【摘要】：大電導(dǎo)鈣激活鉀離子通道（large conductance calcium-activated potassiumchannels，BK）通道廣泛分布于各種細(xì)胞和組織中，參與調(diào)控眾多生理功能，，其表達(dá)量和功能異常與多種人類(lèi)疾病密切相關(guān)，本論文著重研究了martentoxin（一個(gè)自主研發(fā)的新型BK通道阻斷劑）的在體抗驚厥藥效及其與BK通道靶標(biāo)互作的分子機(jī)制。 1. Martentoxin對(duì)大鼠PTZ模型的抗驚厥效應(yīng)：本研究發(fā)現(xiàn)，martentoxin可抑制由PTZ誘導(dǎo)的大鼠驚厥發(fā)作和復(fù)發(fā)。海馬內(nèi)注射martentoxin能緩解PTZ誘導(dǎo)的大鼠驚厥癥狀，低劑量的martentoxin（0.05μg）能延長(zhǎng)驚厥首次發(fā)作的潛伏期，martentoxin （0.05μg和0.5μg）能劑量依賴地減少癲癇樣放電的總時(shí)程，但對(duì)于發(fā)作級(jí)數(shù)沒(méi)有明顯作用。低劑量的martentoxin不但可以延長(zhǎng)PTZ復(fù)發(fā)性模型的驚厥潛伏期，且能顯著地抑制PTZ復(fù)發(fā)模型的驚厥發(fā)作時(shí)程和級(jí)數(shù)。 2. Martentoxin獨(dú)鐘神經(jīng)型BK通道的分子基礎(chǔ)：神經(jīng)型BK通道被證明對(duì)經(jīng)典阻斷劑charybdotoxin和iberiotoxin不敏感，卻可被martentoxin有效地抑制（IC50為72nM）。本章節(jié)采用丙氨酸掃描等方法鑒定了位于BK通道孔區(qū)的Y294為識(shí)別martentoxin的關(guān)鍵性位點(diǎn)，D261和E276對(duì)于識(shí)別martentoxin并不重要。此外，BK通道的單一α亞基對(duì)martentoxin不敏感（即便濃度高達(dá)1μM），由此強(qiáng)烈提示，神經(jīng)型BK通道的β4亞基參與了對(duì)martentoxin的識(shí)別。于是，將BK通道（α+β4）和（α+β1）的胞外環(huán)相互替換構(gòu)建嵌合體。經(jīng)電生理檢測(cè)，BK（α+β1Lβ4）構(gòu)成的嵌合體對(duì)martentoxin敏感，其特征與野生型BK通道（α+β4）吻合；由α+β4Lβ1構(gòu)成的BK通道的電流可被martentoxin顯著增加，其特征與野生型BK通道（α+β1）相似。以上結(jié)果清晰地表明，α亞基的孔區(qū)和β4亞基的胞外環(huán)是神經(jīng)型BK通道識(shí)別martentoxin的關(guān)鍵位點(diǎn)。 3. Martentoxin對(duì)膠質(zhì)瘤型BK和BK（α+β1）通道的增強(qiáng)效應(yīng)：膠質(zhì)瘤BK（gBK）和BK（α+β1）通道在已知BK通道亞型中對(duì)胞內(nèi)鈣離子最為敏感。本研究運(yùn)用電生理記錄、細(xì)胞增殖和鈣成像等技術(shù)檢測(cè)了martentoxin調(diào)制這兩種通道的藥理動(dòng)力學(xué)參數(shù)。當(dāng)胞內(nèi)存有Ca2+時(shí)，martentoxin能夠劑量依賴性地增強(qiáng)膠質(zhì)瘤BK和BK（α+β1）通道的活性，EC50分別為46.7nM和495nM，但未使該兩種通道的穩(wěn)態(tài)激活曲線發(fā)生偏移。Martentoxin調(diào)制膠質(zhì)瘤BK和BK（α+β1）通道電流幅值的增大比率與胞內(nèi)鈣濃度的變化無(wú)關(guān)。iberiotoxin（選擇性BK通道孔區(qū)阻斷劑）能夠完全抑制martentoxin對(duì)這兩種BK通道的增強(qiáng)效應(yīng)。在胞內(nèi)缺乏內(nèi)鈣時(shí)，martentoxin能夠有效地抑制膠質(zhì)瘤BK通道的活性，而對(duì)BK（α+β1）通道沒(méi)有明顯的作用。以上結(jié)果提示，martentoxin能以有別于其他已知BK通道阻斷劑的作用方式增強(qiáng)膠質(zhì)瘤型BK和BK（α+β1）通道的活性。 4. Martentoxin對(duì)β1亞基去糖基化BK通道的藥理調(diào)制：β1亞基的糖基化是否參與BK通道的功能調(diào)節(jié)目前似乎鮮有觸及。對(duì)此，我們運(yùn)用電生理方法，結(jié)合分子突變、生物化學(xué)的技術(shù)，研究發(fā)現(xiàn)，一旦β1亞基經(jīng)N80A/N142A或β1N80Q/N142Q雙突變?nèi)ヌ腔琲beriotoxin對(duì)BK通道的抑制效應(yīng)被顯著增強(qiáng)。相比martentoxin則顯示出了不同的敏感性。在一定內(nèi)鈣條件下，martentoxin可顯著增強(qiáng)糖基化的BK通道活性；而去糖基化的通道則不受影響。有趣的是，在缺乏內(nèi)鈣的情況下，martentoxin有效地抑制去糖基化通道活性；而對(duì)糖基化的通道卻無(wú)此效應(yīng)。同時(shí)，β1亞基N-糖基化也參與了BK通道動(dòng)力學(xué)特征的形成。盡管BK通道激活曲線的V1/2和斜率沒(méi)有發(fā)生改變，但β1亞基去糖基化可顯著加快BK通道的激活速率。因此，β1亞基的N-糖基化程度是決定BK通道功能特性的不可輕視要素之一。 本論文揭示martentoxin是一種以神經(jīng)型BK通道為靶點(diǎn)的新型抗癲癇分子、甄別BK通道亞型的獨(dú)有探針工具，其識(shí)別靶通道的機(jī)制豐富了鉀通道與配體的互作模式，對(duì)于深入解析BK通道的生理功能、研發(fā)該通道相關(guān)疾病的藥物均至關(guān)重要。
[Abstract]:Large conductance calcium-activated potassium channels (BK) are widely distributed in a variety of cells and tissues and participate in the regulation of many physiological functions. Their expression and dysfunction are closely related to a variety of human diseases. This paper focuses on the study of martentoxin (a new BK channel developed independently). The mechanism of anticonvulsant activity in vivo and its interaction with BK channel targets are discussed.
