【作者】 姚镜；

【导师】 丁久平；

【作者基本信息】 华中科技大学 ， 生物物理学， 2009， 博士

【摘要】 现代生命科学的发展改变着人们对生命获得过程的认识,而离子通道的研究不仅与信号发生、传递和转导紧密关联,同时也涉及到诱发各种遗传或非遗传疾病的分子机制。了解离子通道的精细结构,探讨离子通道的门控机制和动力学特性,弄清楚离子通道结构和功能之间的关系意义重大。本研究工作由两部分组成:第一部分探讨了钙和电压激活的大电导钾离子通道（Maxik通道,又名BK通道）的β2亚基N端失活域在由α亚基组成的孔道中的作用位点;文章第二部分则是讲述痛觉敏感型通道TRPV1的适应性调节的分子机制的。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,电压依赖型钾离子通道（Kv通道）的研究已取得较大进展。BK通道与Kv通道在结构和功能上既存在某些相似之处又各具特点。如它们都结合有β辅助亚基,并由β亚基的N端疏水性氨基酸残基堵塞孔道引起N型失活。然而现有实验结果表明,K通道的孔道阻断剂（如TEA、QX-314等）能够减慢Kv通道的失活过程,然而却不能减慢BK通道的失活。为什么这两者如此不同?BK通道的B2亚基N端失活域是否进入由α亚基组成的BK孔道?两者究竟发生了怎样的相互作用?阻断剂为什么不和失活域在孔道中竞争结合位点?本课题的研究目的之一即是要回答和解决这些目前仍然困扰我们的问题。我们将hβ2 N端的前三个氨基酸FIW突变为FWI,发现hβ2的这一突变体hβ2-FWI对BK通道的失活没有影响,但通道的失活恢复曲线却由单指数转变为双指数。利用hβ2-FWI的双指数恢复特性变化作为两者间相互作用的判定依据。通过丙氨酸扫描（Alaninescanning）,即将α亚基形成孔道的S6区疏水性氨基酸依次突变为疏水性较弱的丙氨酸,然后通过膜片钳实验记录分析失活后通道的恢复特性变化,进而了解两者间的相互作用。借助作为通道孔道阻滞剂的麻醉剂QX-314和TEA等药物进一步解释了BK通道失活的非竞争机制。我们得到了如下几个结论:（1）位于mSlo1孔道中的氨基酸Ile-323是在失活过程中与β2亚基N端失活域有相互作用的主要位点。孔道内的另外两个氨基酸M314和V319也参与了这一过程。根据hβ2-FWI亚基N末端的线性结构特点,我们推测mSlo1孔道与hβ2-FWI在BK通道失活过程中存在如下的相互作用关系:1323-I,V319-W,M314-F,其中1323起着主导性的作用。同时进一步明确了β2亚基的失活域的确进入了BK通道的孔道从而引起通道的失活。（2）通道阻滞剂QX-314和TEA在BK孔道内无特异性的结合位点。因此胞内阻断剂不会像在Kv通道中那样减慢通道的失活过程,这是因为β2 N端失活域的作用位置比较靠近孔道口,即孔道内的最后一个疏水性氨基酸Ile-323,而QX-314的作用位置位于孔道内,因此QX-314不会与失活域竞争相同的作用位点,从而不会影响β2引起的通道失活过程。根据实验结果,我们提出了一个全新的非竞争模型,该模型不仅可以很好的解释我们的数据,而且为进一步阐释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）我们的实验结果还显示:与野生型mslo1通道电流相比,突变型1323A不管是单通道还是宏观电流均有明显差异,表现为出现了野生型通道所不具有的外向整流特性。形式上突变型1323A的单通道出现了非常像噪声的电流,同一电压下可以同时记录得到几个不同大小的单通道电流,即是说Ile-323A还引起了BK通道电导的改变。据此我们推测Ile-323对BK通道具有双重作用,既是β2失活域在孔道中的作用位点,同时还能调节BK通道的门控特性。另外还发现一个非常有趣的现象,312位点的Leu亮氨酸残基的突变能够极大地改变BK通道的电压依赖性。我们研究小组在后续的两篇文章中分别证明了这两点推测:Ile-323确实为BK通道的关节所在,323点的氨基酸残基疏水性降低,会使得“关节”变得松弛,进而导致通道开放时四个亚基间协同性的不一致和开放几率的改变（Guo et al,Biophys J.2008 May 1;94（9）:3714-25）。而Leu312位点则可以极大地增强BK通道的电压敏感性（Wu et al.,In review）。适应性调节是大部分感觉器官共有的一种性质,但是痛觉适应性产生的根源和机理仍然不为人们所知。文章的第二部分在受体水平探讨了伤害感受器TRPV1通道的适应性调节的分子机制。我们发现通道在脱敏反应发生后,仅改变了通道对激动剂的敏感性,而不影响最大电流的响应,据此,我们在脱敏感念的基础上提出了痛觉适应性调节概念。该部分我们主要得到以下几个主要结论:（1）Ca²⁺经由开放的TRPV1通道内流引起脱敏反应的发生,脱敏反应发生后仅改变了通道对辣椒素的敏感性,浓度敏感性降低了14倍,但这一改变并不影响通道的功能。（2）通过联合应用全内反射荧光显微技术和膜片钳记录技术,我们证实了PIP2参与了TRPV1通道脱敏反应的发生,PIP2分解的速率和量的变化都足以改变通道对激动剂的响应。借助药物Rapamycin专一性降低细胞内PIP2含量作为研究工具,通过测量通道对辣椒素浓度依赖性曲线,我们进一步量化了PIP2对脱敏反应的贡献,约占60%。（3）我们还发现TRPV1通道对激动剂敏感性的改变依赖于刺激强度,不同的刺激强度得到不同程度的脱敏响应。我们推测有两种可能性:一是通道蛋白有记忆功能,这种可能性比较小;二是不同刺激强度作用下的TRPV1通道离子通透性不同,因此,内流Ca²⁺的量和分布特性不同,导致PIP2分解量不同,从而引起不同程度的脱敏反应。更多还原

【Abstract】 The developments of modern life science and technology have greatly influenced our understanding in the evolution of human lives.The studies on ion channels are not only related to the signal occurrence and transmission,but also involved in the molecular mechanisms of hereditary or inhereditary diseases.To explore the relationships between the structure and function of the ion channels and study on the mechanisms of gating and inactivation of ion channels are very important,and also will benefit the society and human health.There are two parts in this study.The first part is to study on the interaction ofβ2 subunit and the