Iks显性负抑制机制

1、lqt1型型camp依赖性依赖性iks的上调受到显性负调节的上调受到显性负调节iks:slowly activating delayed-rectifier k+ 缓慢激活延迟整流钾电流缓慢激活延迟整流钾电流显性负机制显性负机制(dominant negative)是指是指kcnq1突变型通过一种突变型通过一种“毒性毒性”作用作用干预正常野生型的功能使电流密度降低，而其他电流的动力特征没有大的改干预正常野生型的功能使电流密度降低，而其他电流的动力特征没有大的改变从而使心肌复极化时程延长。变从而使心肌复极化时程延长。显性负突变（显性负突变（dominant negative mutation）是

2、指突变基因的产物与原来）是指突变基因的产物与原来正常基因的产物相拮抗的一种突变。在多聚体蛋白质中，典型的显性负突变正常基因的产物相拮抗的一种突变。在多聚体蛋白质中，典型的显性负突变是由氨基酸的替换或者缺失造成多肽构象的改变，当这个突变多肽与野生型是由氨基酸的替换或者缺失造成多肽构象的改变，当这个突变多肽与野生型亚基以多聚体形式存在时，它降低和破坏了多聚体的活性。在广义上亚基以多聚体形式存在时，它降低和破坏了多聚体的活性。在广义上,凡一凡一对等位基因中因其中一个突变或丢失所致的另一个正常等位基因的功能活性对等位基因中因其中一个突变或丢失所致的另一个正常等位基因的功能活性丧失丧失,都称为显性负突变

3、都称为显性负突变.换言之换言之,显性负突变即杂合的突变产生了纯合突变的显性负突变即杂合的突变产生了纯合突变的效应效应 lqts综合征是一种少见的致死性心律失常，综合征是一种少见的致死性心律失常，以以ecg上上qtc延长为特征，伴有晕厥、癫痫延长为特征，伴有晕厥、癫痫性抽搐、及室性心律失常导致的心脏性猝死，性抽搐、及室性心律失常导致的心脏性猝死，常见的诱发因素有劳累、情绪激动或听觉刺常见的诱发因素有劳累、情绪激动或听觉刺激，因不同的亚型诱发因素可不同。其基本激，因不同的亚型诱发因素可不同。其基本的分子学基础为心脏离子通道缺陷，目前已的分子学基础为心脏离子通道缺陷，目前已经在经在10多个不同的多个

4、不同的lqts易感基因中发现了数易感基因中发现了数百个突变位点，主要包括钾通道功能缺失的百个突变位点，主要包括钾通道功能缺失的突变及钠通道功能获得性突变。突变及钠通道功能获得性突变。lqtilqti是是lqts最常见的类型最常见的类型（30%-35%），其编码基因为），其编码基因为kcnq1，定位于，定位于11p15.5，编，编码电压门控性钾离子通道，该码电压门控性钾离子通道，该通道在心脏高表达。部分通道在心脏高表达。部分lqt1突变者在肾上腺素刺激时呈现突变者在肾上腺素刺激时呈现出反常的出反常的qt间期延长。间期延长。iks电流及电流及iks通道通道iks电流主要在动作电位平台期的后期起作用

5、。膜去极化时, iks激活非常缓慢,在膜电位复极时，它的去激活也慢 。iks通道:目前认为iks通道分子是由4个同源kcnq1成孔道蛋白（-亚单位）及2个附属的kcne1蛋白（-亚单位）、调节亚基yotiao组成。iks通道能对肾上腺素刺激产生反应，从而在运动时通过缩短qtc来增加心率，而lqt1突变株则可破坏其维持心率的机制，使得qtc在运动时进行性延长。 in 2005, brink reported the loss-of-function mutation a341v in kcnq1 in a large south african founder. the mutation a34

6、1v is in the s6 transmembrane segment of kcnq1 and predisposes to a severe long-qt1 syndrome with sympathetic-triggered ventricular tachyarrhythmias and sudden cardiac death. . .kcnqi-a341v objective: the authors aimed to elucidate the molecular mechanisms underlying the pronounced repolarization ph

7、enotype in a341v patients, particularly during -adrenergic receptor stimulation. methods and results1.chinese hamster ovary (cho) cells were transiently transfected with human kcnq1 (wt, mutant or 1:1 wt plus mutant), human kcne1, human yotiao, and gfp. use whole-cell patch-clamp analysis.figure 1a

