中枢神经系统酸敏感离子通道的调控

【作者】 王炜；

【导师】 徐天乐；

【作者基本信息】 中国科学技术大学 ， 神经生物学， 2007， 博士

【摘要】 酸敏感离子通道（acid-sensing ion channels，ASICs）是一类由氢离子（H⁺）激活的配体门控阳离子通道。ASIC通道电流可以被阿米洛利（amiloride）可逆的阻断。有四种编码ASICs的基因：ASIC1、ASIC2、ASIC3和ASIC4。它们编码了6种ASIC亚基蛋白：ASIC1a、ASIC1b、ASIC2a、ASIC2b、ASIC3和ASIC4。这6种ASIC亚基在体内广泛分布，提示ASICs可能参与机体的多种功能。越来越多的研究表明：ASICs参与了神经中枢多种生理、病理过程，包括：情绪、学习和记忆等脑高级功能、视网膜光信息处理、脑缺血损伤以及脑肿瘤等等。而ASICs和突触功能的关系也越来越多的得到了阐明。而ASICs的功能可以被诸多内、外源性分子调控，包括多种必需金属离子如：Ca²⁺、Zn²⁺、Mg²⁺，和对机体有毒性的金属离子如：Ni²⁺、Cd²⁺、Gd³⁺等。研究金属离子对ASICs的调控可以帮助我们更多的了解ASICs诸多生理、病理功能。此外，研究金属离子与通道相互作用，有助于深入认识ASICs的结构及其功能变化。这样我们就可以针对性的设计靶点特异性药物作用于ASICs，为一些顽症的临床治疗带来新的希望。本研究选取了机体必需金属离子Cu²⁺，和一种最重要的毒性金属离子Pb²⁺，运用全细胞膜片钳技术结合神经细胞急性分离、神经细胞原代培养、细胞转染、Ca²⁺成像等技术，研究了它们对ASICs功能的调控，并探讨了这些调控的作用机制。此外，我们还运用全细胞膜片钳技术初步研究了下丘脑中的ASICs。本研究所包括的主要实验结果如下：1．Pb²⁺对ASICs的调控Pb²⁺作为最重要的毒性金属离子，其对神经膜受体和离子通道的调控也广受关注。我们的研究发现：Pb²⁺以浓度依赖的方式，快速、可逆的抑制大鼠脊髓背角（spinal dorsal horn，SDH）和海马CA1区神经细胞的ASIC电流。异源表达系统的结果表明：ASIC1a、ASIC1b和ASIC3亚基介导的电流可以特异的被Pb²⁺抑制。因此，ASICs是Pb²⁺的分子靶点。由于ASIC1b和ASIC3主要分布在外周神经系统，而ASIC1a在中枢神经系统有分布，因此我们的结果提示：ASIC1a亚基是Pb²⁺在中枢神经系统主要的ASIC通道靶点。在中枢神经细胞进行的Ca²⁺成像和电流钳实验表明：Pb²⁺抑制ASIC介导的细胞外Ca²⁺内流和膜兴奋性升高。由于ASICs特别是ASIC1a和突触功能相关，所以我们认为：Pb²⁺对ASICs的抑制可能是pb²⁺神经毒性的一种分子机制之一。我们还研究了pb²⁺对以ASIC1a电流为主的SDH上ASICs的调控机制。我们发现：pb²⁺以电压不依赖的方式抑制ASIC电流幅值，并且这种抑制和细胞内分子信号无关。这些结果提示：pb²⁺的作用靶点位于ASIC通道细胞膜外侧、离开ASIC通道口一定距离的细胞外环上。pb²⁺与amiloride对ASICs调控的非竞争性关系则进一步证实了这一推测。此外，我们发现：pb²⁺使ASICs激活曲线右移，但是不改变电流幅值的最大值和Hill系数，说明pb²⁺通过一种竞争性的方式减小ASICs对细胞外H⁺的敏感性，从而抑制ASIC电流。有趣的是：Ca²⁺对ASICs的调控也有pH依赖性。通过三种不同顺序的给药方式，我们发现：尽管pb²⁺和Ca²⁺都是二价金属离子，但是它们对ASICs的调控方式不尽相同。最近，Paukert等人研究提示：ASIC1a上有至少两个Ca²⁺的作用位点。一个是通道阻断位点，另一个据推测是调节性位点。借用这个模型，我们认为在细胞膜外侧、离开ASIC通道口一定距离的细胞外环上也存在pb²⁺的两个作用位点，即：一个通道阻断位点，pb²⁺与之结合可以阻断开放状态下的ASIC电流；另一个是调节性位点，pb²⁺与之结合可以引起ASIC蛋白构象的改变，影响ASICs对H⁺的亲和力。pb²⁺结合到阻断位点和（或）调节性位点，都可能影响H⁺对ASICs的调控。2．Cu²⁺对ASICs的调控Cu²⁺是人体必需微量元素，对于维持正常的神经功能发挥重要的作用。但是，Cu²⁺含量异常也伴随一些神经系统疾病。我们在培养的下丘脑神经细胞上发现：Cu²⁺快速而可逆的抑制ASIC电流的幅值，减慢ASICs的脱敏。在海马和皮层神经元上，我们也能观察到类似的现象。这些结果说明：Cu²⁺是中枢ASICs活性的内源性调节剂。我们的结果还显示：Cu²⁺对ASICs的调控不受膜电位改变影响，说明Cu²⁺在ASICs上的作用位点离开ASIC通道外口有一定距离。另一方面，从Cu²⁺对ASICs激活曲线的影响来看，Cu²⁺和H⁺对ASICs的调控作用是一种非竞争性的关系。这种非竞争性的关系提示一种变构调控的机制。此外，Cu²⁺延长了ASICs的脱敏时程，进一步支持了变构调控的存在。我们进一步通过三种不同顺序的给药方式发现：预给药方式下，Cu²⁺对ASICs抑制的幅值与共给药和按顺序给药方式所得到的电流抑制幅值的线性叠加，在统计学上是没有显著差异的。综合以上发现，我们推测：在ASICs的细胞外环上有两个彼此独立的Cu²⁺作用位点。一个位点是通道阻断位点；另一个是调节性位点。静息状态下，Cu²⁺与之结合引起变构调节，使ASICs对细胞外H⁺敏感性下降。从而使ASIC电流的幅值减小、通道脱敏时程延长。由于通道阻断位点和调节性位点是彼此独立的，因而，我们可以观察到Cu²⁺分别结合到这两个位点所导致的电流抑制叠加。我还通过电流钳的实验，发现Cu²⁺可逆的抑制由于ASICs激活升高的神经膜兴奋性。这种负反馈调控可能有助于保持正常的神经膜兴奋性。另外，我们还明确：ASIC1a亚基是Cu²⁺的分子靶点。由于微摩浓度的Cu²⁺抑制ASIC1a活性，因此Cu²⁺在ASIC1a介导的脑缺血酸毒过程中可能有保护性的作用。3．下丘脑ASICs的初步研究我们在培养的大鼠下丘脑细胞中记录到一种由细胞外酸溶液诱导的快速激活而又快速失活的阳离子电流。这种离子电流可以被amiloride可逆的阻断、不受膜电压变化的调控、并且主要由Na⁺离子构成。当细胞外液pH值低于6.8-7.0时，这种酸电流就可以被记录到。根据这些电生理学和药理学特性，对比既往在天然神经细胞和异源表达系统的研究，我们得出结论：下丘脑的这种酸电流是由ASICs介导的。进一步分析实验结果，我们认为：下丘脑ASIC电流主要由ASIC1a同聚体或者ASIC1a+2a异聚体介导的。我们也不排除ASIC1a+2b介导下丘脑ASIC电流的可能。我们这项研究的一个新发现就是：功能性ASIC3亚基可能也出现在中枢神经细胞中。我们所得到的RT-PCR的初步结果对上述推断提供了一个佐证。此外，下丘脑ASICs激活可以升高神经膜兴奋性，这可能与调控激素分泌有关。由于下丘脑调控许多重要的生命活动过程，而不同的ASIC亚基承当不同的功能，因此多种ASIC亚基出现在下丘脑提示ASICs参与了下丘脑的多种生理、病理功能。更多还原

