【作者】 赵志英；

【导师】 张海林；

【作者基本信息】 河北医科大学 ， 药理学， 2008， 博士

【摘要】 内向整流钾离子通道(Inwardly Rectifying K Channel, Kir)是在各种组织中广泛分布的一种钾通道,因其电压电流关系中所具有的内向整流特性而得名。Kir2.0钾通道的特征就是具有很强的内向整流特性,这种整流作用有利于维持细胞的静息电位使之接近钾离子的平衡电位并参与动作电位复极化过程。Kir2.3是Kir2.0家族中的重要一员,它具有内向整流性钾离子通道的普遍特征:(1)钾离子向内流较向外流容易。(2)内向整流性钾离子通道的功能均依赖于膜磷脂PIP2。(3)内向整流性钾离子通道可以被多种因素调节,各种因素对不同的Kir通道调节情况不同。(4)Kir具有共有的基本结构,即:N末端起始于细胞内,经两次跨膜折叠后,C末端结束于细胞内。现已知蛋白激酶C(PKC)的激活可以对Kir通道的功能(如Kir2.3、Kir3.x和Kir6.x)产生抑制作用。PKC是依赖钙、磷脂(phospholipid, PL)和二酰基甘油(diacylglycerol, DAG)激活的丝氨酸/苏氨酸(Ser/Thr)蛋白激酶的多基因超家族,通过催化多种蛋白质上Ser/Thr磷酸化,调节细胞的代谢、生长、增殖和分化。佛波醇-12-豆蔻酰-13-乙酸(PMA)是PKC的激活剂。PMA通过激活PKC而对内向整流钾离子通道产生抑制作用,但是对于不同的内向整流钾离子通道及其所表达细胞的不同,PMA产生的作用却很不相同。PMA抑制Kir2.3通道但对Kir2.1通道没有作用。Kir2.3通道表达在爪蟾卵母细胞时,对于PMA引起的抑制作用是非常敏感的,但Kir2.3通道表达在CHO细胞时,应用PMA却看不到通道功能的抑制。虽然目前PMA通过激活PKC而对Kir2.3抑制的相关研究较多,但是有关PKC抑制Kir2.3的机制却有很多疑点,因此本实验对其机制进行了进一步探讨。植物雌激素(genistein)是一种广泛应用的酪氨酸激酶抑制剂,在研究通道的酪氨酸磷酸化调节通路时广泛应用。在实验中我们发现genistein能够抑制Kir2.3通道电流,因此本实验对genistein抑制Kir2.3通道电流的机制也进行了细致研究。一、佛波醇酯通过激活蛋白激酶C抑制Kir2.3通道目的:研究表达在非洲爪蟾卵母细胞中的Kir2.3通道的电生理学特性,同时研究PKC激活剂PMA对Kir2.3通道功能的调节作用。方法:(1)cRNA的体外转录:Kir2.3通道克隆在质粒载体pGEMHE中。将质粒线性化后,体外转录Kir2.3通道的cRNA。(2)爪蟾卵母细胞的分离与注射:在爪蟾冰冻麻醉状态下,取出卵母细胞。在含2mg/ml胶原酶的OR2液中振荡消化1.5-2小时。待细胞消化为单细胞后清洗细胞,进行Kir2.3通道cRNA注射,注射后的细胞在含2.5 mM丙酮酸钠的ND96中18℃培养。(3)双电极电压钳(two-microelectrode voltage clamp,TEVC)方法检测Kir2.3通道在卵母细胞中的表达情况和电流特性以及PMA对Kir2.3电流的影响。结果:(1)分子生物学实验结果。用琼脂糖凝胶电泳分析结果。质粒电泳显示两条或两条以上的泳带;而线性化完全的质粒DNA电泳显示一条泳带。体外转录RNA并进行电泳分析,可得到一条清晰的RNA条带,条带均匀整齐无拖尾现象,经RNA定量,RNA浓度为200 ng/μl。(2)Kir2.3通道电流特性。用TEVC方法检测Kir电流。钳制电压为-90 mV逐渐变至+90 mV,灌流液为ND96K液,从Kir2.3电流的电压电流曲线可以看出Kir通道的内向电流较大且随着电压增加而增大,而外向电流很小且没有明显的电压依赖性。Kir通道的内向与外向电流转换时的电流为零,此时的电位为反转电位,反转电位的位置与细胞外钾离子浓度有关,在ND96K溶液灌流的情况下,其反转电位接近0 mV。用钾通道的阻断剂BaCl2可以阻断Kir电流。(3)PMA抑制Kir2.3通道电流。在ND96K中加入100 nM PMA,能够明显抑制Kir2.3通道的电流,抑制率为46.3±5.2%。PMA的抑制作用起效较慢,在给药后大约2到5分钟开始看到电流的减小,给药后大约5到10分钟电流的抑制作用到达稳态并不再变化。PMA抑制Kir2.3通道的电流作用是不可逆的,在停用PMA并用对照液长期冲洗的情况下,被抑制的Kir2.3通道电流无任何恢复。电流电压关系曲线反映PMA没有改变Kir2.3通道的内向整流特性。PMA对Kir2.3通道电流的抑制具有浓度依赖性,采用Hill方程进行拟合得到半数最大抑制浓度为17.7±0.13 nM。(4)PMA通过激活PKC抑制Kir2.3通道电流。PMA是公认的PKC激活剂,因此PMA抑制Kir2.3通道电流很可能通过激活PKC。用PKC的另一个激活剂PDBu 100 nM,也能抑制Kir2.3通道电流,抑制率为40.8±3.4%。4α-PMA是PMA的无效结构类似物,100 nM 4α-PMA对Kir2.3通道电流无作用,抑制率为0.6±0.0%。用PKC的特异性抑制剂Bisindolylmalimide(5μM)预处理卵母细胞2小时,并在Bisindolylmalimide持续存在的情况下再给予PMA明显降低了PMA对Kir2.3通道电流的抑制程度,抑制率为11.8±2.0%。上述结果说明PMA是通过激活PKC途径对Kir2.3通道电流产生抑制作用的。结论:以爪蟾卵母细胞为表达系统,将体外转录得来的RNA以微注射方式注入卵母细胞可表达Kir2.3通道。用双电极电压钳方法可观察卵母细胞内表达Kir通道的电生理特性。Kir2.3通道电流具有内向整流特性,Ba2+可通过与Kir通道的孔区结合而阻断Kir通道钾电流。PMA作为PKC的激活剂可以浓度依赖性抑制Kir2.3通道电流。PKC的抑制剂可以阻断PMA的抑制作用。二、PKC抑制Kir2.3通道的机制研究目的:PMA通过激活PKC抑制Kir2.3通道,但对其具体机制还有很多争议。本实验结合利用分子生物学和电生理学方法,对其机制进行细致探讨。方法:(1)分子生物学方法:Kir2.3,Kir2.1和一些突变体通道均克隆于在质粒载体pGEMHE中,转录为cRNA并纯化后注射入卵母细胞。嵌合体通道采用重叠延伸聚合酶链式反应技术构建。点突变采用美国stratagene公司的site-directed mutagenesis kit。所有的序列均经基因测序验证。(2)细胞膜蛋白的提取与免疫印迹:将表达通道的卵母细胞匀浆后,4℃,5000 g,离心5min。取上清,4℃,20万g,离心1h,弃上清,用裂解缓冲液重悬沉淀(1μl/细胞)。将提取的膜蛋白进行十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE),电转移至PVDF膜上,封闭后,用特异性一抗和荧光标记的二抗进行检测,显色用Odyssey9120双色红外激光成像系统仪器扫描。(3)细胞表面膜蛋白的提取:应用生物素标记法提取细胞表面膜蛋白,表达通道的卵母细胞在PMA处理后,用甲醛固定,NHS-SS-biotin生物素孵育过夜,匀浆后加入Neutravidin-linked beads,洗涤珠子,进行免疫印迹试验。(4)免疫共沉淀:将提取的膜蛋白加入特异性一抗过夜,加入protein-G beads,洗涤珠子,进行免疫印迹试验。(5)免疫细胞化学:表达通道的卵母细胞用甲醛固定后,用PBS配制含3%BSA和0.2% tritonX-100的封闭液进行封闭,加入特异性一抗和荧光标记的二抗,用激光扫描共聚焦显微镜观察标本显色情况。(6)放射自显影:表达通道的卵母细胞用同位素32P正磷酸盐孵育过夜,提取膜蛋白,免疫共沉淀,SDS-PAGE并电转移至PVDF膜上,压胶片,进行放射自显影。(7)电生理学方法:按前述方法用双电极电压钳记录电流。结果:(1)PKC抑制Kir2.3通道电流与PIP2有关。