The Cloning And Distribution Analysis Of Nav1.5 Sodium Channel In Rat Brain

ObjectiveAs a sort of transmembrane proteins, voltage gated sodium channels (VGSC) are responsible for the regulation of sodium ion permeability of excitable cells during the initial stage of action potential, whose functions are characterized by voltage dependent activation, rapid deactivation and selective ion conduction. So far it has been clarified that mammalian VGSC are composed of numerous protein subfamilies, including an a subunit and fourβsubunits (β1,β2,β3 andβ4), among which the former subunit is able to function independently while the latter group can only play auxiliary and regulative roles in physiological action. Furthermore, the a subunit genes of such VGSC subfamilies as Nav1.1-Nav1.9 have been successively cloned and named by an international unified denomination. Classified as the TTX resistant type and once recognized as principal ion channels distributed in cardiac muscle cells, Nav1.5 channels have been detected in high expression level in neuroblastoma cells and brain.The Nav 1.5 sodium channels have been successfully cloned in rat cardiac muscle and human neuroblastoma cells. However, there are no reports upon the issues of amino acids subsequence of Nav 1.5 a subunit in the brain, homology to cardiac sodium channels as well as distribution in cerebral lobes of rats during different developmental stages. In this paper, we, therefore, cloned a subunit of Nav 1.5 sodium channel from brain of rat and inspected its distribution in each cerebral lobe during various developmental stages.Materials and Methods Materials and ReagentsMale wistar rats, purchased from Liaoning Laboratory Animal Center; RANOUT Kits, Tiandz, Inc.(Mianyang, Sichuan Province, China); RT-PCR Kits and Restriction Endonuclease Kits, TAKARA BIO INC (Japan); DNA Purification Kits, pGEM-TEasy Kits, Cloning Vector, T4 ligase, competent cells and Plasmid Extraction Kits were all purchased from Pharmacia.Methods1 .RNA Extraction and Reverse TranscriptionAccording to relative protocols mentioned in references, every five male rats were killed by cervical dislocation at day 0, 9, 18, 36 and 72 after birth and the brain tissue including cerebrum, cerebellum, brain stem and hippocampus were rapidly taken out and samples of cardiac cells were prepared at day 9 and 72 after birth, respectively for RNA extraction. By using AMV reverse transcriptase, reverse transcription solutions were prepared for the synthesis of first-strand cDNA.2.Cloning of Nav1.5α-Subunit of Rat BrainAccording to the known Navl.5 gene sequence of rat myocardium cells and human neuroblastoma cells, total 8 pairs of PCR (polymerase chain reaction) premiers, A1-A8 were designed. All cDNAs were extracted from the cerebrum of 72-day old rats. PCR products were run by 1% agarose gel electrophoresis and the prospective PCR fragments were spliced and purified in the same agarose gel medium. Fragments A1-A8 containing the whole encoding domain of Navl.5 a subunit gene sequences of rat were cleaved by proper restriction enzymes. The eight fragments were then cleaved in their overlapping sections to obtain prospective fragments that would be subsequently linked by T4 ligase until the formation of complete DNA sequences. The sequences were analyzed by DNA sequence meter, about which the detailed procedure could be referred to relevant references.3.Competitive Polymerase Chain Reaction The same method of PCR mentioned above was performed in competitive PCR for No 5 premier (A5) of Nav1.5αsubunit rN1 (769bp) and its isomer rN1-2 through 30, 35 and 40 cycles. PCR products were run by 1% agarose gel electrophoresis and then analyzed by Scion Image Software for observation of the signal intensity in brain tissues.4.Distribution of Nav1.5α-Subunit in Each Lobe of Rat Brain during Different Developmental StagesBased on the known Nav1.5α-subunit sequence of myocardial cells of rat, the primer B1 (779bp) was designed. Its sequence could be selected different from that of other sodium channel's. In the same PCR system as mentioned above, cDNA templates were synthesized from the hippocampus, brainstem, cerebellum, and cerebrum of male rats at days 0, 9, 18, 36 and 72 after birth. As for comparative analysis, the myocardial cDNA of rat was simultaneously selected at days 9 and 72 after birth. All amplifications would reach 35 and 40 cycles. While PCR was performed, theβ-actin primer was adopted as a positive controller. And the PCR products were examined in 1% agarose gel electrophoresis and then analyzed by Scion Image Software.Statistical AnalysisBy SPSS 15.0 statistics software, One-Way ANOVA was used to assess the results. P <0.05 was considered distinctly different.Results1.Cloning