【作者】 高英；

【导师】 罗建红；

【作者基本信息】 浙江大学 ， 神经生物学， 2008， 博士

【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Ca²⁺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。更多还原

【Abstract】 ASICs are cationic channels activated by extracellular protons,widely expressed in nervous system.They play multiple roles in both physiological and pathological conditions,such as mechanical perception,taste,vision,nociception,synaptic plasticity,memory,ischemia and seizure etc.ASICs belong to the ENaC/DEG （epithelial amiloride-sensitive Na+ channel and degenerin）family of ion channels. They share the same overall structure with other members of this family:two hydrophobic transmembrane regions（TM1 and TM2）,a large extracellular loop comprising many conserved cysteines,and the C and N termini facing the intracellular space.So far,six ASIC subunits encoded by four genes have been cloned, ASIC1a and its splice variant ASIC1b,ASIC2a and its splice variant ASIC2b,ASIC3 and ASIC4.In the CNS,neurons mainly express ASIC1a,2a and 2b subunits,while ASIC1a is the predominant functional subunit.Homomeric ASIC1a channels mediate a fast and transient inward current,conducting Na⁺ and Ca²⁺ions.The pH for half maximal activation(pH_0.5)in homomeric ASIC1a channels was 6.2.Homomeric ASIC2a channels have a low sensitivity to protons with a pH_0.5of～4.4.ASIC2b subunits do not form functional homomeric channels,but they may associate with other subunits to form heteromeric ASICs with distinct properties.Functional ASICs are usually believed to be tetrameric assemblies of homomeric or heteromeric subunits.However,recent Gouaux and colleagues’ crystal structure shows that there are three subunits in ASIC1 homomeric channel.Recent studies have shown that ASICs play an important role in the CNS, contributing to synaptic plasticity,learning and memory and axonal degeneration. Welsh lab（2002）generated ASIC1 knockout mice and found that loss of ASIC1 impaired hippocampal-dependent LTP,spatial learning and eye-blink conditioning. Then this group also found that ASIC1 knockout mice showed a reduced fear response to both cued and context fear conditioning.Conversely,overexpressing ASIC1 in the amygdala and elsewhere in the brain enhanced context fear conditioning. A recent study using organotypic hippocampal slices shows that ASIC1a,localized in dendritic spines,can affect the density of spines.Decreasing ASIC1a reduced the number of spines,whereas overexpressing ASIC1a had the opposite effect.The mechanism is through influencing intracellular Ca²⁺concentration and CaMKII phosphorylation.Friese et al.（2007）found that compared to wild-type mice,ASIC1 knock out mice showed both a markedly reduced clinical deficit and reduced axonal degeneration after induction of experimental autoimmune encephalomyelitis（EAE）, which suggested that ASIC1 contributes to axonal degeneration in autoimmune inflammation of the central nervous system.In this dissertation,we studied the assembly of ASICs in living cells and the role of ASICs in dendritic development in hippocampal neuron.1.Fluorescence resonance energy transfer analysis of subunit assembly of the ASIC channelHomo-and heteromeric ASIC complexes have been verified by classical methods such as co-immunoprecipitation,or functional assays applying electrophysiology.However,the exact subunit assembly and stoichiometry of heteromeric ASIC channels in living cells are largely unknown.In the present study, we have used a biophysical approach based on Fluorescence Resonance Energy Transfer（FRET）to address these questions directly in living cells.For FRET measurement is non-invasive,it allows analysis of the interaction between proteins in living cells.In addition,FRET has high spatial resolution.It can serve as "a molecular ruler" and has been successfully applied in the investigations of stoichiometry and assembly of many types of receptors and channels.We fused ASICs subunits with CFP and YFP,and transfected these constructs into CHO cells. We get the FR value through three-cube FRET imaging,then FRET efficiency between neighboring subunits was estimated according to the tetramer or trimer FRET model.Our results showed that when either tetrarner or trimer FRET model was used,co-expression of the same ASIC protein fused with CFP and YFP produced significant FRET signals,consistent with the homomeric assembly of ASIC channels. When two different ASIC proteins fused with CFP and YFP,respectively,were co-expressed in the cell,significant FRET signals were also produced,suggesting the occurrence of hetermeric assembly of ASIC proteins.In addition,we studied the stoichiometry of hetermeric ASIC complex.Our results suggested that the channel contains two of each subunit and the stoichiometry is apt to 2:2 if hetermeric ASIC channel is a tetramer,while the assembly of ASICs is random,the stoichiometry is not fixed（2:1 and 1:2 coexist）if hetermeric ASIC channel is a trimer.This study provided interesting new data supporting the occurrence of the homomeric and heteromeric assembly of ASICs.The existence of many isoforms and subunits creates functional heterogeneity of ASICs,which to some extent determines the variable and complicated biological properties that ASICs have in the brain and peripheral sensory neurons. 2.The role of ASIC channel in dendritic development in hippocampai neuronRegulated growth and arborization of dendritic processes are critical to the formation of functional neuronal networks.However,the role of ASICs in dendritic development is still elusive.In the present study we transfected F-GFP into cultured hippocampal neurons at 5 days in vitro（DIV 5）.Then ASICs antagonists were applied to the neuronal medium to inhibit the function of ASICs.Dendritic growth and arborization of cultured hippoeampal neuron were observed in DIV8 and DIV14. We attempted to determine whether and how ASICs regulate dendritic development. Our results showed that the total dendrite branch length and arborization of hippocampal neuron were not affected by the inhibition of ASICs for a short time, while the total dendrite branch length was reduced and dendrite development was impaired if ASICs were inhibited for a long time.It suggested that ASICs play a role in the dendrite development,which may help understand how ASICs affect the formation of neuronal networks and participate in higher brain fuctions such as learning and memory.更多还原