6β-acetoxy-7α-hydroxyroyleanone對於Kv1.2通道之阻斷有C-型鈍化之依賴性

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題名:	6β-acetoxy-7α-hydroxyroyleanone對於Kv1.2通道之阻斷有C-型鈍化之依賴性 Dependence of 6β-acetoxy-7α-hydroxyroyleanone block of Kv1.2 channel on C-type inactivation
作者:	林佳慧;Chia-Huei Lin
貢獻者:	神經科學與認知科學研究所碩士班
關鍵詞:	C-型鈍化;離子通道;Kv通道;C-type inactivation;ion channel;Kv channels
日期:	2009-07-28
上傳時間:	2016-01-08 15:24:09 (UTC+8)
出版者:	中國醫藥大學
摘要:	在處於興奮狀態的細胞可以藉由電壓門控鉀離子通道(Kv通道)的開啟使胞內鉀離子流出胞外而讓細胞膜再極化回到平靜的狀態，因此Kv通道在調控細胞的生理機轉上扮演了一個重要的角色。而大部分的Kv通道受到去極化的刺激而活化的同時，其對鉀離子的通透性也會逐漸下降，產生了慢速鈍化的現象(slow inactivation)，或稱為C-型鈍化，其產生原因是離子通道外側的過濾器壓縮或塌陷造成，但其分子機制至今尚未完全了解。目前已發現對慢速鈍化現象之探測藥物，仍有不足。本文發現從台灣衫萃取之化合物6β-acetoxy-7α-hydroxyroyleanone (AHR)可以加速Kv1.2通道之慢速鈍化之過程。在Kv通道的家族中，Kv1.2通道普遍表現於各種類型的神經元上，並且在持續去極化的過程中產生慢性鈍化的現象。本文利用cDNA轉染的方式讓Kv1.2通道大量表現在HEK293以及H1355細胞株上，以電生理的方式測量AHR對於Kv1.2電流之影響。實驗結果顯示，在細胞外側加入AHR可加速Kv電流C-型鈍化的現象，並且其作用必須是在通道開啟的階段，而自細胞內加入AHR並不能加速電流的C-型鈍化。而AHR抑制Kv1.2電流的IC50為17.7 μM，其阻斷作用並非直接堵住通道孔，對於Kv1.2的活化閥門亦沒有影響。將Kv1.2通道上的Val370突變為Gly370使通道的慢速鈍化產生缺陷，則AHR加速C-型鈍化的效果大幅減弱。再者，AHR對於不具有C-型鈍化特性的ATP-敏感型鉀離子通道(KATP channel)並沒有產生抑制電流的作用。這樣的結果說明了AHR阻斷Kv1.2電流的效果需依賴C-型鈍化的加速。根據上述，AHR可以有效的加速Kv通道的C-型鈍化又不影響活化閥門，因此可以作為一個探測慢速鈍化的工具。　　Voltage-gated K+ (Kv) channels repolarize excitable cells by providing a pathway for K+ efflux. Kv1.2 channels are present in many neurons and exhibit slow inactivation during continuous depolarization. Here we reported that 6β-acetoxy-7α-hydroxyroyleanone (AHR) could affect slow inactivation of Kv1.2 channels heterologously expressed in HEK293 cells. Extracellular AHR (50 μM) dramatically speeded up the slow decay of Kv currents and left-shifted the steady-state inactivation curve. Intracellular dialysis of AHR did not accelerate the slow current decay, suggesting that this drug acted extracellularly. AHR did not affect at all the kinetics and voltage-dependence of Kv1.2 channel activation. Blockade of currents by AHR required an open state of channels. AHR inhibited Kv1.2 currents with an IC50 of 17.7 μM. In addition, % block of Kv currents by AHR was independent of the intracellular K+ concentration. Therefore, AHR did not appear to directly block the outer channel pore; rather, results suggest that it drastically accelerated Kv channel slow inactivation. The effects of AHR on a slow inactivation defective Kv1.2 mutant (V370G) were much attenuated. In addition, AHR did not affect ATP-sensitive potassium (KATP) channels, which do not display slow inactivation. These results suggest that AHR effects are dependent on Kv channel slow inactivation. Thus, our results show that AHR selectively accelerate Kv slow inactivation without affecting the activation gate, and could be used as a pharmacological probe for Kv slow inactivation gate.
顯示於類別:	[神經科學與認知科學研究所] 博碩士論文

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