of Nav1.5α-Subunit of Rat BrainIn this experiment, we obtained the cDNA with full-length of 6093 bp and named it after rN1 (GenBank Accession No: EF618549). Open reading frame (ORF) of rN1 encodes 2016 amino acids and Its Amino acid sequence analysis of rN1 showed the structural characteristics of voltage-dependent sodium channels, literally containing four similar transmembrane domains (D I～DIV) including six transmembrane segments (S1-S6). In each domain, the transmembrane segments (S4) of voltage receptor possessed positive charge residues. And in rN1, there were 70 different amino acids with 96.53% similarity to rH1; 78 different amino acids with 96.13% similarity to hNbR1.2.Alternative Splicing in Nav 1.5α-Subunit in Rat BrainBy using premier couple A1, it has been detected that rN1 possessed obviously different nucleotide sequence in the corresponding domain of exon 8 of rH1, i.e. T206V, T207S, F209N, V210I, D211K, V215L and S234P. By comparing with Nav1.5/SCN5A genome sequence, exon 8 of rHl is located on chromosome 8 with its genome sequence arranging among 20731-20822, while the corresponding sections of rN1 exist on chromosome 8 with its genome sequence within 20975-21066, suggesting the presence of another kind of alternative splicing. From the comparative results of genome sequences, the sequence section of rN1 is located in the exon 7 of Nav1.5/SCN5A that shows the typical characteristics of intron/exon boundaries. In addition, its 3' end (C/T) AG and 5' end GT (A/G) indicated that it was certainly a new exon and then named after exon 7. This result showed that rN1 encoded exon 7 instead of exon 8 by alternative splicing. After application of A5 primers, electrophoresis showed another band with slightly less molecular mass than the expected which was a new isomer after exon 20 splicing (53 amino acids) and was subsequently demonstrated by gene sequencing and named after rN1-2 (GenBank Accession No: EF618550 ). We adopted the A5 primer couple for 30, 35, 40 cycles of competitive PCR. And the electrophoresis confirmed that no significant differences presented in the expression of rN1 and rN1-2 in the rat brain.3.Distribution of Nav 1.5α-Subunits in Each Lobe of Rat Brain during Different Development StagesNav1.5 sodium channels did not showed significantly different expression levels in cerebrum, cerebellum and brainstem of rat during the same developmental stage. However, expression of Nav1.5 gradually increased with development (P <0.01). On the other side, through postnatal days 0-9, the expression was significantly higher in the hippocampus than in other brain lobes, although it showed no significant increase during different developmental stages and tended to slightly decrease in the postnatal day 72. Meanwhile, by the selectingβ-actin as internal reference, no significant difference about theβ-actin expression was detected in the various brain lobes during different developmental stages (P >0.05).4.Comparison of Nav 1.5 Expression in Brain and Heart of RatIn order to demonstrate the difference between Nav 1.5 expression in the brain and heart tissue of rat during various developmental stages, samples of these two organs from child and adult rats were used for PCR test by 40 cycles. The electrophoresis results showed that Nav 1.5 was expressed in distinct levels in different tissues of rat by the following trend as: adult heart > child heart > adult cerebrum >child cerebrum.Conclusion1. All clones of Nav1.5 a subunit gene from rat brain fully confirm that Nav1.5 is not the ion channel uniquely located in cardiac tissue.2.The exon 7 and exon 20 of alternative splicing that in rat brain cells maybe the one of structure foundations which related with the diversity of function between brain and cardiac muscle .3.Nav 1.5 sodium channels were widely distributed in the entire brain of rat, however, the distribution appeared in remarkably different level in various brain lobes during developmental stages. And at the same developmental stage, Navl.5 sodium channel expression was found in cerebrum, cerebellum and brainstem without significant differences, but it tended to gradually increase with development (P <0.05).4. By comparison with other lobes during various developmental stages, Navl.5 sodium channels were significantly expressed in different level in hippocampus of rat, therefore, hippocampus maybe bring into play a special function in rat brain.5. Navl.5 is indeed the major cardiac sodium channel since it is expressed significantly more in the myocardium cells than in the brain. Furthermore, the expression levels of Nav1.5 in different tissues tend to be as follows: adult rat cardiac myocytes > child rat cardiac myocytes> adult rat cerebrum > child rat cerebrum.