PMA可以抑制表达于非洲爪蟾卵母细胞的Kir2.3通道电流,抑制率为46.3±5.2%,对Kir2.1通道电流没有作用;PMA对Kir2.3(I213L)的抑制减弱,抑制率为16.9±3.1%,而PMA对Kir2.1(L222I)的抑制作用增强,抑制率为8.9±1.7%。前期实验结果表明:Kir2.3通道与PIP2亲和力要弱于Kir2.1通道与PIP2亲和力,而Kir2.3(I213L)明显的增强了Kir2.3通道与PIP2的亲和力,Kir2.1(L222I)则明显减弱了Kir2.1通道与PIP2的亲和力。结合这些结果,我们的上述实验结果表明PKC抑制Kir2.3通道电流和通道与PIP2亲和力有关。(2)PKC抑制Kir2.3通道电流和通道第53位与213位氨基酸有关。Kir2.3和Kir2.1通道属于同一个Kir2.0亚家族,他们的氨基酸序列具有58%的同源性,构建Kir2.3和Kir2.1通道的嵌合体极有可能产生功能性通道。Kir通道的结构有两个跨膜区(M1,M2)和位于胞内的一个较短的氨基末端(N)和一个较长的羧基末端(C)。构建Kir2.3和Kir2.1通道的嵌合体,通道命名原则如下:数字1和3分别代表Kir2.1和Kir2.3通道,字母N代表氨基末端,字母C代表羧基末端,字母P代表两个跨膜区和孔区。将嵌合体通道表达于卵母细胞,给予100 nM PMA,发现PMA抑制N3P1C3,抑制率为42.8±3.8%,对N1P3C1无作用,这表明Kir2.3通道的氨基末端和羧基末端在PKC对Kir2.3通道的抑制中起决定作用。PMA抑制N1P3C3,抑制率为12.1±2.0%;PMA抑制N3P1C1,抑制率为33.6±2.5%;PMA可以抑制N3P3C1,抑制率为25.3±4.1%;PMA也抑制N1P1C3,抑制率为13.9±2.7%。这些结果表明Kir2.3通道的氨基末端和羧基末端都在PKC对Kir2.3通道的抑制中起决定作用,二者缺一不可。为了进一步找到PKC的作用位点,我们构建了两个突变体:Kir2.3(T53I)和Kir2.1(I79T)。结果表明PMA抑制Kir2.3(T53I),抑制率为36.3±3.1%,与PMA对Kir2.3的抑制有显著性差异(p<0.05),PMA不能抑制Kir2.1(I79T)。我们在上述结果已经表明Kir2.3第213位异亮氨酸在PKC对Kir2.3通道的作用中很重要,因此我们又进一步构建了两个双突变通道:Kir2.3(T53I,I213L)和Kir2.1(I79T,L222I)。给予100 nM PMA,发现PMA不能抑制Kir2.3(T53I,I213L),抑制率仅为4.2±9.3%,同PMA对Kir2.1的抑制无差异(p>0.05);PMA可以抑制Kir2.1(I79T,L222I),抑制率为13.8±1.5%,与PMA对Kir2.1的抑制有显著性差异(p<0.05)。这个结果表明Kir2.3通道第53位苏氨酸与第213位异亮氨酸是PKC的作用位点。(3)Kir2.3通道第53位与213位氨基酸也是决定通道被其他因素调节的关键位点。我们前期结果表明:a:叠氮化钠(Azide)和碳酸氢钾(KHCO3)都能使细胞内pH降低,由此抑制Kir2.3通道电流,而对Kir2.1通道电流没有作用;b:渥漫青霉素(Wortmannin)(PI-4K(磷脂酰肌醇-4激酶)抑制剂,通过抑制PI-4K而阻断PIP2合成)可抑制功能依赖PIP2的钾通道功能,抑制程度与通道和PIP2亲和力负相关。我们进一步观察了上述因素对Kir2.3和Kir2.1及突变体功能的影响,并与PMA对通道功能的影响结果进行比较。灌流Azide(3 mM)或KHCO3-ND96K 2min,或10μM Wortmannin孵育表达不同通道的卵母细胞2小时后,记录电流大小,同未孵育的表达相同通道的卵母细胞电流大小进行比较。结果表明Azide和KHCO3及Wortmannin等调控因子对Kir2.3及这些突变体的作用与PMA的作用相一致。即:与Kir2.3相比,Kir2.3(T53I)、Kir2.3(I213L)和Kir2.3(T53I,I213L),都对这些调控因子的反应减弱,且减弱的程度以Kir2.3(T53I,I213L)为最大;而与Kir2.1相比,Kir2.1(I79T)、Kir2.1(L222I)、Kir2.1(I79T,L222I)都增强了对这些调控因子的反应,且增强的程度以Kir2.1(I79T,L222I)为最大。结合以上(2)、(3)两方面的结果,我们认为与其他因素一样,PMA很可能是通过影响通道与PIP2的相互作用而影响Kir2.3功能。(4)PKC抑制Kir2.3通道电流与通道蛋白磷酸化无关。我们在实验中发现Kir2.3通道的氨基末端在PKC对Kir2.3的抑制作用中相对更重要,在通道氨基末端只有两个可能的磷酸化位点:S36和S39,我们把它们同时突变为为甘氨酸后发现Kir2.3 (S36G,S39G)仍然能被PMA抑制,抑制率为56.0±2.6%,与Kir2.3相比无差异。Okadaic acid (OA)是蛋白磷酸酶的抑制剂,若PKC使Kir2.3通道蛋白磷酸化从而抑制通道电流,那么蛋白磷酸酶活性的抑制则应该使PKC抑制Kir2.3通道电流程度增加。然而,向卵母细胞注射20 nM OA没有改变PMA对Kir2.3的抑制,抑制率为42.2±1.7%。我们又直接观察了Kir2.3通道蛋白的磷酸化情况。用32P孵育卵母细胞后,提取卵母细胞膜蛋白进行放射自显影。结果显示在Kir2.3通道蛋白量一致的情况下,PMA处理前后Kir2.3通道蛋白放射自显影的量化分析无差异(p>0.05)。我们同时以PKC本身作为磷酸化反应的阳性对照,PMA处理后PKC放射自显影程度明显增强。这些结果表明PMA可以激活并磷酸化PKC,但PKC没有使Kir2.3通道蛋白磷酸化,也就是说PKC抑制Kir2.3通道电流与通道蛋白磷酸化无关。(5)PKC抑制Kir2.3通道电流可能与通道蛋白转位有关。Phalloidin能够牢固地结合于细胞的F actin,使之不能解聚,从而使肌动蛋白变得非常僵硬;Latrunculin A能够破坏肌动蛋白网络结构,抑制微丝蛋白调控的过程。Phalloidin和Latrunculin A因而能影响蛋白转运过程。结果表明Phalloidin 10μM注射卵母细胞2小时后,降低了PMA抑制Kir2.3电流的作用,PMA的抑制率降为7.5±1.6%;Latrunculin A 1μM预孵育卵母细胞2小时后,PMA对Kir2.3通道电流抑制作用也降低,抑制率为31.3±3.3%。上述结果提示PKC抑制Kir2.3通道电流可能与通道蛋白转位有关。采取细胞免疫化学的方法,结合激光扫描共聚焦显微镜对细胞膜Kir2.3进行了定量分析,发现卵母细胞膜Kir2.1在PMA处理前后荧光强度量化分析无差异(p>0.05);而卵母细胞膜Kir2.3在PMA处理后荧光强度下降到51.1±8.9%,同对照相比有显著性差异(p<0.05)。这说明在PMA作用后细胞膜上Kir2.3通道蛋白数量减少,通道极有可能进行了内吞。最后我们采用生物素法特异性提取细胞跨膜蛋白,结果显示PMA作用后细胞膜上Kir2.3通道蛋白数量少于PMA作用前,免疫印迹结果表明条带灰度下降到61.2±10.4%。这也进一步提示PKC抑制Kir2.3通道电流可能与通道蛋白转位有关。结论:PKC抑制Kir2.3通道电流可能与通道和PIP2亲和力有关, Kir2.3通道的第53位与213位氨基酸是影响Kir2.3通道被包括PMA在内的多种调节因素调节的关键位点。PKC抑制Kir2.3通道电流与通道蛋白磷酸化无关。PKC抑制Kir2.3通道电流可能与通道蛋白转位有关。Kir2.0家族钾离子通道与膜PIP2反应特征可能也决定其在细胞内和细胞膜间的转运过程。三、植物雌激素抑制Kir2.3通道电流的分子机制研究目的:观察酪氨酸激酶抑制剂植物雌激素genistein对表达在卵母细胞和HEK293细胞中的Kir2.3和Kir2.1通道的作用,并进一步研究genistein的作用机制。方法:(1)分子生物学方法:Kir2.3,Kir2.1和一些突变体通道按前述方法表达于非洲爪蟾卵母细胞。(2)细胞膜蛋白的提取与免疫印迹和免疫共沉淀实验如前述。