摘要
利用膜片钳技术之内面向外式,对培养2～24h不同类型的中华绒螯蟹(Eriocheirsinensis)血淋巴细胞表达的钙激活钾(KCa)通道进行种类鉴别和特征研究。结果表明:在高对称钾溶液(200mmol/L)中,只有小颗粒细胞表达大电导钙激活钾(BKCa)通道和中电导钙激活钾(IKCa)通道。其中BKCa通道电导为396pS,通道的开放活动表现为矩形方波。同一钳制电压下,当浴液中[Ca^(2+)]在0.3～3.0 μmol/L范围内,通道开放概率和开放数目随Ca^(2+)浓度增加而增加,当浴液中[Ca^(2+)]达到100.0 μmol/L,通道开放受到抑制,表现明显的钙依赖性失活;当浴液中Ca^(2+)浓度为3.0 μmol/L,钳制电压分别为-40mV和-80mV时,通道平均开放时间分别为(0.03233±0.00741)s和(0.05585±0.01299)s(P<0.01),平均开放概率分别为0.596±0.125和0.961±0.012(P<0.01),说明通道开放具有电压依赖性。IKCa通道电导为88pS,开放活动也表现为矩形方波。当浴液中Ca^(2+)浓度为0.3、1.0μmol/L时,通道不开放;而Ca2+浓度为3.0μmol/L时通道开放,说明通道具有明显的钙依赖性,钳制电压分别为-40mV和-80mV时,通道平均开放时间分别为(0.01736±0.00403)s和(0.02262±0.01616)s(P>0.05),平均开放概率分别为0.699±0.065和0.790±0.114(P>0.05),说明通道开放不具有明显的电压依赖性。药理学研究结果表明,40mmol/L四乙铵(TEA)灌流15min能完全阻断KCa通道活动。这提示钙激活钾通道在甲壳动物细胞免疫中发挥重要作用。
Inside-out patch clamp technique was used to identify and characterize Calcium-activated potassium channels （Kca） from cultured Eriocheir sinensis haemocytes 2-24 h after plating. Calcium-activated potassium channels were observed in high symmetrical potassium solutions （200 mmol/L）. Only small granule cell （SG） expressed BKc, and IKca channels. The conductance of BKca channel was 396 pS with open rectangle waves. At constant potential, the open-rate and open-number of this channel were increased with [Ca2＋] from 0.3 to 3.0 μmol/L, but decreased when [Ca2＋] was increased up to 100.0μmol/L, manifesting Ca2＋-dependent inactivation of BKca channel. When the potential was held at -40 or -80 mV in the presence of 3.0 lamol/L [Ca2＋], the mean open-duration were （0.032 33±0.007 41） s or （0.055 85±0.012 99） s （P〈0.01）, respectively and the mean open probability were 0.596±0.125 or 0.961±0.012 （P〈0.01）, respectively, indicating that BKca channel is volt- age-dependent. The conductance of IKca channel was 88 pS, with open rectangle waves. IKca channel was open when [Ca2＋] was kept at up to 3.0 pmol/L, showing that IKca channel is Ca2＋-dependent. When the potential was held at -40 or -80 mV, the mean open-duration were （0.017 36±0.004 03）s or （0.022 62±0.016 16） s （P〉0.05）, re spectively and the mean open probability were 0.699±0.065 and 0.790±0.114 （P〉0.05）, respectively, indicating that IKca channel is not obviously voltage-dependent. In addition, the Calcium-activated potassium channel could be completely be blockaded by a 15-min perfusion of tetraethylammonium （40 mmol/L）, suggesting that the KCa channels have a pivotal function in cellular immunity of crustacean.
出处
《海洋科学》
CAS
CSCD
北大核心
2010年第5期55-61,共7页
Marine Sciences
基金
国家自然科学基金项目(30571421
30871907)
国家科技支撑计划项目(2006BAD09A11)
