Molecular Cloning And Functional Analysis Of SpVv1.3from Sea Perch, Lateolabrax Japonicus

Voltage-gated potassium (Kv) channels on cell plasma membrane play animportant role in both excitable cells and non-excitable cells. Kv1.3is mostextensively studied channel in lymphocytes of mammalian cells. This potassiumchannel was reported to control processes inside mammalian T lymphocytes such ascell proliferation and volume regulation. It has been shown that Kv1.3performs a keyfunction in innate immune system, initiating and controlling cytokine synthesis oftumor necrosis factor-alpha (TNF-α) and interleukin (IL). However, little is knownabout Kv1.3channel in fish.Electrophysiological tools open the way toward the understanding of the role ofK+channels in cell proliferation but this technique is limited due to lack of specificitywhen identifying individual K+channels. Furthermore, Electrophysiologicalapproaches often fail to give deep insight into the molecular mechanisms. More directevidence regarding the role of K+channels on immune response will be possible afterthe molecular entities underlying K+currents make clear. Here, we combine those twotechniques and provide information about the cloning and characterizations of avoltage-gated potassium channel isolated from sea perch, Lateolabrax japonicus.Employing specific primers and RNA template, we cloned a segment of a novelgene from sea perch blood sample and subsequently obtained a full cDNA sequenceusing RACE approach. Bioinformatic analysis revealed structural and phylogeneticcharacteristics of a novel Kv channel gene, designated as spKv1.3, which exhibitshomologous domains to the members of Kv1.3family. Full-length of spKv1.3cDNAis2152bp with a1440bp open reading frame encoding a protein of480amino acids.The deduced molecular weight was60.8kDa and pI was9.22. Phylogenetic treeconstruction showed that spKv1.3was most closely related to the Kv1.3shaker family.The Oreochromis niloticus was the nearest species from spKv1.3in phylogenetic distance, which also belongs to Osteichthyes. SpKv1.3was homologous to Kv1.3ofother species. The amino acid sequence deduced from spKv1.3was95%identity withthe Nile tilapia (Oreochromis niloticus). Structural prediction showed that spKv1.3had six transmembrane regions and six strong hydrophobic domains. We also foundthat spKv1.3gene was expressed in all tested organs and tissues such as gill, brain,head kidney, muscle, liver, spleen, intestine, skin and blood with a highest level inbrain, indicating that it was a constitutive gene in sea perch lymphocytes and involvedin multiple-functions in different tissues. Furthermore, genomic DNA analysisdemonstrated that the channel was intronless, there was no introns presented withinthe coding sequence.To assess the postulated immune function of spKv1.3, we stimulatedlymphocytes with LPS and/or channel blocker4-AP. Expression levels of messengerRNA (mRNA) of spKv1.3and cytokine IL-β and TNF-α under stimulation conditionswere measured by quantitative RT-PCR. The results showed that LPS can motivate theup-regulations of spKv1.3、IL-β and TNF-α expression significantly at different time.Two hours after LPS stimulation, IL-β and TNF-α are2.46-fold and3.36-foldcompared with the control group respectively. Four hours after LPS stimulation,spKv1.3is2.98-fold higher than that in control. Interestingly, in addition tosuppression of channel activity, the4-AP can also increase the spKv1.3、IL-β andTNF-α mRNA expression levels in presence of LPS during the time course, especiallyIL-β and TNF-α mRNA expression levels. Four hours after LPS+4-AP stimulation,IL-β and TNF-α mRNA expression levels are20.82-fold and16.44-fold higher thanthat in control respectively. Although4-AP could block potassium channels physically,we speculated that the blockage of potassium channel may start up an alternativemechanism which feed back and evoke the spKv1.3IL-β and TNF-α mRNAexpression when the organism non-specific immune system was mobilized by LPSchallenge.The electric character of spKv1.3was studied by transiently expressed spKv1.3cDNA in Chinese hamster ovary cells using the patch-clamp technique. In whole-cellrecordings, spKv1.3displayed a typical rapidly activating outward rectifier potassium current with the2057.5±590.85pA peak current at+70mV. The4-AP accelerated thedecay rate of inactivation of spKv1.3and thus decreased the current amplitude at theend of the pulse at the final concentration of5mM. The inhibition displayed strongvoltage dependence, increasing at more positive potentials. The degree of inhibitionincreased from23.4±12.77%at-10mV to62.2±23.07%at+70mV. The activationvoltage was not affected by4-AP, which was-20mV too.