【作者】 刘波；

【导师】 杨祥良； 甘璐；

【作者基本信息】 华中科技大学 ， 生物制药工程， 2009， 博士

【摘要】 大电导钙依赖性钾通道(Large conductance calcium and voltage-dependentpotassium,BK_Ca通道)分布广泛,受氧化、磷酸化、胞内钙浓度等因素的调节,在血管、神经内分泌等生理、病理过程中发挥重要的作用。过氧化氢（Hydrogen peroxide,H₂O₂）属于活性氧（ROS）的重要成员,既是细胞损伤的因素,又是胞内的第二信使。PTEN具有负调控PI3K的作用,能调节细胞的大小、生长、凋亡、迁移等,是当前研究的热点之一。本研究以膜片钳技术为基础,结合分子、细胞生物学技术,从H₂O₂对BK_Ca通道的作用,PTEN对H₂O₂诱导的BK_Ca通道激活的作用和PTEN磷脂酶活性对H₂O₂诱导的BK_Ca通道激活的调节等三个方面阐释PTEN、BK_Ca通道和H₂O₂之间的相互关系,旨在阐明H₂O₂激活BK_Ca通道、舒张血管的分子机制,为进一步探索氧化诱发的心血管疾病机制提供基础。本文以鼠源BK_Ca通道α亚基（Slo）基因（mSlo）在HEK 293细胞表达,运用inside-out和cell-attached两种不同的膜片钳记录模式检测BK_Ca通道电流,研究H₂O₂对BK_Ca通道的作用。在inside-out模式下,发现以Oμmol Ca²⁺电极外液灌流时H₂O₂能明显抑制BK_Ca通道电流,且DTT对抑制的电流有部分恢复作用。而以10μmolCa²⁺电极外液灌流时,H₂O₂、DTT对BK_Ca通道电流均无明显作用。说明H₂O₂对BK_Ca通道的作用主要依靠氧化还原效应影响通道对Ca²⁺的敏感性。在cell-attached模式下,我们发现H₂O₂能显著、快速增强BK_Ca通道活性,表明H₂O₂刺激BK_Ca通道能产生两种完全不同的作用。而巯基特异性的氧化剂DTNB在两种模式下均抑制BK_Ca通道活性,说明在cell-attached模式下H₂O₂和DTNB对BK_Ca通道的作用机制不同。H₂O₂对BK_Ca通道的影响受作用方式和H₂O₂浓度的影响。为了研究PTEN在H₂O₂激活BK_Ca通道电流中的作用,我们构建了PTEN-Tdimer2的嵌合基因克隆表达质粒。通过将PTEN_wt与mSlo或mSlo和hβ1在HEK 293细胞共转染,在cell-attached模式下记录BK_Ca通道电流,发现在PTEN_wt过表达的细胞上,不管有无β1的表达,H₂O₂刺激初始10 min都不会明显增加BK_Ca通道的活性。此结果不仅说明PTEN能抑制H₂O₂诱导的BK_Ca通道激活,而且证实β1亚基的存在并不影响PTEN的作用。进一步以PI3K抑制剂LY294002或Wortmannin孵育细胞,在cell-attached模式下记录电流,发现H₂O₂刺激初始10 min也并不明显增强BK_Ca通道活性,说明PI3K参与了H₂O₂诱导的BK_Ca通道激活过程。为进一步阐明PI3K/PTEN通路对H₂O₂诱导BK_Ca通道激活的生理、病理学意义,我们观测了LY294002对H₂O₂诱导的血管舒张的作用,发现LY294002能明显抑制H₂O₂诱导的血管舒张,但当BK_Ca通道阻断剂Iberiotoxin存在时,LY294002的抑制作用消失,说明PI3K是通过BK_Ca通道的调节参与H₂O₂诱导的血管环舒张作用。为了进一步阐明PTEN对H₂O₂激活BK_Ca通道的抑制作用机制,将mSlo与磷脂酶活性缺失的PTEN_C124S或PTEN_G129E在细胞共表达,发现H₂O₂诱导的BK_Ca通道激活并没有被抑制,说明PTEN对H₂O₂诱导的BK_Ca通道激活的抑制作用主要是通过其磷脂酶活性实现的。针对PTEN蛋白的磷脂酶活性,我们应用Western Blot分析了H₂O₂刺激PTEN_wt、PTEN_C124S、PTEN_G129E过表达细胞产生的p-AKT表达变化,发现H₂O₂刺激初期并不影响PTEN对p-AKT表达的抑制作用,但是在刺激30 min后p-AKT的表达增加,可能与H₂O₂对PTEN的氧化有关。通过激光共聚焦扫描显微镜检测H₂O₂刺激PTEN_wt、PTEN_C124S、PTEN_G129E过表达细胞后胞内钙浓度变化,发现与对照组细胞相比,在PTEN_wt过表达细胞中H₂O₂引起胞内钙浓度增加明显降低,时间上显著延长,而在磷脂酶活性缺失的PTEN_C124S和PTEN_G129E过表达细胞中却没有明显变化。说明H₂O₂诱导的BK_Ca通道激活是受PTEN的磷脂酶活性调节,p-AKT、钙浓度的变化可能是作用于BK_Ca通道的重要途径。更多还原