pore forming by mslo 1 subunit of Calcium- and voltage- activated large conductace potassium channel（Maxik channel,also termed BK channel）,the other one is focus on the molecular mechanisms of desensitization of pain receptors TRPV1 channel.BK channel extensively exists in excitable cells,especially in neural system,which plays a significant physiological role in mediating the concentration of intracellular calcium ions and the membrane potential.To date,the voltage dependent potassium channel（Kv channel） has been deeply studied.BK channel shares structural and functional similarities with Kv channel,i.e.,they both can combine auxiliaryβsubunits, and have a typical N-type inactivation induced by the N terminals ofβsubunits.However there are also some differences between them.Unlike Kv channels,the pore blockers （such as TEA and QX-314） of K channels can not slow down the inactivation rate of BK channels.Why are they so different in this issue? Do the N terminals ofβsubunits indeed enter into the pore forming by mslol? Where or what are the interaction sites between them during inactivation? To address these questions,we presented this study here.When mutating the first three amino acids of hβ2 N terminal FIW into FWI,we surprisingly found that the property of recovery curve was changed into bi-exponent from mono-exponent,but the FWI mutant has no effects on the inactivation process of the BK channels.Using this behavior of recovery of FWI,we tried to elucidate the relationship between them during inactivation by mutating the hydrophobic amino acids within the BK pore to less hydrophobic one,alanine.At last,we found that Ile 323 is the main interaction site betweenαsubunit andβ2 subunit during the process of inactivation.Our data also support that:（1） Among all the S6 mutants,mSlol-I323A coexpressed with hβ2-FWI,the slow component of the recovery was maximally reduced.That is,its recovery curve can be almost fitted with a mono-exponential function.It indicates that I323 is the main interaction site withβ2 subunit during the inactivation.In addition,the fast components of M314A and V319A were extrodinarily faster,sugesting that the two points have certain effects during the process of inactivation.Based on the linear structure relationship of FWI, we inferred the following interaction relationships:I323-I,V319-W,M314-F,and I323 played the leading role.（2） Despite the sensitivity of V319A to QX-314 is less higher than others,there is no specific binding site for QX-314 in the pore.A one step non-competition model can well explain our data other than a two-step model.The reason is that Ile-323 is the last residue in the pore,the interaction sites ofβ2 inactivation domain are located on the outer entrance of the pore,the interaction sites of QX-314 are inside of the pore,and therefore, QX-314 might not impact the process of inactivation.This non-competitive model explained well that why intracellular blockers do not slow down the inactivation process of BK channels.These data can help us understanding the structure and the gating mechanisms of BK channels.