(3)电生理学方法:按前述方法用双电极电压钳记录电流。用脂质体转染的方法在HEK293细胞表达Kir2.3和Kir2.1通道,用全细胞膜片钳记录通道电流。结果:(1)Genistein特异性抑制Kir2.3电流。Genistein特异性抑制Kir2.3电流,100μM genistein对Kir2.3抑制率为44.8±2.1%。对Kir2.1和Kir3.4*(Kir3.4-S143T)没有作用。在卵母细胞,Genistein浓度依赖性抑制Kir2.3电流,半数最大抑制浓度为16.9±2.8μM;在HEK293细胞,半数最大抑制浓度为19.3±3.2μM。在卵母细胞,Genistein起效很快,在90.7±4.2秒就可到达平台期,并且抑制作用易于被洗脱。Genistein的这种抑制作用没有电压依赖性。(2)Genistein对Kir2.3的抑制作用不是通过改变通道的酪氨酸磷酸化状态起作用。Daidzein是genistein的无效结构类似物;tyrphostin 23是Genistein的功能类似物,但与genistein结构不同,他们都不能够抑制Kir2.3电流。Vanadate,酪氨酸磷酸酶的抑制剂,也没有改变genistein对Kir2.3电流的抑制作用。应用抗酪氨酸磷酸化抗体PY99进行免疫共沉淀实验,结果表明genistein没有改变通道的酪氨酸磷酸化状态。这些结果都说明genistein不是通过抑制酪氨酸激酶而抑制Kir2.3电流。(3)Genistein对Kir2.3的抑制作用不是通过改变通道与PIP2的亲和力起作用。Kir2.3(I213L)明显地增强了Kir2.3通道与PIP2的亲和力,但genistein对其抑制作用同Kir2.3相比没有明显差异(p>0.05),100μM genistein对Kir2.3(I213L)抑制率为47.5±2.6%。Kir3.4与PIP2有较弱的亲和力,但genistein对其没有作用。(4)Kir2.3通道的跨膜区和孔区在genistein抑制作用中起重要作用。构建Kir2.3与Kir2.1的嵌合体通道,表达于卵母细胞,灌流genistein,发现通道的跨膜区和孔区在genistein抑制作用中起重要作用,二者缺一不可。结论:植物雌激素(genistein)特异性抑制Kir2.3通道电流,这种抑制作用没有改变通道的酪氨酸磷酸化状态,通道的跨膜区和孔区在genistein抑制作用中起重要作用。本研究对于研究Kir2.3通道的结构与功能的关系和研发Kir2.3通道特异性的调节剂有重要意义。总结1用双电极电压钳方法可观察卵母细胞内表达Kir通道的电生理特性。Kir2.3通道电流具有内向整流特性,Ba2+可阻断Kir通道的钾电流。PMA可以浓度依赖性抑制Kir2.3通道电流。PKC的抑制剂可以阻断PMA的抑制作用。PMA是通过激活PKC而抑制Kir2.3通道电流的。2 PKC抑制Kir2.3通道电流与通道和PIP2亲和力有关,Kir2.3通道的第53位与213位氨基酸是影响Kir2.3通道被包括PMA在内的多种调节因素调节的关键位点。PKC抑制Kir2.3通道电流与通道蛋白磷酸化无关。PKC抑制Kir2.3通道电流可能与通道蛋白转位有关。Kir2家族钾离子通道与膜PIP2反应特征可能也决定其在细胞内和细胞膜间的转运过程。3植物雌激素genistein特异性抑制Kir2.3通道电流,这种抑制作用没有改变通道的酪氨酸磷酸化状态,通道的跨膜区和孔区在genistein抑制作用中起重要作用。本研究对于研究Kir2.3通道的结构与功能的关系和研发Kir2.3通道特异性的调节剂有重要意义。更多还原

【Abstract】 Inwardly Rectifying K+ Channels (Kir) are a group of potassium channels extensively distributed in many kinds of tissues. It is known for its inwardly rectifying property. Kir2.0 is characteristic for its strong inward rectifying property. This property of Kir2.0 confers it the role to help to maintain the resting membrane potential of the cell by keeping the membrane potential near to the K+ equilibrium potential. Kir 2.0 is also involved in the process of repolarization of membrane action potential. Kir2.3 is an important member of Kir2.0 family. It shares the common characters of inward rectifying potassium channel: (1) K+ influx is easier than its efflux. (2) The channel function depends on membrane phospholipid—specifically PIP2. (3) The channel is targets of modulation of many types of factors. (4) All Kir channels share the same basic structure. The N-terminus of the channel starts from the interior of the cell, and after twice transmembrane foldings, the C-terminus also ends in the interior of the cell.It is known that PKC activation can lead to the inhibition of some Kir channel (Kir2.3、Kir3.x and Kir6.x) functions. PKC is a Ser/Thr protein kinase whose activation depends on calcium, phospholipid and diacylglycerol. PMA is an activator of PKC. It has been reported that PMA has an inhibitory effect on some Kir channels. However, the effects of PMA on Kir channels are variable depending on the channel type studied or the tissues they expressed. It has been reported that PMA inhibits Kir2.3 channel, and that Kir2.1 is not sensitive to PMA. Kir2.3 channels are very sensitive to the inhibitory effect of PMA when it is expressed in Xenopus oocytes. However no inhibition can be seen when Kir2.3 channels are expressed in CHO cells. Thus although there have some reports about PMA inhibition of Kir2.3, the underlying mechanism is not fully elucidated. This study will