【Abstract】 Large conductance calcium and voltage-dependent potassium(BK_Ca) channels are ubiquitously distributed and play a pivotal role in physiological and pathological condition, such as vascular tone,neuronal secretion.In general,the BK_Ca channel function is regulated by oxidation,phosphorylation,intracellular calcium concentration and others. H₂O₂ is one of members of Reactive Oxygen Species（ROS） and also plays an important role in physiological and pathological processes.It not only causes oxidative damages to cellular function,but also acts as a second messenger.PTEN negatively controls the activity of PI3K to regulate cellular function,such as cell size,cell growth,apoptosis and migration.In this study,the relationship between H₂O₂,BK_Ca channels and PTEN was elucidated by studying the effect of H₂O₂ on BKca channel activity,PTEN and its lipid phosphatase activity on H₂O₂- induced BK_Ca channel activation.This study is to prove the molecular mechanism of H₂O₂-induced BK_Ca channel activation or vasodilation and provide evidences for discovering the mechanism of oxidation induced vascular diseases.In this study,BK_Ca channel encoded by mouse Slo was heterologously expressed in HEK 293 cells and the typical BKc,channel currents were recorded to study the effects of H₂O₂ on BK_Ca channel in inside-out and cell-attached configurations.In inside-out configurations,our results showed that BK_Ca channel current was inhibited after exposure to H₂O₂ at 0μmol Ca²⁺ bath solutions,and the inhibition could be reversed partially by DTT.However,the effects of H₂O₂ and DTT on BK_Ca channel were abolished at 10μmol Ca²⁺ bath solutions.These data suggested that the effect of H₂O₂ on BK_Ca channel is in a voltage and Ca²⁺-dependent manner and H₂O₂ could decrease the Ca²⁺ sensitivity of channels by thiol oxidation.In cell-attached configuration,we found that NP_o of single BK_Ca channels were significantly increased after application of H₂O₂.However,the specific sulfhydryl oxidant DTNB inhibited BK_Ca channel NP_o in two different configurations,suggesting that the regulation of BK_Ca channels by DTNB and H₂O₂ is different.It also elucidate that the effects of H₂O₂ on BK_Ca channels depend on different action modes and H₂O₂ concentrations.To explore the effect of PTEN on H₂O₂-induced BK_Ca channel activation,the PTEN-Tdimer2 plasmids were constructed in our studies.The PTEN-Tdimer2 plasmids and mSlo in the presence or absence of hβ1 were coexpressed in HEK 293 cells and the typical BK_Ca channel currents were recorded in cell-attached configuration.Contrary to H₂O₂-induced BK_Ca activation,the BK_Ca channel currents and conductance in the absence or presence of hβ1 were not changed in PTEN overexpressing cells during the initial 10 min treatment with H₂O₂.It not only suggested that PTEN inhibited H₂O₂-induced BK_Ca channel activation,but also verified that the inhibition of PTEN was not affected byβ1. Similarly,the typical BK_Ca channel currents were recorded in cell-attached configuration when HEK293 cells expressing mSlo channels were pre-incubated with the PI3K inhibitor LY294002 or Wortmannin for 1 hour.The results showed that the effect of LY294002 or Wortmannin on H₂O₂-induced BK_Ca channel activation was same as PTEN,suggesting that PI3K activity is involved in H₂O₂-induced BK_Ca channel activation.In order to elucidate the effects of PI3K/PTEN pathway on H₂O₂- induced BK_Ca channel activation in physiological and pathological processes,H₂O₂-induced relaxation of isolated rat thoracic aortas was investigated.The results showed that H₂O₂-induced relaxation of aortal rings was decreased by PI3K inhibitor LY294002.However,the effect of LY294002 was abolished in the presence of BK_Ca channel inhibitor Iberiotoxin.It suggested that PI3K activity is involved in H₂O₂-induced relaxation of aorta tings via BK_Ca channel activation.To further elucidate the mechanism of PTEN on the inhibiton of H₂O₂-induced BK_Ca channel activation,mSlo was coexpressed with catalytically inactive PTEN_C124S/ PTENG_G129E mutants that lack lipid phosphatase activity.We found that the mutants produced no regulation on the H₂O₂-induced BK_Ca channel activation,suggesting that PTEN regulates H₂O₂-induced BK_Ca channels activation by acting as a phosphatidylinositol 3-phosphatase.Meanwhile,the p-AKT expression in PTEN_wt, PTEN_C124S and PTEN_G129E overexpressing cells after H₂O₂ application was investigated by Western Blot analysis.We found that the p-AKT expression inhibited by PTEN was not changed after H₂O₂ addition for 10 min.However,the inhibition was changed after H₂O₂ addition for 30 min.The results suggested that the change of p-AKT expression may be through oxidation of PTEN by H₂O₂.The cytoplasmic free calcium concentrations ([Ca²⁺]_i) in PTEN_wt,PTEN_C124S and PTEN_G129E overexpressing cells afeer H₂O₂ application were also detected by laser scanning confocal microscopy.We found that the increase of[Ca²⁺]_i induced by H₂O₂ was also inhibited in PTEN_wt overexpressing cells. However,the results were not detected in PTEN_C124S and PTEN_G129E overexpressing cells. These data suggested that the lipid phosphatase activity of PTEN is involved in H₂O₂-induced BK_Ca channel activation,p-AKT and[Ca²⁺]_i may be important downstream signal molecules for H₂O₂-induced BK_Ca channel activation.更多还原