【作者】 闾彩霞；

【导师】 丁久平； 徐涛；

【作者基本信息】 华中科技大学 ， 生物物理， 2008， 博士

【摘要】 离子通道作为一种膜蛋白在动物、植物、单细胞或多细胞生物的细胞膜上广泛存在,产生细胞生物电现象。钙依赖、电压激活的大电导钾通道(BK通道)作为钾离子通道家族中的一员,在线虫、昆虫和哺乳动物的肌肉、脑、胰腺、背根神经节上都有表达。BK通道的α亚基不同剪接变体和四种β亚基一起促成了其电流和功能的多样性。β2亚基通过其N端的三个疏水性氨基酸FIW产生了失活的BK电流。本文以BK通道β2亚基为研究对象,以HEK293细胞为工具,利用免疫荧光技术,共聚焦显微镜技术,结合分子生物学和电生理学方法,研究了β2亚基的转运机制,探索了其与α亚基的结合位点。主要内容如下:(1)通过在β2的N端增加三个疏水性的氨基酸FIW(dFIW),我们发现没有记录到预期的失活加强的BK电流,而是记录不到任何电流。通过荧光实验我们发现这种突变体dFIW造成了其本身和共表达的α亚基的ER滞留。通过突变其N端的带电荷的氨基酸D16E17和E44D45,我们发现突变体E44D45能使大部分的α亚基上膜,同时能记录到没有失活的BK电流。我们推断E44D45是结合位点之一。(2)通过在β2亚基的胞外部分加入抗原决定簇c-myc,利用荧光素耦联的抗体检测发现β2亚基不同于β1亚基,它单独不能被运输到HEK细胞膜上,而是被滞留在内质网(ER)。但与α亚基共表达可以被α亚基带上膜。通过切掉其N端或C端的一些序列我们发现β2亚基N端的一段α螺旋(18-31)造成了其ER滞留。通过在其loop的不同位置加抗原,我们发现K137附近是最容易被抗体接近的区域。小鼠mslo1形成的BK单通道电流有较长的开放,但线虫dslo(A2/C2/E2/G5/10)在开放时却非常flickery。本文还以BK通道α亚基为研究对象,以非洲爪蟾卵母细胞为工具,利用电生理技术研究BK通道的门控机制。主要内容如下:我们发现mSlo的突变体I323T会产生跟dslo(A2/C2/E2/G5/10)类似快速开放的单通道电导,并且还有外向整流的性质。而突变dslo(A2/C2/E2/G5/10)相应位置的亲水性氨基酸T337I,发现该通道开放的单通道电导不再flickery。通过单通道电导分析我们发现突变体I323T存在不同亚态的亚电导,这是因为四个亚基之间的协同性被破坏造成的。通过对mSlo的I323位点做一系列的突变,我们发现越疏水性的氨基酸会产生越长的单通道电导。分子动力学模拟表明位于BK通道孔道的四个疏水性氨基酸I323在通道门控过程中起着“开关”作用。因此,我们证明BK通道的亚电导是由于323位置的四个氨基酸残基的协同性降低和开放几率的降低造成的。更多还原

【Abstract】 Large-conductance voltage- and Ca2+-activated K+ (BK) channels encoded by the msloαandβ2 subunits exist abundantly in rat chromaffin cells, pancreaticβcells and DRG neurons. The extracellular loop of hβ2 acting as the channel regulator influences the rectification and toxin sensitivity of BK channels and the inactivation domain at its N-terminus induces rapid inactivation. However, the regulatory mechanism, especially, the trafficking mechanism of hβ2 is still unknown. With the help of immunofluorescence and patch-clamp techniques, we found that the hβ2 subunit could be completely restrained within the cytoplast of cells by inserting three amino acids FIW at the upstream of its N-terminus termed double FIW or dFIW-hβ2. The dFIW-hβ2 also prevented mSloαsubunit from trafficking onto membrane surface and thus suppressed BK currents to null. Consequently, a novel method has been developed to identify the binding sites between the mSloαand hβ2 subunits of BK channels in this study. Our results reveal that the mSloαsubunits associate with the hβ2 subunits before they traffic to membrane surface; the residues E44D45 (hβ2) is a major binding site for mSloαand hβ2 subunits of BK channels and the residues D16E17 (hβ2) is a candidate as a preinactivation site. Our results also demonstrate that the weak binding sites ofαand hβ2 subunits of BK channels exist.We determine that the hβ2 subunit alone resides in the endoplasmic reticulum (ER), suggesting that trafficking mechanism of hβ2 differs from that of hβ1 opposite to what we predicted previously. We further demonstrate that a four-turnαhelical segment at the N terminus of hβ2 prevents the surface expression of hβ2, that is, the helical segment itself is a retention signal. Using the c-myc epitope tagged the extracellular loop of hβ2, we reveal that the most accessible site by antibody is located at the middle of the extracellular loop, which might provide clues to understand how the auxiliaryβsubunits regulates the toxin sensitivity and the rectification of BK-type channels.Single large-conductance calcium-activated K+ (BK) channels encoded by mSlo gene usually have a concerted gating, but a Drosophila--dSlo (A2/C2/E2/G5/10) splice variant (dSlo1A)--exhibits very flickery openings. To probe this difference in the gating, we constructed a mutant I323T. This channel exhibits four subconductance levels similar to that of dSlo1A. Rectification of the single channel current-voltage relationship of I323T decreased as [Ca2+]in increased from 10 to 300μM. Mutagenesis suggests that the hydrophobicity of the residue at the position is important for the wild-type gating i.e. increasing hydrophobicity prolongs open duration. Molecular dynamics simulation suggests that four hydrophobic, pore-lining residues at position 323 of mSlo act cooperatively in a“shutter-like”mechanism gating the permeation of K+ ions. We suggest that the appearance of rectification and substates of BK-type channels arise from a reduction of the cooperativity among these four residues and a lower probability of being open.更多还原