Martentoxin抗惊厥及其锚定BK通道靶标的分子机制研究
发布时间：2018-09-03 07:40
【摘要】：大电导钙激活钾离子通道（large conductance calcium-activated potassiumchannels，BK）通道广泛分布于各种细胞和组织中，参与调控众多生理功能，，其表达量和功能异常与多种人类疾病密切相关，本论文着重研究了martentoxin（一个自主研发的新型BK通道阻断剂）的在体抗惊厥药效及其与BK通道靶标互作的分子机制。 1. Martentoxin对大鼠PTZ模型的抗惊厥效应：本研究发现，martentoxin可抑制由PTZ诱导的大鼠惊厥发作和复发。海马内注射martentoxin能缓解PTZ诱导的大鼠惊厥症状，低剂量的martentoxin（0.05μg）能延长惊厥首次发作的潜伏期，martentoxin （0.05μg和0.5μg）能剂量依赖地减少癫痫样放电的总时程，但对于发作级数没有明显作用。低剂量的martentoxin不但可以延长PTZ复发性模型的惊厥潜伏期，且能显著地抑制PTZ复发模型的惊厥发作时程和级数。 2. Martentoxin独钟神经型BK通道的分子基础：神经型BK通道被证明对经典阻断剂charybdotoxin和iberiotoxin不敏感，却可被martentoxin有效地抑制（IC50为72nM）。本章节采用丙氨酸扫描等方法鉴定了位于BK通道孔区的Y294为识别martentoxin的关键性位点，D261和E276对于识别martentoxin并不重要。此外，BK通道的单一α亚基对martentoxin不敏感（即便浓度高达1μM），由此强烈提示，神经型BK通道的β4亚基参与了对martentoxin的识别。于是，将BK通道（α+β4）和（α+β1）的胞外环相互替换构建嵌合体。经电生理检测，BK（α+β1Lβ4）构成的嵌合体对martentoxin敏感，其特征与野生型BK通道（α+β4）吻合；由α+β4Lβ1构成的BK通道的电流可被martentoxin显著增加，其特征与野生型BK通道（α+β1）相似。以上结果清晰地表明，α亚基的孔区和β4亚基的胞外环是神经型BK通道识别martentoxin的关键位点。 3. Martentoxin对胶质瘤型BK和BK（α+β1）通道的增强效应：胶质瘤BK（gBK）和BK（α+β1）通道在已知BK通道亚型中对胞内钙离子最为敏感。本研究运用电生理记录、细胞增殖和钙成像等技术检测了martentoxin调制这两种通道的药理动力学参数。当胞内存有Ca2+时，martentoxin能够剂量依赖性地增强胶质瘤BK和BK（α+β1）通道的活性，EC50分别为46.7nM和495nM，但未使该两种通道的稳态激活曲线发生偏移。Martentoxin调制胶质瘤BK和BK（α+β1）通道电流幅值的增大比率与胞内钙浓度的变化无关。iberiotoxin（选择性BK通道孔区阻断剂）能够完全抑制martentoxin对这两种BK通道的增强效应。在胞内缺乏内钙时，martentoxin能够有效地抑制胶质瘤BK通道的活性，而对BK（α+β1）通道没有明显的作用。以上结果提示，martentoxin能以有别于其他已知BK通道阻断剂的作用方式增强胶质瘤型BK和BK（α+β1）通道的活性。 4. Martentoxin对β1亚基去糖基化BK通道的药理调制：β1亚基的糖基化是否参与BK通道的功能调节目前似乎鲜有触及。对此，我们运用电生理方法，结合分子突变、生物化学的技术，研究发现，一旦β1亚基经N80A/N142A或β1N80Q/N142Q双突变去糖基化，iberiotoxin对BK通道的抑制效应被显著增强。相比martentoxin则显示出了不同的敏感性。在一定内钙条件下，martentoxin可显著增强糖基化的BK通道活性；而去糖基化的通道则不受影响。有趣的是，在缺乏内钙的情况下，martentoxin有效地抑制去糖基化通道活性；而对糖基化的通道却无此效应。同时，β1亚基N-糖基化也参与了BK通道动力学特征的形成。尽管BK通道激活曲线的V1/2和斜率没有发生改变，但β1亚基去糖基化可显著加快BK通道的激活速率。因此，β1亚基的N-糖基化程度是决定BK通道功能特性的不可轻视要素之一。 本论文揭示martentoxin是一种以神经型BK通道为靶点的新型抗癫痫分子、甄别BK通道亚型的独有探针工具，其识别靶通道的机制丰富了钾通道与配体的互作模式，对于深入解析BK通道的生理功能、研发该通道相关疾病的药物均至关重要。
[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.
【学位授予单位】：上海大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R965
本文编号：2219345
[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.
【学位授予单位】：上海大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R965
【参考文献】
相关期刊论文 前1条
1 李明华,王跃丰,陈学勤,张乃霞,吴厚铭,胡国渊;东亚钳蝎中新分离的毒素BmTx3B抑制大鼠海马神经元延迟整流性钾电流(英文)[J];Acta Pharmacologica Sinica;2003年10期
本文编号：2219345
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