酸敏感离子通道的装配及其在海马神经元树突发育中的作用

发布时间：2018-09-01 14:58

【摘要】： 酸敏感离子通道(ASICs)是一类胞外质子激活的阳离子通道,在神经系统中分布广泛并具有多种生理病理功能,如参与触觉、味觉、视觉、痛觉、突触可塑性、学习记忆、脑缺血和癫痫等等。ASICs属于ENaC/DEG家族,与该家族的其他成员一样,它有两个疏水跨膜区,一个具有很多保守的半胱氨酸的大胞外区,氨基端和羧基端朝向胞内侧。现已克隆得到四个基因编码的6个ASIC亚单位,分别是ASIC1a及其剪接变体ASIC1b,ASIC2a及其剪接变体ASIC2b,ASIC3和ASIC4。中枢神经元中主要表达ASIC1a,2a,2b,其中ASIC1a是主要的功能性亚基,ASIC1a同聚体通道介导一种快速,瞬时的内向电流,通透钠离子和钙离子,半最大激活pH值(pH_(0.5))为～6.2。ASIC2a同聚体通道对质子的敏感性很低,pH_(0.5)为～4.4。ASIC2b不能形成功能性同聚体通道,但是可以和其它亚单位装配在一起,形成具有不同性质的异聚体通道。功能性的ASICs通常被认为是由相同或不同的亚单位形成的四聚体,然而最近Gouaux等的晶体结构研究表明ASIC1同聚体由三个亚单位装配而成。近来研究表明ASICs在中枢神经系统中起很重要的作用,参与突触可塑性、学习记忆和轴突退化等。2002年Welsh实验室利用ASIC1敲除小鼠发现ASIC1的缺失可以损伤海马依赖的LTP,损伤空间记忆和眨眼反射;随后该小组又发现ASIC1敲除小鼠对线索和背景条件性恐惧反应有所降低,然而在杏仁核和其他脑区过表达ASIC1,则可以增强背景条件性恐惧。最近海马脑片上的一项研究表明定位在树突棘上ASIC1a亚基可以影响树突棘的密度,抑制ASIC1a的表达降低树突棘的数目,而过表达ASIC1a则有相反的作用,其机制是通过影响胞内Ca~(2+)浓度和CaMKⅡ磷酸化。Friese等(2007)发现与野生型相比,在ASIC1敲除小鼠中,实验性自身免疫性脑脊髓炎(EAE)所产生的临床缺陷和轴突退化明显减轻,表明ASIC1参与中枢神经系统自身免疫炎症中的轴突退化。在本论文中,我们研究了活细胞中酸敏感离子通道的装配,并且初步探讨了酸敏感离子通道在海马神经元树突发育中的作用。 1.用荧光共振能量转移(FRET)方法研究酸敏感离子通道的装配用经典的方法如免疫共沉淀或电生理分析,认为大多数亚单位可以形成同聚体或异聚体复合物,然而关于活细胞中ASICs亚单位装配以及ASICs异聚体中各亚单位分子比的情况并不清楚。在本研究中,我们采用了一种生物物理的方法——荧光共振能量转移(Fluorescence Resonance Energy Transfer,FRET),在活细胞中直接研究ASICs的装配情况。因为FRET技术具有非侵袭性,可以在活细胞中分析蛋白—蛋白之间的相互作用,还具有高空间分辨率,被称为“分子尺”,已经被成功应用于多种受体和通道的分子比、装配等研究中。我们将各种ASICs亚单位的羧基端标记上CFP和YFP,然后将这些构建物转染到CHO细胞中,通过三通道FRET成像,先计算FR值,再根据建立的四聚体或三聚体FRET模型推测出相邻亚单位之间的FRET效率。实验结果显示,不管用四聚体FRET模型,还是用三聚体FRET模型,标记了CFP和YFP的同一种亚单位共表达时都可以检测到显著的FRET信号,表明ASIC亚单位可装配成同聚体通道;标记了CFP和YFP的不同亚单位共表达在CHO细胞中时,也可以检测到显著的FRET信号,表明ASIC亚单位可以装配成异聚体通道。另外,我们还对ASIC异聚体的分子比情况进行了初步的探讨,结果发现如果所形成的ASICs异聚体通道是四聚体,那么每种亚单位有两个,分子比倾向于2∶2;如果是三聚体,则提示ASICs的装配是随意的,分子比是不固定的,2∶1和1∶2这两种情况都存在。这项研究为ASIC同聚体和异聚体装配提供了一些新的证据,这种亚单位组成的差异形成了ASICs的功能异质性,并很大程度上成为ASICs多样而复杂的生理功能的内在结构基础。 2.酸敏感离子通道在海马神经元树突发育中的作用树突的生长和分支对功能性神经网络的形成非常重要,然而有关ASICs在树突发育中的作用则鲜有报道。在本研究中,我们在体外培养第五天(days in vitro,DIV5)的原代海马神经元中转染定位在膜上的GFP(F-GFP),随后加入ASICs拮抗剂抑制ASICs的功能,观察DIV8和DIV14这两个时间点海马神经元的树突生长、分支复杂程度,来研究酸敏感离子通道是否会影响海马神经元的树突发育。结果发现短时间抑制ASICs的功能并不影响海马神经元的树突总长度和分支,而长时间抑制ASICs的功能则会降低树突总长度,影响树突的发育,表明ASICs参与调节树突的发育,这将有助于理解酸敏感离子通道如何影响神经网络的形成,并进而参与学习记忆等高级脑功能。
[Abstract]:Acid-sensitive ion channels (ASICs) are a class of extracellular proton-activated cationic channels that are widely distributed in the nervous system and possess various physiological and pathological functions, such as tactile, taste, vision, pain, synaptic plasticity, learning and memory, cerebral ischemia and epilepsy. Six ASIC subunits encoding four genes, ASIC1a and its spliced variants ASIC1b, ASIC2a and their spliced variants ASIC2b, ASIC3 and ASIC4, were cloned and expressed in central neurons. ASIC1a is the main functional subunit. ASIC1a homopolymer channel mediates a fast, instantaneous inward current that permeates sodium and calcium ions. Semi-maximum activation pH (pH_ (0.5)) of ~6.2. ASIC2a homopolymer channel is insensitive to protons, and pH_ (0.5) of ~4.4. ASIC2b does not form a functional homopolymer channel, but can form other channels. Functional ASICCs are generally considered to be tetramers formed by the same or different subunits. However, recent crystal structure studies such as Gouaux have shown that ASIC1 homomers are assembled by three subunits.