8、12-year-old male a341v carrier at rest(left) and during exercise (right) apd is significantly prolonged in a341v het conditions during ar stimulation compared with wt.figure 2combined figure 1 with figure 2,a341v het disrupted camp sensitivity predominantly duringar stimulation .these suggested that

9、 iks modulation is under dominant-negative control.what is the mechanism underlying the suppressed camp responsiveness of a341v iks? steven have showed thatar modulation of iks requires targeting of adenosine 3,5-monophosphate (camp) dependent protein kinase (pka) and protein phosphatase 1 (pp1) to

10、hkcnq1 through the targeting protein yotiao and indentified ser27 as the unique site of pka phosphorylation on hkcnq1(science. 2002;295:496 499). based on that theory ,the authors presented three hypothesis as following. (1)disruption of the conformational changes occurring after phosphorylation of

11、kcnq1-s27; (2)disruption of kcnq1 interaction with yotiao thereby reducing local pka availability; (3)disruption of phosphorylation of s27, even in the presence ofyotiao.s27d led to an upregulation of iks-tail amplitudes and a significant leftward shift of the half-maximal activation potential in wt

12、 conditions. s27d substitution resulted in a significant upregulation leftward shift in half-maximal activation potential both in a341v heterozygous and homozygous conditionsthese data refuted hypothesized mechanism 1.figure 32.using the phosphomimetic substitution kcnq1-s27d in combination with wil

13、d-type (wt) or mutant kcnq1 to study the effects of mimicked phosphorylation.3. cho cells were transfected with kcnq1-wt or kcnq1-a341v-kcne1-yotiao. western blot analysis of whole-cell lysates or immunocomplexes was performed with anti-kcnq1 and anti-yotiao antibodies.yotiao was detected in both wt

14、 and a341v but not in negative controls. in the presence of a341v, kcnq1/yotiao interaction was not statistically different from wt. a341v-mutant iks was markedly increased by the s27d substitution when yotiao was cotransfected, there was no such increase in the absence of the anchoring proteinthis

15、indicated that hypothesized mechanism 2cannot explain it.figure 44.cells were transfected with kcnq1-wt+kcne1+yotiao or kcnq1-a341v+kcne1+yotiao. western blot analysis was performed and membranes were probed with anti-kcnq1-phospho-s27 and anti-kcnq1 antibodies. a significant increase phosphorylated

16、 kcnq1 in response to stimlulation with camp/oa in kcnq1-wt. although increased phosphorylation was also observed in a341v ex-pressing cells, the fraction of phosphorylated ikschannels was significantly lower than that in wt cells (by 25%), both for a341vhom and a341vhetthus, defective phosphorylati

17、on of kcnq1-a341v is responsible, at least partly, for the loss of camp-dependent iks upregulation.figure 55.cho were transfected with kcnq1-myc, kcnq1-a341v-gfp, kcne1, and yotiao cdnas. stainings were made with mouse monoclonal anti c-mycconjugated and texas redconjugated goat anti-mouse antibodie

18、s. cells were analyzed under a confocal microscope.the overlay of these signals indicates strong colocalization (yellow color) of wt and a341v subunits in the cell membrane. the cross-section profile of both signal intensities shows enhanced intensity at the membrane.kcnq1-wt and kcnq1a341v are both

19、 expressed in the membrane figure 6compared kcnq1-a341v with others mutations of lqt1 .wt, heterozygous and homo-zygous k557e, and heter-ozygous and homozygous a344v all showed a pronounced responsiveness to camp. in contrast,a341v and g589d iks were unresponsive to stimulation,even when coexpressed

20、 with wt iks.the very similar mutation a344v( same amino acid substitution, only 3 residues apart)that showed a pronounced responsiveness to camp dis-tinguished from a341v.both a341v and heterozygous g589d show dominant-negative suppression of camp-dependent iks upregulation .figure 7 whether altera

21、tions in kcnq1-s27 alone could exert dominant-negative control of camp/pka-dependent iks upregulation or not？ to further ivestigation, the author analyzed a to further ivestigation, the author analyzed a 1:1 coexpression of wt kcnq1with kcnq1-s27a 1:1 coexpression of wt kcnq1with kcnq1-s27a (togethe

22、r with kcne1 and yotiao). (together with kcne1 and yotiao). this heterozygous s27a condition will disable this heterozygous s27a condition will disable the pka phosphorylation site.the pka phosphorylation site.in contrast to the pronounced increase observed with camp/oa treatment in wt iks, there was no significant increase in iks-tail amplitude