Study On The Function Of Potassium Channels In The Innate Immune System From Japanese Sea Perch(Lateolabraxjaponicus) Macrophages

Potassium channels form potassium-selective pores that span cell membranes. They are the most widely distributed type of ion channel and are found virtually in all kind of living organisms. Potassium channels play an important role in cell signal transduction, proliferation, differentiation, maintaining normal functions in non-immune cells. So far quite a few potassium channel genes have been cloned from a number of mammalian species. Little is known about the diversity and role of potassium channel in mediating the immune cell function in lower vertebrates, such as fish. Present study identified four different types of voltage-gated potassium channels (spKvl.1, spKv1.2, spKvl.5and spKv3.1) in Japanese sea perch, which belong to shaker and shaw subfamily.Based on this, we studied the distribution and expression pattern of the voltage-gated potassium channels gene in the differnent tissues, such as spleen, head kidney, liver, muscle, brain, intestines skin and blood after LPS activation. Result showed that the expression of spKvl.l, spKv3.1increased significantly in immune-ralated tissues, which indicate that the potassium channels involve in the innate immune system. The levels of transcriptional expression of these genes after macrophages activation by LPS challenge were also examined. The expressions of all investigated channel genes have increased two hours after LPS activation. Among them, the spKvl.2and spKv1.5expressions increased almost6times and the spKvl.1expression was most significant which increased nearly35times compared with the control, indicating that these channels participated in the nonspecific immune response of Japanese sea perch macrophages. In the presence of LPS plus potassium channel blocker, the expression ofspKvl.5mRNA was still up-regulating and kept increasing until12hours after treatment. It suggest that the cell may start an alternative mechanism to upregulate the gene transcription in order to compensate the malfunction after blockage of channel protein. Concomitant immune responses such as cell proliferation, reactive oxygen intermediate production and cytokine secretion were also studied. Results revealed that, in addition to high expressions of TNF-a and IL-β gene in macrophages after LPS challenge, the cytokines gene expressions significantly upregulated with a4to10fold increases after4h to7h treatment of channel blocker, although the inhibitory effects of blocker on cytokines secretion in mammalian immune-related cell were well documented. We also showed that the channel blockers can inhibit the proliferation and reactive oxygen intermediates production in the Japanese sea perch macrophages. The two blockers can inhibit the respiratory burst activity at all experimental time points. These results suggested that4kind of voltage-gated potassium channels we cloned participate in the activity of nonspecific immune system, channel blocker may trigger the up-regulation of channel and cytokine genes at the transcription level, and the spKvl.1might play a more active role among tested potassium channels of Japanese sea perch macrophage. These research may enrich the understanding1of marine fish immunology and provides a theoretical basis for aquaculture fish disease prevention.