focus on the mechanism of PMA action on Kir2.3 channels. We also studied the effects of genistein on Kir2.3 channels. Genistein is a widely used inhibitor of protein tyrosine kinase. When we studied the tyrosine phosphorylation of Kir2.3 channels, we found that genistein inhibited Kir2.3 currents. We decided to have a through investigation into the mechanism of genistein action.1. PMA inhibited Kir2.3 currents through PKC pathway.Aim: To express Kir2.3 channels in Xenopus oocyte and study the properties of Kir2.3 channels. To study the effects of PMA, an activator of PKC, on Kir2.3 currents.Methods: (1) Transcription of Kir2.3 channels in vitro. cDNA coding Kir2.3 was inserted into the pGEMHE vector. The cRNA was transcribed after linearizing the DNA constructs. (2) Preparation and microinjection of oocytes. Oocytes from adult female frogs were used. Xenopus oocytes were treated with 2 mg/ml collagenase (Type II, Sigma) in the OR2 solution for 90 min. After washes with the OR2 solution, the oocytes were incubated at 18°C in the ND96 solution and injected. (3) Currents recording. Whole cell currents were recorded under two-electrode voltage clamp. PMA was applied in the bath solution of ND96K.Results: (1) Molecular biology. A restriction enzyme was chosen to cut the circular plasmid and the linearized plasmid DNA were analyzed by agarose electrophoresis. The plasmid DNA showed more than one bands in the electrophoresis, whereas the linearized DNA showed only one band. The band of cRNA in vitro transcribed is uniform, clean, and has no sign of degradation. (2) The property of Kir2.3 currents. Kir currents can be recorded using two-electrode voltage clamp method. A series of ramp voltage-clamp from -90 mV to +90 mV was used to evoke the Kir2.3 currents. Oocytes were constantly perfused with ND96K. ND96 was used to reduce most of the Kir currents at -80 mV. The current-voltage relationship (I-V) curve of Kir2.3 channel currents showed the significant inward rectifier property. When ND96K solution was used, the reversal potential of Kir2.3 currents was around 0 mV. Ba2+ blocked these currents effectively. (3) PMA inhibited Kir2.3 currents. Bath application of 100 nM PMA inhibited the currents amplitude by 46.3±5.2%. The inhibition developed relatively slowly with a delay for 2-5 min, and reached a stable level within 5-10 min. Upon washing out of PMA for 10-20 min, the Kir2.3 currents showed no sign of recovery. PMA did not alter the rectification property of Kir2.3 currents. PMA concentration-dependently inhibited the currents of Kir2.3 channels expressed in Xenopus oocytes with an IC50 of 17.7±0.13 nM. (4) PMA inhibited Kir2.3 currents through PKC pathway. PDBu, another activator of PKC, also inhibited the Kir2.3 currents by 40.8±3.4%; 4α-PMA was an inactive analog of PMA and had no inhibition on the channel currents. Bisindolylmalimide, a PKC blocker, almost abolished the PMA effect. These results showed that PMA affected Kir2.3 currents by PKC activation.Conclusion: Kir2.3 channels could be expressed in Xenopus oocytes by microinjecting the transcribed cRNA. By using two-electrode voltage clamp, the currents through Kir2.3 could be observed with the characteristics of inwardly rectification. Ba2+ blocked Kir2.3 currents. PMA, an activator of PKC, concentration-dependently inhibited the currents of Kir2.3 channels. PKC blocker prevented the effect of PMA.2. The study on the mechanism of