the study, four full-length cDNAs encoding the a-subunits of the Navl.5 channels in human and rat cerebral cortexes were found and cloned, which were designated hB1, hB2, rN1 and rN2. The full size of the human Navl.5 cDNA was 6201 nucleotide long and was designated hBl.The longest open reading frame of hBl encoded 2016 a.a residues and it was highly homologous with hHl (>98% a.a identity) and hNbRl (>99% a.a identity).The hBl differed from hHl by 28 a.a. They were distributed in domainⅠ(16), loop 1-Ⅱ(2), domainⅡ(1), loopⅡ-Ⅲ(5), domainⅢ(1), loopⅢ-Ⅳ(0), domainⅣ(3). However, when compared with hNbRl, there was 1996 a.a. identity with only 20 a.a difference. They were distributed in domain I (8), loopⅠ-Ⅱ(2), domainⅡ(1), loopⅡ-Ⅲ(4), domainⅢ(1), loopⅢ-Ⅳ(0), domain IV (4).The hB2 was 6147 nucleotides long, lacking exon24 compared with hB1, and encoded 1998 amino acid residues. The missing exon24 encodes 18 amino acids in the extracelluar loop between S5 and S6 of domain III.The open reading frame of rNl also encoded 2016 amino acid residues and sequence analysis indicated that it was highly homologous with mHlwith>96% amino acids identity. Alternative splicing of exon24 was also found in the cloning of Nav1.5/SCN5A from the rat brain and the new variant was designated rN2, which encoded 1998 amino acid residues. There were four different nucleotides between exon24 of human Nav1.5/SCN5A and that of rat Nav1.5/SCN5A, but they encoded identical amino acid residues. 3. The expression patterns of Nav1.5 mRNA in different part of developing rat brainsIn order to investigate whether the expression levels of Nav1.5 mRNA in CNS change with age development, rat brains and spinal cords at five different developmental stages (PI, P9, P40, P80, P120) were used. We detected the expressions of Nav1.5 mRNA in cerebral cortex, hippocampus, cerebellum, brain stem and spinal cord (cervical) using RT-PCR method. The results indicated that the expression levels of Nav1.5 mRNA in different part of the brain showed a similar expression pattern with age development. In cerebral cortex, hippocampus, brain stem and cerebellum, the high expression levels were detected in neonatal rats (P1, P9), whereas expression decreased with age development up to P120.4. The expression of total Nav1.5 mRNA in 16 different tissue types of Wistar ratsRT-PCR method was used to detect the expression of total Nav1.5 mRNA in 16 different tissue types of Wistar rats (P80). As reported previously, Navl.5 mRNA showed the strongest expression in the heart. And our results indicated that it could also be detected in all tissues assayed. Compared to its extremely high expression in the heart, the Nav1.5 mRNA showed high expression levels in the adult and fetal rat brains, spinal cord and testis, moderate levels in the kidney, adrenal gland, lung, skeletal muscle, spleen, stomach and bladder, and low levels in the liver and pancreas.Conclusion1. Nav1.5 channels in the brain are encoded by new variants of Nav1.5/SCN5A> but their functions remain unknown.2. Two novel alternative splicing variants of Nav1.5/SCN5A are found in our study (alternative splicing of exon6A/exon6 and exon24). And their expression levels in different tissues are different.3. The expression levels of Nav1.5 mRNA in different part of the brain are different, and the high expression levels were detected in neonatal rats (PI, P9), whereas expression decreased with age development (up to P120).4. Nav1.5 channels are more widely distributed and expressed than previously thought, but its specific encoding genes and functions in different tissues remain unknown, and the relationship between Navl.5 channels and ion channel diseases merits further investigation.