Potassium channel in peripheral blood lymphocytes of turbot ( Scophthalmus maximus )
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Potassium channel in peripheral blood lymphocytes of turbot (Scophthalmus maximus) CHAI Yingmei1,2 , HUANG Xiaohang2∗ , CONG Bailin2 , LIU Chenlin2 , LIU Shenghao2 1 2
College of Marine Life Sciences, Shandong University at Weihai, Weihai 264209, China Marine Bioactive Substances Laboratory, the First Institute of Oceanography, State Oceanic Administration, Qingdao 266061, China
Received 11 December 2008; accepted 13 July 2009 c The Chinese Society of Oceanography and Springer-Verlag Berlin Heidelberg 2010
Abstract In order to provide pertinent evidence of ion channel with immune response in the fish, whole cell patch-clamp technique was employed for potassium ion channel study in turbot (Scophthalmus maximus). Lymphocytes were isolated by Percoll density gradient centrifugation from peripheral blood samples, and electrophysiological characters of the channel were analyzed. In the recorded cells, activated voltage of the channels was –42.5±3.7 mV and the average peak current was 313.12±28.2 pA. The channel was identified as voltage dependent, the current was outward and it could be inhibited by 10 mmol/dm3 TEA or 5 mmol/dm3 4-AP, a specific potassium channel inhibitor, identifying the existence of potassium channel in peripheral lymphocytes of the turbot. Key words: patch-clamp technique, potassium channel, turbot peripheral blood lymphocyte
1 Introduction Various ion channels exist in mammal lymphocytes and they are located on the plasma membranes, one kind of them is potassium (K+ ) channel. Ion channels are critical for initiation and transmission of intracellular signal, lymphocytes differentiation and proliferation (Rader et al., 1996), and activation of whole immune system in T cells (DeCoursey et al., 1984; Matteson and Deutsch, 1984). The predominant K+ ion channels are voltage-gated K+ channels under resting state which are necessary for maintaining the normal function of the mammalian cells. Lymphocytes exist in teleost as those in mammals (Richard et al., 2004) and they play certain important roles in specific immune response as well (Lunden and Bylund, 2000). However as a critical regulator of metabolism, whether K+ channels are present in the teleost lymphocytes and what are their main characters still remain poorly undersood. We used whole-cell patch-clamp technique to record the membrane current from turbot lymphocytes and analyzed its characters. Preliminary data identified that the recorded membrane current was the K+ channel current. This research will provide basic information for further investigation of the K+ channel in order to learn the ion channels function in teleost
lymphocytes activation and of their role in teleost disease prevention. 2 Materials and methods 2.1 Animal Healthy marine teleost turbot (Scophthalmus maximus) (800 g mean mass) were obtained from an aquaculture farm in Qingdao (Shandong, China) and kept in a 260 L seawater tank at 20 ◦ C for later studies. 2.2 Cell separation The separation of lymphocytes was in three stages. First, the fish were anaesthetized with Benzocain (
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