PKC inhibition of Kir2.3 currents.Aim: To explore the mechanism of PKC inhibition of Kir2.3 currents by the methods of molecular biology and electrophysiology.Methods: (1) Molecular biology. cDNA coding Kir2.3, Kir2.1 and the mutants was inserted into the pGEMHE vector. Chimeric constructs were prepared by the overlap extension polymerase chain reaction. Site-specific mutants were produced with a QuickChange kit (Stratagene, La Jolla, CA). Orientation of the constructs and correct mutants were confirmed with DNA sequencing. (2) Membrane protein extraction and Western blotting. The injected or uninjected oocytes were lysed with a homogenizer. Homogenate was spun at 5000 g for 5 min at 4°C and the resulting supernatant was further spun at 200,000 g for 60 min. The pellet containing the membrane fraction was resuspended in the lysis buffer (1μl/oocyte). The membrane protein were resolved by 10% SDS-PAGE and transferred to PVDF membranes. Standard Western blottings were performed. Specific primary antibody and IR dye680-conjugated secondary antibody were used. Blots were then scanned using the Odyssey Infrared Imaging System (LiCor, Lincoln, NE). (3) Detection of proteins on the surface of oocytes. Immediately after pretreament with PMA, oocytes expressing Kir2.3 channels were fixed in 4% paraformaldehyde, and incubated with NHS-SS-biotin at 4°C overnight. After being homogenized and centrifuged, Neutravidin-linked beads were added to the supernatant. The beads were washed and western blottings were performed. (4) Immunoprecipitation. The pellet was resuspended in the lysis buffer, and the primary antibodies were added. After rotating at 4°C overnight, protein G beads were added. After washing the beads, western blottings were performed. (5) Immunocytochemistry. Immediately after pretreament with PMA, oocytes expressing Kir2.3 channels were fixed in 4% paraformaldehyde, and were placed in blocking buffer. The primary antibodies were added and incubated overnight. Fluorescein isothiocyanate secondary antibody was used to recognize the primary antibody and visualized using the laser scanning confocal microscopy. (6) autoradiography. Oocytes expressing Kir2.3 channels were incubated with 32P (0.5 mCi/ml) overnight. After pretreament with PMA, the membrane protein were extracted. Kir2.3 protein were immunoprecipited, and transferred to PVDF membrane. The membranes were subjected to autoradiography using Kodak film at -80°C. (7) Electrophysiology: The currents were record as described above.Results: (1) Characteristic interactions with PIP2 determined the inhibition of Kir2.3 currents by PKC. 100 nM PMA inhibited the currents of Kir2.3 channels expressed in Xenopus oocytes by 46.3±5.2%, whereas Kir2.1 is not sensitive to PMA inhibition. PMA had a weaker inhibition on Kir2.3(I213L) of 16.9±3.1% than on Kir2.3 and a stronger inhibition on Kir2.1(L222I) of 8.9±1.7% than on Kir2.1. It had been shown that Kir2.3 had a weaker interaction with PIP2 than Kir2.1. Kir2.3(I213L) is a mutant that has a stronger interaction with PIP2 and Kir2.1(L222I) is a mutant that has a weaker interaction with PIP2. So the characteristic interactions with PIP2 determined the modulation of Kir2.0 channels by PKC. (2) T53 and I213 were both important for PMA-induced inhibition of Kir2.3 currents. Amino acid sequences of Kir2.3 shared high homology of 58% with those of Kir2.1, which suggests that recombinant channel proteins