1. Anticonvulsive effect of Martentoxin on PTZ model in rats: This study found that martentoxin could inhibit the seizure and recurrence of PTZ-induced convulsions in rats. Low dose of martentoxin not only prolonged the convulsive latency of PTZ recurrence model, but also significantly inhibited the convulsive duration and progression of PTZ recurrence model.
2. Molecular Basis of Martentoxin Uniclockwise Neural BK Channel: Neural BK Channel has been shown to be insensitive to classical blockers charybdotoxin and iberiotoxin, but can be effectively inhibited by martentoxin (IC50 is 72nM). In this chapter, Y294 located in the pore region of BK channel was identified as a key site for identifying martentoxin by alanine scanning. D261 and E276 are not important for recognizing martentoxin. In addition, the single alpha subunit of BK channel is insensitive to martentoxin (even at a concentration of up to 1 mu M), which strongly suggests that the beta 4 subunit of neural BK channel is involved in the recognition of martentoxin. Thus, the extracellular rings of BK channel (alpha+beta 4) and (alpha+beta 1) are substituted for each other to construct chimeras. The results showed that the chimera composed of BK (alpha+beta 1L beta 4) was sensitive to martentoxin and its characteristics were consistent with that of wild type BK channel (alpha+beta 4). The current of BK channel composed of alpha+beta 4L beta 1 could be significantly increased by martentoxin, which was similar to that of wild type BK channel (alpha+beta 1). The BK channel identifies the key loci of martentoxin.
3. The enhancement effect of Martentoxin on BK and BK (alpha+beta 1) channels in glioma: BK (gBK) and BK (alpha+beta 1) channels are most sensitive to intracellular calcium ions in known BK channel subtypes. The pharmacokinetic parameters of these two channels modulated by martentoxin were measured by electrophysiological recording, cell proliferation and calcium imaging. In the presence of Ca2 +, martentoxin could increase the activity of BK and BK (a + beta 1) channels in a dose-dependent manner. EC50 was 46.7 nM and 495 nM respectively, but the steady-state activation curves of these two channels were not offset. The increase rate of Martentoxin modulating the amplitude of BK and BK (a + beta 1) channels in glioma was not related to the change of intracellular calcium concentration. N (selective BK channel blocker) can completely inhibit the enhancement effect of martentoxin on these two BK channels. In the absence of intracellular calcium, martentoxin can effectively inhibit the activity of BK channels in glioma, but has no obvious effect on BK (alpha + beta 1) channels. These results suggest that martentoxin can block BK channels differently from other known BK channels. The action of these agents enhanced the activity of glioma type BK and BK (alpha + beta 1) channels.
4. Martentoxin's pharmacological modulation of the deglycosylated BK channel of the beta 1 subunit: Whether the glycosylation of the beta 1 subunit is involved in the regulation of BK channel function seems to be seldom touched. In this regard, we used electrophysiological methods, combined with molecular mutation, biochemical techniques, and found that once the beta 1 subunit was deglycosylated by N80A/N142A or beta 1N80Q/N142Q double mutation. The inhibitory effect of iberiotoxin on BK channel was significantly enhanced. Compared with martentoxin, martentoxin showed different sensitivities. Under certain internal calcium conditions, martentoxin significantly increased the activity of glycosylated BK channel, while the channel of de-glycosylation was not affected. Interestingly, in the absence of internal calcium, martentoxin effectively inhibited de-glycosylation. The N-glycosylation of the beta-1 subunit is also involved in the formation of the kinetic characteristics of the BK channel. Although the V1/2 and slope of the activation curve of the BK channel have not changed, the N-glycosylation of the beta-1 subunit can significantly accelerate the activation rate of the BK channel. One of the most important elements of the BK channel's functional characteristics is not to be despise.
This paper reveals that martentoxin is a novel antiepileptic molecule targeting neuronal BK channels. It is a unique probe tool for identifying BK channel subtypes. Its mechanism of identifying the target channels enriches the interaction patterns between potassium channels and ligands. It is very important for further understanding the physiological functions of BK channels and developing drugs for BK channel-related diseases.
【學(xué)位授予單位】：上海大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R965

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相關(guān)期刊論文 前1條

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