（3） Another intriguing phenomenon was seen with the mutant I323A.In comparison to the wide-type BK channels,the mutation I323A showed an obiviously outward rectification in both single channel recordings and macro patches.And also the single channel current of I323A is much flickery,even comparing to that of the single channel current of dSlo1.These data support that the residue Ile-323 also mediates the gating of BK channel.The corresponding residue in dSlo1 is Thr-337.Ile is a higher hydrophobic residue in comparison to Ala and Thr.In the following studies,we found that mutation I323T induced a similarly flickery single channel current similar as wild-type dSlo1 channel,which was reported by Guo et al in the Biophys J.（Guo et al,Biophys J. 2008 May 1;94（9）:3714-25）.This mechanism may also help us to understand the behavior of dSlo single channel current.Adaptation is a common feature of many sensory systems.But its occurrence to pain sensation has remained elusive.In the second part of this study,we address the problem at the receptor level and show that the capsaicin ion channel TRPV1,which mediates nociception at the peripheral nerve terminals,processes properties essential to the adaptation of sensory responses.Ca²⁺ influx following the channel opening caused a profound shift（～14 folds） of the agonist sensitivity,but did not alter the maximum attainable current.The main points we got are as following:（1） Ca²⁺ influx following the channel opening caused a profound shift of the agonist sensitivity,about 14 folds,but did not alter the maximum attainable current.The shift was adequate to render the channel irresponsive to normally saturating concentrations,leaving the notion that the channel became no longer functional after desensitization.（2） By simultaneous patch-clamp recordings and total internal reflection fluorescence（TIRF） imaging,it was shown that the depletion of phosphatidylinositol 4,5-bisphosphate（PIP2） induced by Ca²⁺ influx had a rapid time course synchronous to the desensitization of the current.The extent of the depletion was comparable to that by rapamycin-induced activation of a PIP2 5-phosphatase,which also caused a significant reduction of the agonist sensitivity without affecting maximum response.The change induced by rapamycin accounted for～57%of the overall shift resulting from the desensitization by Ca²⁺ influx,indicating that the depletion of PIP2 constitutes a prominent component of the adaptation of the channel.（3） We showed that the adaptation of the channel appeared to be stimulus-dependent. It appeared that the desensitized channel could only be reactivated by a stimulus higher than used for desensitization.We do not know how the channel "remember" the desensitizing conditions,and hypothesize the causal is the transient profile of the submembrane[Ca²⁺]could differ with different stimuli.The dynamic of Ca²⁺ waves are known to affect a variety of cellular processes.Conceivably,it may also contribute to the regulation of ion channels.更多还原