were likely to produce functional channels. According to the widely accepted transmembrane topology, both Kir2.3 and Kir2.1 channels have their N and C terminus inside the membrane, two transmembrane-folding domains (M1 and M2) and putative pore regions (P). Chimeras were named based on the following rules: numbers 1 and 3 indicated Kir2.1 and Kir2.3, respectively, letter N and C represented N and C terminus, respectively, and the P letter for the rest sequence (transmembrane domains, M1 and M2, and pore region). 100 nM PMA inhibited N3P1C3 by 42.8±3.8% and had no effect on N1P3C1. This result indicated that the N and C termini distinguished the difference of Kir2.3 and Kir2.1 on their sensitivity to PMA inhibition. PMA inhibited N1P3C3 by 12.1±2.0% and N3P1C1 by 33.6±2.5%; PMA inhibited N3P3C1 by 25.3±4.1% and N1P1C3 by 13.9±2.7%, Thus both the N and C termini are essential for a full sensitivity of Kir2.3 to PMA regulation. To further find the sites of PKC action, we constructed two mutants: Kir2.3(T53I) and Kir2.1(I79T). These mutations had been shown to be important for PMA inhibition of Kir channels. PMA inhibited Kir2.3(T53I) by 36.3±3.1%, which was significantly different from PMA inhibition on Kir2.3 (p<0.05). PMA did not inhibit Kir2.1(I79T). As it has been showen, I213 was also important for PMA inhibition on Kir2.3 currents. We constructed two other mutants: Kir2.3(T53I, I213L) and Kir2.1(I79T, L222I). Kir2.3(T53I, I213L) was almost fully avoid of PMA inhibition, with an inhibition of only 4.2±9.3%, which was not significantly different from PMA inhibition on Kir2.1 (p>0.05). PMA inhibited Kir2.1(I79T,L222I) by 13.8±1.5%, which was significantly different from PMA inhibition on Kir2.1 (p<0.05). Thus both the T53 and I213 are essential for a full action of PMA on Kir2.3. (3) T53 and I213 in Kir2.3 are also the key amino acids in determining the modulation of Kir2.3 by other factors. As we reported before, a: azide and KHCO3 both could lower the intracellular pH and inhibit Kir2.3 currents, but had not effects on Kir2.1 currents. b: wortmannin (a widely used PI-4K inhibitor and thus could block the synthesis of PIP2) inhibited potassium channel currents and the degree of the inhibition were negatively correlated with PIP2-channel interactions. We further tested the effects of azide, KHCO3 and wortmannin on Kir2.1, Kir2.3 and their mutants and compared the results with those of the inhibiton effects of PMA on these channels. Azide, KHCO3 and wortmannin all inhibited the currents through Kir2.3(T53I), Kir2.3(I213L) and Kir2.3(T53I, I213L) to a less degree than on the currents through Kir2.3; on the other hand, azide, KHCO3 and wortmannin all inhibited the currents through Kir2.1(79T), Kir2.1(L222I) and Kir2.1(I79T, L222I) to a greater degree than on the currents through Kir2.1. Based on the above results from (2) and (3), we believe that like other modulators, PMA possibly inhibits Kir2.3 currents through an alteration of channel-PIP2 interaction. (4) Kir2.3 channels were not phosphorylated under the condition of PMA treatment. We found that N terminus was more important for PMA inhibition. There were only two possible phosphorylation sites: S36, S39. We made a mutant Kir2.3 (S36G, S39G) and found that PMA could inhibit Kir2.3 (S36G, S39G) by 56.0±2.6%, which was not differet from inhibion of Kir2.3 Okadaic acid (OA) was a phosphatase inhibitor, and it did not change the