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Title: 神经元离子通道、动作电位和递质释放的关系
Author: 何林玲
Degree Level: 博士
Issued Date: 2003
Degree Grantor: 中国科学院上海生命科学研究院神经科学研究所
Place of Degree Grantor: 中国科学院上海生命科学研究院神经科学研究所
Supervisor: 周专
Keyword: 颈上神经节神经元 ; 突触终扣 ; 钙库 ; 分泌 ; 乙酰胆碱
Alternative Title: The relationships of ionchannels, action potentials and secretion inneurons
Abstract: 化标记、电镜和FMI-43荧光成像技术等方法证明在急性分离的SCG神经元胞体上存在突触终扣。结合碳纤电极(CFE,可以用来实时地记录量子化分泌的电流信号),fura-2(测钙信号)和FMI-43(观测突触)等方法,我们发现并证明了在单个突触终扣上存在有钙库,并进一步研究了其功能。IP3敏感和RyR敏感的钙库可以诱导量子化肾上腺素分泌,我们发现这些肾上腺素是从附着在胞体上的突触终扣分泌来的,而不是来自于胞体。ATP是中枢和外周神经系统中的一种快速神经递质,结合CFE、膜片钳和钙成像技术我们研究了ATP诱导的突触终扣量子化释放。ATP诱导的分泌是由PZX受体通道本身的钙离子内流引起的,而不依赖于电压依赖性钙通道和胞内钙库;PZY/G一蛋白诱导的胞内钙库释放不能引起量子化释放,但它们可以调节由ATP产生的分泌。 不同神经递质受体之间存在相互作用(crosstalk),这里我们主要研究了SCG神经元和RACC中mAChRs和nAChRs的crosstalk。我们发现mAChRs的激动剂MCh(1mM)几乎完全阻断nAChRs介导的电流(M阻断),M阻断具有时间和温度依赖性;用Pertussis toxine(PTX)过夜处理、胞内灌注BaPta或GDP βs都可以减弱M阻断;胞内灌注GTP γs可以模拟M阻断;PKA和PKC的激动剂或结抗剂对M阻断均有影响,这些实验都说明MCh对nAChRs电流的阻断是由G蛋白偶联的胞内第二信使介导的。 神经递质的释放依赖于动作电位的编码,而动作电位的编码依赖于质膜上的离子通道,包括钙通道和钙依赖性钾通道。RACC存在两种类型的钙依赖型钾通道,大电导的钙依赖型钾通道(BKCa)是负责动作电位的复极化和快速的后超级化,可以调节动作电位的宽度而改变动作电位期间钙离子的内流;小电导的钙依赖型钾通道(sKCa)是负责一串动作电位的慢速后超级化,也可以调节动作电位的频率。高频刺激下,BKCa通道失活,SKCa通道被激活,起负反馈作用,使细胞发放频率降低,避免受高频刺激的伤害。KCa通道可以通过调节动作电位的频率和模式来调控细胞分泌,因此研制影响KCa通道的特异性的工具药,如生物毒素(包括某些蝎毒素)是很有意义的。我们主要研究了BmP05和MortenToxin等蝎毒素的结构和功能的关系,主要结果如下:BmP饰可以专一性阻断SKCa通道;通过研究BmP05突变体对通道的阻断作用,我们发现N末端α螺旋区域内的第6和13位的赖氨酸和精氨酸对毒素与SKCa的相互作用起必要和协同的作用,而C-末端的连接两个β-折叠的β-转角是负责毒素作用的专一性。
English Abstract: Single synaptic terminal attached to freshly isolated superior cervical ganglion (SCG) neurons, or "soma-attached terminal", were identified using hmmuno staining, electron microscopy (EM) and fluorescent FM1-43 imaging. Using electrochemical carbon fiber electrode (CFE) (for secretion), fura-2 (for hntracellular [Ca~+]j) and FM 1-43 (for synapse visualization), we have examined the existence and role of Ca2+ stores in a single terminal. Activation of either IP-3 or ryanodine Ca2+ stores was sufficient to trigger quantal secretion from tbe terminals. ATP is a fast neurotransmitter in both the central and peripheral neuron systems. CFE, patch clamp, and together with fura-2 Ca2+ imaging were employed to investigate the ATP-induced quantal secretion from free pre-synaptic terminals. We concluded that ATP-induced Ca2+ influx through P2X channels located in free pre-synaptic terminals alone was sufficient to trigger secretion, while ATP-induced intracellular Ca2+ release via P2Y/G-protein pathway played an additional modulation role. We investigated the mechanisms of mAChRs inhibition on nAChRs. lmM methacoline (Mch), the agonist of muscarinic receptors, almost completely suppressed lOOuM nicotine-induced current (M-inhibition). The results of temperature experiments supposed that the inhibition of nicotine current mediated by Mch involved pathway linked to G protein. Pertussis toxine (PTX) pretreatment overnight, or whole-cell dialysis with Bapta (20mM) or GDP 3 s attenuated M-inhibition. The agonists or antagonists of PKA and PKC had effects on M-inhibition. We concluded that the inhibition effect of Mch on nicotine current was mediated by intracellular second messages activated by G-proteins. Release of neurotransmitter is dependent on encoding of action potentials (AP) in single cells. Pattern of APs are dependent on ionic channels on plasma membrane, including Ca2+ channels and Ca2+-activated K+ channels. The two types of Ca2+ dependent K+ channels regulate APs pattern differently. The large conductance Ca2+-activated K+ (BKCa) channels are responsible for the repolarization and the fast afterhyperpolarization of AP, can regulate the duration of AP, and change the Ca2+ influx during APs. The small conductance Ca2+-activated K+ (SKCa) channels are responsible for the slow afterhyperpolarization of a train APs, and also can regulate APs pattern. The subsequence of inactivation of BKCa current and enhancement of SKCa current during the higher electrical firing would result in the action potential adaptation to protect cells from the harm of excess high frequency stimulation. KCa channels can regulate neurotransmitters release, so it is important to study the antagonists inhibition of KCa channels. Here we investigated the inhibition of BmPOS and BmTX3 on KCa channels.
Language: 中文
Content Type: 学位论文
URI: http://ir.sibs.ac.cn/handle/331001/2263
Appears in Collections:神经所(总)_学位论文

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Recommended Citation:
神经元离子通道、动作电位和递质释放的关系.何林玲[d].中国科学院上海生命科学研究院神经科学研究所,2003.20-25
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