【作者】 李卉；

【导师】 丁久平；

【作者基本信息】 华中科技大学 ， 生物物理学， 2008， 博士

【摘要】 钙和电压激活的大电导钾离子通道(Maxik通道,又名BK通道)广泛地存在于可兴奋细胞,特别是神经系统中,具有调节胞内钙浓度和膜电位等重要生理功能。目前,人们对电压依赖的钾离子通道(Kv通道)的研究已较为深刻。BK通道与Kv通道在结构和功能上既存在某些相似之处又有各自的特点。如它们都能结合β辅助亚基,并通过其N端的疏水氨基酸引起N型失活。然而,BK通道的胞内阻断剂(如TEA、QX-314等)却不能像Kv通道那样减慢失活过程。因此,BK通道的α亚基在失活过程中如何与其β亚基的N端相互作用,它们的相互作用位点怎样,这些问题自被提出后至今仍未解决。我们将hβ2 N端的前三个氨基酸FIW突变为FWI,发现hβ2的突变体hβ2-FWI对α亚基的失活没有影响,但使其的失活恢复时间曲线从单指数转变为双指数特征。利用hβ2-FWI的这一特性,我们将α亚基形成孔道的S6区的疏水性氨基酸依次突变为不太疏水的丙氨酸。S6的突变体与FWI共表达来检测失活过程中hβ2亚基与α亚基孔道的相互作用位点。本课题的主要研究成果如下:(1)在S6突变中,mSlo1-I323A与hβ2-FWI共表达,它们的失活恢复双指数成分中的慢相得到最大地消减,即能最好拟合成单指数恢复曲线,说明I323是在失活过程中与β2亚基相互作用的主要位点。另外,M314A和V319A的快相变得异常地快,说明这两点也在失活过程中起着一定的作用。根据FWI的线性结构关系,我们推测如下的相互作用关系:I323-I,V319-W,M314-F;I323起着主导性的作用。(2)虽然V319A对QX-314的敏感性强于其他位点,但QX-314在孔道内并没有特异性的结合位点。我们提出了一个非竞争模型。I323是孔道内的最后一个氨基酸,既β2 N端失活域的作用位置是在孔道口,而QX-314的作用位置位于孔道内,因此QX-314不会影响β2引起的失活过程,这就解释了胞内阻断剂为什么不减慢BK通道的失活。这为进一步了解BK通道的结构和门控机制提供了重要的实验和理论支持。(3)I323A不管是单通道还是宏观电流都有外向整流现象,而野生型的mSlo1没有;I323A的单通道出现非常像噪声的电流,就像dSlo1A的单通道电流一样。这说明了Ile-323还调节BK通道的门控。在dSlo1A上与之对应的氨基酸是Thr-337。Ile是疏水氨基酸,而Ala和Thr都是亲水氨基酸。我们把野生型mSlo1的Ile-323突变为Thr,发现I323T导致与dSlo相似的噪声单通道电流。这个机制可能帮助我们了解dSlo1A单通道的行为。(4)计算机模拟mSlo1孔道与β2 N端相互作用的结构,得到了部分可供参考的两蛋白分子共价键之间相互作用的细节。更多还原

【Abstract】 Calcium- and voltage- activated large conduction potassium channel (Maxik channel, also termed BK channel) extensively exists in excitable cells, especially in neural system. BK channel plays a significant physiological role in mediating the concentration of intracellular calcium ions and the membrane potential. At present, the voltage dependent potassium channel (Kv channel) has been deeply studied. BK channel shares structural and functional similarities with Kv channel and also has its own characteristics. For example, they all can combine auxiliaryβsubunits, and all have N-type inactivation induced by the hydrophobic amino acids of the N terminals. However, unlike Kv channels, the intracellular blockers (such as TEA and QX-314) of BK channels can not slow the process of inactivation. Therefore, how does theαsubunit of BK channel interact with theβsubunit during inactivation, and what are they interaction sites, these questions are still unsolved since being presented.We mutated the first three amino acids of hβ2 N terminal FIW into FWI, found that FWI mutant had no impact on the inactivation ofαsubunit, but its time curve of recovery was changed into bi-exponential characteristic from mon-exponent. Utilizing this feature of FWI, we mutated the hydrophobic amino acids in the pore formed S6 segment ofαsubunit into less hydrophobic alanine. The interaction sites betweenαsubunit andβ2 subunit in the process of inactivation were detected by the coexpression of S6 mutants and FWI.The main research achievements in this study are as follows:(1) Among the S6 mutants, mSlo1-I323A coexpressed with hβ2-FWI, their slow component of the bi-exponent was maximally reduced. That is, its recovery curve can be almost fitted into mon-exponent. It demonstrated that I323 is the main interaction site withβ2 subunit during the inactivation. In additional, the fast components of M314A and V319A were extrodinarily faster, sugesting that the two points have certain effect in the process of inactivation. Based on the linear structure relationship of FWI, we inferred the following interaction relationship: I323-I, V319-W, M314-F; I323 played the leading role.(2) Although the sensitivity of V319A to QX-314 is higher than other sites, there is no specific binding site for QX-314 in the pore. We presented one step non-competition model. For Ile-323 is the last residue in the pore, the interaction site ofβ2 inactivation domain is on the entrance of the pore, the interaction sites of QX-314 are in the pore, therefore, QX-314 would not impact the process of inactivation. This model explained that why intracellular blockers do not slow the inactivation process of BK channel. We provided important experimental and theoretical support for understanding the structure and the gating mechanism of BK channel.(3) I323A had outward rectification phenomena in both single channel level and microcurrent level, but wide type mSlo1 did not; the single channel current of I323A is flickery, like the single channel current of dSlo1A. These demonstrated that Ile-323 can also mediate the gating of BK channel. The corresponding residue in dSlo1A is Thr-337. Ile is hydrophobic residue, but Ala and Thr are hydrophilic ones. We mutated the Ile-323 of wide type mSlo1 into Thr, found that I323T induced the flickery single channel current similar to dSlo1A. This mechanism may help us to understand the behavior of dSlo single channel current.(4) From the computer simulation of the interaction structure between the mSlo1 pore andβ2 N terminal, we can obtain part details of interaction between the two proteins.更多还原