inhibition effect of PMA on Kir2.3 currents in our study. We also directly studied the phosphorylation state of Kir2.3 channels. The results of autoradiograph showed that Kir2.3 channels were not phosphorylated directly in vivo by PMA. On the other hand, PKC, as a positive control, was autophosphorylated by treatment with PMA. Thus the results showed that activation of PKC by PMA also phosphorylate PKC itself but did not phosphorylate Kir2.3 channels. (5) The protein interlization might be involved in the inhibition of Kir2.3 currents by PKC. Phalloidin’s toxicity is attributed to its ability to bind to F actin and prevented its depolymerization. Latrunculin A is an inhibitor of the microfilament-mediated processes of fertilization and early development and it can disrupt microfilament-mediated processes. We tested the effects of phalloidin and Latrunculin A. After application of phalloidin (PLD), PMA inhibited Kir2.3 by 7.5±1.6%; After application of Latrunculin A (LAT), PMA inhibited Kir2.3 by 31.3±3.3%. Thus both phalloidin and latrunculin A reduced the inhibitory effect of PMA on Kir2.3 currents. We also used confocal microscopy to examine the effect of PMA on the Kir2.3 surface expression. Quantitation of the images shows that the immunofluorescence in the oocytes expressing Kir2.3 significantly decreased to 51.1±8.5% after PMA treatment. For the oocytes expressing Kir2.1, the immunofluorescence was not changed. We directly measured the changes in surface expression of Kir2.3 channels in oocytes using Western blots by labeling cell-surface proteins with NHS-SS-biotin. After PMA treament, PMA significantly decreased the surface protein of Kir2.3 to 61.2±10.4%.Conclusion: Characteristic interactions with PIP2 determined modulation of Kir2.0 channels by PKC. Like other modulators, PMA possibly inhibited Kir2.3 currents through an alteration of channel-PIP2 interaction. Kir2.3 channels were not phosphorylated under the condition of PMA treatment. The protein internalization might be involved in PKC inhibition of Kir2.3 currents. The characteristic interactions between Kir channel-PIP2 might also determine the internalization of Kir channels.3. Molecular basis for genistein-induced inhibition of Kir2.3 currents.Aim: To study the effect of genistein, an inhibitor of protein tyrosine kinase, on Kir2.3 and Kir2.1 channels expressed in Xenopus oocytes and HEK293 cells.Methods: (1) Molecular biology: Kir2.3, Kir2.1 and the mutants were expressed as described above. (2) Membrane protein extraction, Western blotting and immunoprecipitation were done as described above. (3) Electrophysiology: Two-electrode voltage clamp were used to record the currents in Xenopus oocytes. The Kir2.3 and Kir2.1 channels were expressed in HEK293 cells using Lipofectamine kits (Invitrogen). Whole-cell patch clamp recordings were made from HEK293 cells.Results: (1) Genistein significantly inhibited Kir2.3 currents. In this study, we found that among three members of the Kir family (Kir2.3, Kir2.1, and Kir3.4* [a highly active mutant of Kir3.4, Kir3.4-S143T]) we tested, genistein significantly inhibited Kir2.3 currents. Using the two-electrode voltage clamp technique, we have demonstrated that micromole concentrations of genistein concentration-dependently and reversibly inhibited the currents of Kir2.3 channel expressed in Xenopus oocytes with an IC50 of 16.9±2.8μM. 100μM genistein inhibited Kir2.3 currents by 