【作者】 刘杰；

【导师】 徐涛；

【作者基本信息】 华中科技大学 ， 生物物理， 2005， 博士

【摘要】 α-latrotoxin是由黑寡妇蜘蛛分泌的仅作用于哺乳动物的毒素,在二价阳离子的作用下可以形成四聚体。其在细胞膜上存在有三种受体Neurexin,latrophilin和PTPδ。通过和受体的结合,α-latrotoxin可以通过在细胞膜上形成的穿孔以及受体介导的信号通路而引起突触前神经递质的释放以及细胞的分泌。其中在受体介导的信号通路中,α-latrotoxin除了动员细胞内钙库参与作用以外,还可以通过其它特殊途径直接作用于细胞的分泌机制。对于α-latrotoxin以及其作用方式的研究必将有助于我们对细胞囊泡分泌机制以及神经递质的释放机制的认识。本论文中通过细胞膜电容检测、细胞内Ca²⁺荧光测量、Ca²⁺光解释放以及碳纤维电极等技术手段对α-latrotoxin在大鼠胰腺β细胞的[Ca²⁺]i以及β细胞分泌的动力学影响进行了详细研究。研究的主要结果如下: α-latrotoxin可以在β细胞膜上形成具有大电导的通道并导致细胞外液中的Ca²⁺内流,但是α-latrotoxin不会引起细胞内的钙库释放。α-latrotoxin在β细胞的穿孔可被三价阳离子La3+阻断。通过使用荧光持续照射使[Ca²⁺]i缓慢升高来刺激分泌,我们发现α-latrotoxin降低了β细胞分泌时的钙阈值,但是不会对立即可释放囊泡库（RRP）的大小产生影响。通过Ca²⁺光解释放技术,我们也发现分泌的立即可释放囊泡库的分泌动力学明显加快,在同样的[Ca²⁺]i下,α-latrotoxin使β细胞分泌的最大速度加大并且使β细胞的分泌与Ca²⁺依赖关系向低钙方向移动。与之相对应的是,在无Ca²⁺条件下,通过碳纤维电极的检测发现α-latrotoxin可以在β细胞上引发一定的量子释放。在α-latrotoxin作用方式的研究中,α-LTXN4C是一种有效的研究工具。它是一种α-latrotoxin突变体,与受体的亲合力与野生型相同,但是其不能在细胞膜上形成穿孔,在存在二价阳离子(Ca²⁺或Mg2+)的情况下,α-LTXN4C会引起β细胞内的钙库释放,从而导致[Ca²⁺]i升高。使用α-LTXN4C处理β细胞以后,可以在分泌的钙阈值以及RRP动力学上得到与α-latrotoxin一致的结果。不仅如此,α-latrotoxin和α-LTXN4C的作用依赖于细胞外液中二价阳离子(Ca²⁺或Mg2+)的存在。使用不同的PKC阻断剂都可以使更多还原

【Abstract】 α-latrotoxin, a potent neurotoxin from black widao spider venom, is selectively targeted against vertbrates. α-latrotoxin forms dimers in solution that may assemble further into tetramers or higher order oligomers in the presence of divalent cations There are three distinct families of cell-surface receptors, Neurexin,latrophilin and PTPδfor α-latrotoxin. α-latrotoxin cause massive release of neurotransmitters from nerve terminals and cell exocytosis by froming pore and single transdunction mediated by receptor after binding to specific receptors. α-latrotoxin can mobilize the calcium store in cytoplasm and act on exocytotic machinery directly by receptors mediatied. Elucidation of precisely how α-latrotoxin works is likely to provide major insight into how cell vesicle exocytosis and the release machineries of neurotransmitters are regulated. In this study, by using capacitance measurement, microfluorometric measurement of calcium, flash photolysis of caged Ca²⁺, carbon fiber electrode, we studied the regulation mechanism of α-latrotoxin in regulation of the kinetics of exocytosis from single rat pancreatic βcells. Main results of the study are as fellows. We show α-latrotoxin can form pore with a unitary conductance of 0.13 nS on βcell membrane and induce Ca²⁺ influx form extracellular. α-latrotoxin could not release Ca²⁺ from calcium store. Trivalent cations, La3+, block the toxin pores. We find α-latrotoxin lowers the Ca²⁺ threshold required for exocytosis without the RRP change employed the ramp [Ca²⁺]i stimulation generated by continuous UV illumination; Using combined techniques of membrane capacitance measurement and Ca²⁺ uncaging, we show that α-latrotoxin accelerates the fusion kinetics of the readily releasable pool of vesicles, and α-latrotoxin shifts the Ca²⁺-dependence toward lower Ca²⁺ concentrations. In clacium free solution, we also show α-latrotoxin induce quantal release using carbon fiber electrode. The mutant toxin α-LTXN4C is a powerfu tool in the α-latrotoxin research. Despite the fact that this mutant has the same affinity for the receptors as the wild type α-latrotoxin, it fails to incorporate into the membrane and form pores as the wild type toxin does. α-LTXN4C can mimic the action on βcell exocytosis of α-latrotoxin. Moreover, the role of α-latrotoxin and α-LTXN4C dependent on divalent cations (Ca²⁺ or Mg2+). The different Protein Kinase C Inhibitors blocked the effect of either α-latrotoxin or α-LTXN4C in lowering the Ca²⁺ threshold for exocytosis, suggesting that PKC activation is required for the receptor-mediated modulation of the secretory machinery by α-latrotoxin. Our article propose that α-Latrotoxin, by binding to the membrane receptors, sensitizes the fusion machinery to Ca²⁺ and, hence, may permit release at low [Ca²⁺]i level. This sensitization is mediated by activation of protein kinase C.更多还原