Studies On The Effects Of Potassium Channels In The Innate Immune Defense From Starry Flounder (Platichthys Stellatus)

Potassium channel are a kinds of important ion channels located on the cell membrane which only allow potassium ions to pass, they participate in the regulation of various physiological functions and play a very important role in fish innate immune defense. KCNN2 channel belongs to the family of small conductance calcium-activated potassium channel, which has some special characteristics such as high sensitivity of Ca2+ and selectivity of K+. It is also a vital potassium channel located on lymphocytes and neutrophils membrane. Lymphocytes and neutrophils are immune cells of organisms, when the cells are activated by stimulation, potassium channels involve in the activation of lymphocytes and neutrophils, respiratory burst, cytokine secretion and other immune response via regulating the intracellular ion concentration and the level of membrane potential.Starry flounder is a kind of high value commercial flatfish, now it is considered to be one of the great potential cultured fishes after artificial breeding of Psetta maxima and paralichthys olivaceus. Aquaculture diseases frequently break out, which seriously restrict the healthy and sustainable development of aquaculture in our country. In this Study, we find that the effects of potassium channels in the innate immune defense mechanism and regulatory mechanism from starry flounder, which reveal that potassium channels has a close relationship with a variety of the immune function. It has theoretical significance and great practical value on controlling the occurrence of fish diseases, promoting the development of Starry flounder aquaculture and so on.Using cDNA of lymphocytes separated from Starry flounder peripheral blood as template, we obtained three kinds of voltage-gated potassium channels gene’s core fragments. The results suggest that sfKCNT1 belongs to potassium voltage-gated channel subfamily T member 1, sfKCNQ2 belongs to potassium voltage-gated channel subfamily KQT member 2-like, sfKCNQ5 belongs to potassium voltage-gated channel subfamily KQT member 5-like. In addition, each of the three gene segments has the:.:ghest potassium channel gene homology with stegastes partitus, followed by 93%,91%,85%. Structural domains of three gene segments were predicted by SMART server, we found that sfKCNT1 had no obvious structural domain due to the shorter fragment, sfKCNQ2 contained six transmembrane regions and a low complexity area, sfKCNQ5 only existed a low complexity area. Because the voltage-gated potassium channels are highly conservative in core fragments, we can not obtain the full length sequence of potassium channel gene. However, we prove that voltage-gated potassium channels are existed in Starry flounder, which laid the foundation for the function study of subsequent experiments.qRT-PCR analysis show that the expression of KCNN2 mRNA in lymphocytes was promoted after LPS stimulated and the change trend in different time was the same as the expression of KCNN2 in blood after intraperitoneal injection of LPS. The expression of KCNN2 in neutrophils was significantly up-regulated after LPS, PMA and heat-killing vibrio anguillarum challenged, the most significant effect was by LPS. In neutrophils. channel activator and blocker for KCNN2 expression had different levels of inhibition. These results indicate that the KCNN2 gene is involved in the immune response of lymphocytes and neutrophils of Starry flounder.In order to study the effect of KCNN2 channel on respiratory burst activity, we stimulated neutrophils by PMA, CyPPA+PMA and Apamin+PMA. The results suggest that the influence of PMA on neutrophils respiratory burst activity had timeliness, that is to say PMA showed a promotion action in stimulating within 2 hours, then showed the inhibition. Channel activator CyPPA and blocker apamin suppressed the respiratory burst activity of neutrophils. The secretion of IL-1β and TNF-a in lymphocytes were promoted by LPS and inhibited by apamin, but the effect was not obvious in a short period of time. We stimulated neutrophils by LPS, CyPPA+LPS and apamin+LPS and detected the concentration changes of IL-1β, TNF-a and MPO. ELISA analysis reveal that LPS promot secretion of IL-1β, TNF-α and MPO in the neutrophils, CyPPA and apamin significantly reduce the increase of concentration caused by LPS stimulation. Moreover, the inhibitory effect of apamin is stronger than CyPPA. The above results show that the potassium channel KCNN2 could play an important role in the innate immune defense of Starry fl under.Our study suggest that potassium channel KCNN2 is activated and induced a series of immune reactions by LPS, and the change trend of these reactions are nearly same. Besides, we also notice that the response time of cytokine is relatively later than gene expression, which conforms to the basic rule of nucleic acid protein change. Channel activator clearly suppressed LPS-induced gene expression, respiratory burst and cytokine release, we predict CyPPA might protect the cells by counteracting LPS-related inflammatory responses. On the other hand, this illustrates that there is a close relationship between immune response and potassium channels. Furthermore, Apamin, specific channel blocker on KCNN2 channel, prevent a series of immune reaction, which is a direct evidence of potassium channel KCNN2 participating in the immune response.