| 摘要: |
| 利用膜片钳技术之内面向外式, 对培养2～24 h 不同类型的中华绒螯蟹(Eriocheir sinensis)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 (200 mmol/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396 pS, 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[Ca2+]在0.3～3.0 μmol/L范围内, 通道开放概率和开放数目随Ca2+浓度增加而增加, 当浴液中[Ca2+]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Ca2+浓度为3.0 μmol/L, 钳制电压分别为－40 mV 和－80 mV 时, 通道平均开放时间分别为(0.032 33±0.007 41) s 和(0.055 85±0.012 99) 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88 pS, 开放活动也表现为矩形方波。当浴液中Ca2+浓度为0.3、1.0μmol/L 时, 通道不开放; 而Ca2+浓度为3.0 μmol/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－40 mV 和－80 mV 时,通道平均开放时间分别为(0.017 36±0.004 03)s 和(0.022 62±0.016 16)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15 min 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。
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| 关键词: 中华绒螯蟹(Eriocheir sinensis) 血淋巴细胞 钙激活钾通道 膜片钳 内面向外式 |
| DOI： |
| 分类号: |
| 基金项目:国家自然科学基金项目(30571421, 30871907); 国家科技支撑计划项目(2006BAD09A11) |
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| Calcium-activated potassium channels expressed by the haemocytes of Eriocheir sinensis |
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Duan Hu,Wang Xue-hui,Sun Jin-sheng
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| Abstract: |
| 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 BKCa 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 100.0 μmol/L, manifesting Ca2+-dependent inactivation of BKCa channel. When the potential was held at ?40 or ?80 mV in the presence of 3.0 μmol/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 voltage-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 μmol/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), respectively 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.
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| Key words: Eriocheir sinensis Haemocytes Calcium-activated potassium channel Patch clamp Inside-out recording |
