Studies On The Cloning Gene Of Small Conductance Calcium-activated Potassium Channel SKCa2and Its Immune Function From Starry Flounder (Platichthys Stellatus)

Potassium channels are transmembrane proteins located on the cell membrane with highly selective to gate for potassium ions. These channels initiate various important cell signal transduction to participate in regulating cellular functions. SKCa2channel is a strictly voltage-independent K+channel with high sensitivity of Ca2+, which belongs to small conductance calcium-activated potassium channel (SKCa) family. It is also an important channel located on lymphocytes membrane. When lymphocytes stimulated, potassium channels play a crucial role in lymphocyte activation, proliferation, differentiation, and other immune response by regulating intracellular ion concentration and membrane potential.Starry flounder is a high-value marine economic fish. Frequent breeding diseases can cause huge economic losses, which directly restrict the production and healthy development of agriculture industry. Based on the key role of the potassium channel in the immune response, it has a great meaning for healthy agriculture. It can enhance the basic theory of fish immunology to learn about the potassium channel function on regulation of nonspecific immune response. At present, much more concern are paid on the immune function regulation of ion channel proteins.Employing RNA template of Starry flounder brain, the full length sequence of SKCa2gene was obtained and characterized through the RT-PCR and RACE techniques. The full-length cDNA of SKCa2was2346bp, with a5’UTR of153bp, a31UTR of135bp, and an ORF of2055bp encoding a protein of685amino acids. The deduced amino acid sequence of Starry flounder SKCa2had highest homology of96.3%with rainbow trout (Oncorhynchus mykiss) through the analysis of phylogenetic tree construction and amino acid homology. Transmembrane and hydrophobic region of Starry flounder SKCa2were predicted using DAS and protscale server and structural characters were compared with the other species. SKCa2contained a pore region and six transmembrane domains (S1to S6). The pore region lied between the S5and S6, it had a highly conservative sequence—"TXXTXGYG" motif and a binding site of SKCa2blocker—apamin. Other prediction shown that SKCa2has multiple PKA and PKC phosphorylation sites and a calmodulin-binding domain (CaMBD) existing in C terminus.qRT-PCR analysis revealed the presence of SKCa2transcripts in all detected tissues including blood, heart, liver, gill, spleen, intestine, stomach, brain, head kidney, skin and muscle. The relative expression quantity of SKCa2was relatively higher in blood and gill, lower in intestine, brain and stomach. The expression of SKCa2mRNA in the blood, head kidney, spleen, intestine, skin and liver was up-regulated after LPS challenged, which indicated the importance of SKCa2in fish immune response.In order to study the effect of SKCa2channel to lymphocytes proliferation and respiratory burst, we stimulated starry flounder lymphocytes by LPS and LPS+Apamin. The results showed the OD570value of group with LPS+Apamin stimulation was lower than that with LPS stimulation. The SKCa2blocker, apamin, inhibited the lymphocyte proliferation. Subsequently, our results showed that apamin could also inhibite the respiratory burst of lymphocytes. These results suggested that SKCa2could play an important role in the innate immune responses.