Molecular Cloning And Functional Analysis Of The TTX-resistant Sodium Channel In Human Neuroblastoma Cell Line, NB-1

AimNeuroblastoma tumor cells originate usually in the neural crest during neu-rogenesis and express biochemical,pharmacological and functional properties similar to those of neurons.They offer an archetype in vitro system to correlate differentiation with the expression and regulation of ion channels including the voltage- dependent Na⁺ channels.Investigations on the electrophysiological properties of various neuroblastoma cell lines indicate that the amplitude of Na⁺ currents(I_Na)is highly variable among different cell lines.In addition,voltage -dependence of activation and inactivation of I_Naare also variable suggesting that different isoforms of Na⁺ channel gene are expressed in those cell lines. The human neuroblastoma cell line,NB-1,origin in Japan established by Miy-ake et al.in 1973,have been shown to express voltage -dependent Na⁺ and K⁺ channels in both blast and mature types.Thus NB-1 cells provide a useful model system for studies on the expression and regulation of the Na⁺ channels during differentiation or in response to biosignals.Our previous study has re-vealed that I_Nain NB-1 cells exhibit tetrodotoxin-sensitive(TTX-S)and re-sistant(TTX-R)components,suggesting an expression of multiple Na⁺ chan-nel genes.However,molecular properties of these Na⁺ channels have not been investigated.It was reported that the TTX-R sodium channel of mouse neuroblastoma cell line(B104)was encoded by the same gene with mouse heart.And the mu-tations in the TTX-R sodium channel in human heart which produce inward currents during excitation and construction of cardiomyocytes was related to con- genital long QT syndrome type 3(LQT3)and Brugada syndrome.Being a kind of nerve system tumor,whether the TTX-R sodium channel in NB-1 cell en-code the same gene as hH1? So in this study the reverse transcriptase-polymer-ase chain reaction(RT-PCR)was used to clone theαsubunit of TTX-R Na⁺ channel in NB-1 cells and whole cell patch clamp was used to record the sodium currents in order to clarify the mechanism of the tumor.MethodsNB-1 cells were grown in standard medium containing MEM,Total cellu-lar RNA from NB-1 cells was isolated using RNeasy(QIAGEN,Japan)kit, First strand cDNA was synthesized using Sensiscript Reverse Transcriptase Kit (QIAGEN)using oligo-(dT)and random primers.The PCR primers for theα-subunit of Na⁺ channels were designed against highly conserved sequences based on published sequences of Nay1.5/SCN5A (accession No.M77235).After ligation,using appropriate restriction enzymes,the cDNA encoding entire region of the channel was subcloned into mammalian expression vector, pcDNA3.1(+)HEK293 cells were maintained in culture under 5%CO₂ at 37℃in 35 mm culture dishes at a density of 1-5×10⁵ cells/dish in MEM containing 10%fe-tal bovine serum,50 U/ml penicillin,50μg/ml streptomycin,and 2 mM L-glutamine.2μg of plasmids,pcDNA3.1(+),encoding a Na⁺ channel gene were transfected into HEK293 cells by using a transfection kit Transfast Co-transfection with 1μg green fluorescent protein(GFP)expression vector,pEG-FP-F(Clontech,USA),was used for identification of transfected cells in the patch-clamp experiments.The whole-cell patch-clamp technique was used to record I_Nausing CEZ2300 amplifier.The I_Nawere evoked by depolarizing steps from a holding potential of -120 mV with a stimulus intervals of 5 s.Currents were sampled at 20 kHz and stored on a computer for later off-line analysis.The competitive PCR reactions for the amplification of two variants of Nav1.5 were carried out using primer pair A6.Signal intensity of each band was determined using Quantity One 4.5 software.ResultThe longest open reading frame of the clone,designated as hNbR1,en-codes 2016 amino acid residues.Sequence comparisons indicated that hNbR1 is highly homologous to human cardiac Nay1.5/SCN5A,99.1%amino acids iden-tity;The hNbR1 had all the hallmark structural features of a voltage-dependent Na⁺ channel;four homologous domainsâ… -â…£,each of which consists of six putativeα-helical transmemhrane segments(S1-S6);positively charged res-idues in the voltage-sensor S4 of each domain;The presence of a cysteine resi-due(C373)in the pore region of domainâ… suggests that this channel is resistant to TTX;a new exon(exon6A)located in chromosome 3 encode the region be-tween domainâ… S3 and S4;an alternative splicing variant,which is character-ized by a deletion of exon 18 in the intracellular loop between domainâ…¡andâ…¢was found,hNbR1 and its splicing isoform were found to be subtle differences in the kinetics of steady-state inactivation and activation,-74.4 mV;-37.8 mV and -81.5 mV,-43.5 mV for hNbR1 and hNbR1-2 respectively,shif-ted in the negative direction with -5.7mV and -7.1mV in hNbR1 -2.The TTX IC₅₀of these two channels was about 8μmol concentration.ConclusionWe have identified two splicing variants of TTX-R Na⁺ channels(hNbR1 and hNbR1-2)in human neuroblastoma cell line NB-1.These variants are isoforms of cardiac Na⁺ channel Nav1.5/SCN5A.It is possible that Nav1.5 channels may be expressed in neuroblastoma cells in a different pattern of alter-native splicing.These TTX-R Na⁺ channels are predominantly expressed in blast(immature)type.Thus NB-1 cell line provides a model for the regula-tion of Na⁺ channel expression.