44.8±2.1%. Using the whole-cell patch-clamp technique, genistein inhibited the currents of Kir2.3 channel expressed in HEK293 cells with an IC50 of 19.3±3.2μM. In Xenopus oocytes, the inhibitory effect developed relatively fast and reached a stable level after 90.7±4.2 s and could be washed out easily. (2) The inhibitory effect of genistein on Kir2.3 currents did not involve an alteration of protein tyrosine phosphorylation. The effect of genistein on Kir2.3 currents was not affected by vanadate, a potent protein tyrosine phosphatase inhibitor. Furthermore, the effect of genistein was not mimicked by daidzein, an inactive analogue of genistein, or another potent tyrosine kinase inhibitor, tyrphostin 23. In immunoprecipitation experiments, a tyrosine phosphorylation state of Kir2.3 channels was not detected by anti-phosphotyrosine antibody PY99. (3) Channel-PIP2 interactions were not involved in genistein inhibition of Kir2.3 currents. Kir2.3(I213L) is a mutant that has a stronger interaction with PIP2. Genistein at 100μM inhibited Kir2.3(I213L) currents by 47.5±2.6% which was not significantly different from that of Kir2.3 currents (p > 0.05). Kir3.4* has a property of a weak PIP2 interaction but genistein did not have a visible effect on Kir3.4* currents. (5) Chimeras between Kir2.3 and Kir2.1 channels were constructed to identify molecular basis that distinguished the effect of genistein on these channels. It was found that the transmembrane domains and the pore region of Kir2.3 channels were important determinant for high sensitivity for genistein inhibition.Conclusion: Genistein significantly inhibited Kir2.3 currents. The inhibitory effect of genistein on Kir2.3 currents did not involve an alteration of protein tyrosine phosphorylation. Transmembrane domains and pore region of Kir2.3 channels were both important for genistein-induced inhibition of Kir2.3 currents. Understanding the mechanism of genistein action will promote our understanding of structure-function relationship of Kir channels as well as development of potent and specific modulators of Kir channels.SUMMARY1 Kir2.3 channels could be functionally expressed in Xenopus oocytes by microinjecting the transcribed cRNA. The Kir2.3 currents could be observed by using two electrodes voltage clamp technique and the current had the characteristics of inwardly rectification. Ba2+ blocked Kir2.3 currents. PMA, an activator of PKC, concentration-dependently inhibited the currents of Kir2.3 channel. PKC blocker prevented PMA effect. PMA affected Kir2.3 currents by PKC activation.2 Characteristic interactions with PIP2 determined the modulation of Kir2.0 channels by PKC. Kir2.3 channels were not phosphorylated under the condition of PMA treatment. The protein internalization might be involved in PKC inhibition of Kir2.3 currents. The characteristic interactions between Kir channel-PIP2 may also determine the internalization of Kir channels.3 Genistein significantly inhibited Kir2.3 currents. The inhibitory effect of genistein on Kir2.3 currents did not involve an alteration of protein tyrosine phosphorylation. Transmembrane domains and pore region of Kir2.3 channels were both important for genistein-induced inhibition of Kir2.3 currents. Understanding the mechanism of genistein action will promote our understanding of structure-function relationship of Kir channels as well as development of potent and specific modulators of Kir channels.更多还原