Recent studies have shown that ASICs play an important role in the central nervous system, involved in synaptic plasticity, learning and memory, and axonal degeneration. A recent study in the hippocampal slices showed that ASIC1a subunit localized on the dendritic spine can affect the density of the dendritic spine, inhibit the expression of ASIC1a and reduce the number of dendritic spines, while overexpression of ASIC1 in the amygdala and other brain regions can enhance the background-conditioned fear. One year later, the effect was reversed by influencing intracellular Ca ~ (2+) concentration and CaMK II phosphorylation. Friese et al. (2007) found that compared with wild-type mice, experimental autoimmune encephalomyelitis (EAE) significantly reduced clinical deficiency and axonal degeneration, suggesting that ASIC1 participated in central nervous system autoimmune inflammation. The axons degenerate.
In this paper, we investigated the assembly of acid-sensitive ion channels in living cells and the role of acid-sensitive ion channels in hippocampal neuronal burst development.
1. to study the assembly of acid sensing ion channels by fluorescence resonance energy transfer (FRET) method.
The classical methods, such as immunoprecipitation or electrophysiological analysis, suggest that most subunits can form homopolymers or heteromeric complexes. However, it is not clear about the ASICs subunit assembly in living cells and the molecular ratio of each subunit in ASICs heteromers. In this study, we used a biophysical method, fluorescence. Fluorescence Resonance Energy Transfer (FRET) directly studies ASICs assembly in living cells. Because of its non-invasive nature, FRET can analyze protein-protein interactions in living cells and has high spatial resolution, it has been successfully applied in many fields. In the study of receptor-channel molecule ratio and assembly, we labeled CFP and YFP on the carboxyl end of various ASICs subunits, then transfected these constructions into CHO cells. FRET values were calculated by three-channel FRET imaging, and FRET efficiencies between adjacent subunits were deduced from the established tetramer or trimer FRET model. The results showed that significant FRET signals could be detected when the same subunit of CFP and YFP was co-expressed either in the tetramer FRET model or in the trimer FRET model, indicating that ASIC subunits could be assembled into synaptic channels, and significant FRET signals could also be detected when different subunits of CFP and YFP were co-expressed in CHO cells. In addition, the molecule ratio of ASIC heteromers was also preliminarily discussed. It was found that if the ASIC heteromer channel was tetramer, each subunit had two, the molecule ratio tended to be 2:2; if it was trimer, ASICs assembly was random. This study provides some new evidence for ASIC homopolymer and heteromer assembly. This difference in subunit composition forms the functional heterogeneity of ASICs and largely forms the intrinsic structural basis for the diverse and complex physiological functions of ASICs.
2. the role of acid sensing ion channels in the development of dendrites in hippocampal neurons
The growth and branching of dendrites are important for the formation of functional neural networks, but the role of ASICs in tree burst development is rarely reported. Function: To observe the dendritic growth and branching complexity of hippocampal neurons at the time points of DIV8 and DIV14, and to study whether acid-sensitive ion channels affect the dendritic development of hippocampal neurons. ASICs are involved in regulating the development of dendrites, which will help to understand how acid-sensitive ion channels affect the formation of neural networks and thus participate in higher brain functions such as learning and memory.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R33

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