【摘 要】
：
我们先前已报道拟南芥环核苷酸门控的离子通道(CNGC)家族的成员之一,CNGC6,是一个受热和cAMP 激活的质膜钙通道蛋白,介导热诱导的胞外Ca2+的内流,并促进植物的耐热性.然而,CNGC6 钙通道的反馈抑制机制还不知道.动物中的研究表明,CaM 反馈抑制CNGC 通道的开放.生物信息学分析显示,CNGC6 分子中包含CaM 结合位点,然而CNGC6 是否受CaM 的反馈调节还没有实验证据.本
【机 构】
：
河北师范大学生命科学学院;邢台学院 河北师范大学生命科学学院;河北省中医学院
论文部分内容阅读
我们先前已报道拟南芥环核苷酸门控的离子通道(CNGC)家族的成员之一,CNGC6,是一个受热和cAMP 激活的质膜钙通道蛋白,介导热诱导的胞外Ca2+的内流,并促进植物的耐热性.然而,CNGC6 钙通道的反馈抑制机制还不知道.动物中的研究表明,CaM 反馈抑制CNGC 通道的开放.生物信息学分析显示,CNGC6 分子中包含CaM 结合位点,然而CNGC6 是否受CaM 的反馈调节还没有实验证据.本课题通过电生理学、分子遗传学、生物化学和细胞生物学等方法,研究CaM对CNGC6 钙通道活性的调节作用.我们首先通过膜片钳实验研究CaM拮抗剂处理及CaM 基因缺失对拟南芥CNGC6 钙通道活性的影响.用CaM 的拮抗剂CPZ 和W7 处理拟南芥野生型和CNGC6 突变体(cngc6)的原生质体,结果表明处理后,在-200 mV 电压下野生型细胞的质膜钙电流明显增大,而cngc6突变体细胞的质膜钙电流与处理前没有明显的变化,说明CNGC6 钙通道的活性可能受CaM 的反馈抑制.CaMX 突变体和CaMZ 突变体细胞热激后的质膜钙电流明显大于野生型,而CaMY 突变体热激后的钙电流与野生型没有明显差异,表明热激时CNGC6 钙通道的活性可能是受CaMX 和CaMZ 的反馈抑制.然后,我们通过酵母双杂交、pull-down 技术检测了CNGC6 与不同亚型CaM 的结合,结果显示CaMX 和CaMZ 能与CNGC6 结合,而CaMY 不能与CNGC6 结合.进一步我们通过CNGC6 上的CaM 结合位点缺失实验和体外小肽结合实验鉴定了CNGC6 分子上的CaM结合位点,证明了CNGC6 上的IQ 域是CaMX和CaMZ的结合位点.总之,本实验结果证明了热激时CaM 对CNGC6 钙通道的反馈调节作用.
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