【Abstract】 Acid-sensing ion channels （ASICs） are amiloride-sensitive and voltage-insensitive cation channels that are activated by extracellular H⁺. Four genes encoding six ASIC subunits, ASIC1a, ASIC1b, ASIC2a, ASIC2b, ASIC3 and ASIC4, have been cloned and demonstrated to be broadly expressed throughout the body including the central and peripheral nervous system. Increasing evidence has highlighted the role of ASICs in the central nervous system in various physiological and pathological processes such as learning and memory, emotion, retina function, ischemic brain injury and brain tumor. In addition, the contribution of ASICs to synaptic function has been recently elucidated.It is well known that ASICs are functionally regulated by some exo- and endogenous agents such as metal ions including physiologically relevant Ca²⁺, Zn²⁺, Mg²⁺ and toxically relevant Ni²⁺, Cd²⁺, Gd²⁺. Moreover, unraveling the interaction between metal ions and ASICs will provide the structure information, which is helpful to the design of novel drugs that specifically target these channels. Therefore, in our study, we examined the effects of Cu²⁺, an essential element, and Pb²⁺, a well-known neurotoxicant, on ASICs in the central neurons using whole-cell patch clamp combined with cell transfection and Ca²⁺ imaging. Besides, using whole-cell patch clamp, we made preliminary investigation on the ASICs in the hypothalamus, the region of the brain that controls a large number of bodily functions. The major findings are summarized as follows:1. The modulation of ASICs by Pb²⁺At a holding potential of -50 mV, extracellular Pb²⁺ rapidly and reversibly inhibited the amplitude of ASIC currents in the acutely dissociated hippocampal CA1 and spinal dorsal horn （SDH） neurons of the rat in a concentration-dependent manner. The results from the CHO cells transfected with five major ASIC subunits: 1a,1b, 2a, 2b and 3 in combination showed that the ASIC currents mediated by ASIC1a, 1b, and 3 were inhibited by Pb²⁺. Since ASIC1b and 3 have been reported to be expressed in the peripheral nervous system of the rat, we suggest that ASIC1a is the main molecular target of ASICs in the central nervous system. Next, we studied the mechanisms underlying the modulation of ASICs by Pb²⁺ in the SDH neurons in which ASIC currents are mainly mediated by ASIC1a. We found that Pb²⁺-induced inhibition of ASIC currents was voltage-independent, and not modified by intracellular signaling messengers. These results suggest that the action site（s） of Pb²⁺ are located on entracellular domain of these channels. The non-competitive interaction of Pb²⁺ and amiloride with ASIC currents also supports this speculation.Furthermore, we found that Pb²⁺ shifted activation curve of ASICs in a parallel manner without significantly altering the maximal value and Hill coefficient, suggesting that Pb²⁺ reduced the affinity of ASICs for H⁺ in a competitive manner. Notably, the Ca²⁺ modulation of ASICs is also pH-dependent manner. To examine their possible interactions in inhibiting these channels, we co-applied the two divalent cations to ASICs, and found that they modulated ASICs in a distinct fashion. Paukert et al. has ever proposed a model to explain the mechanism underlying the modulation of ASIC1a by Ca²⁺. In this model, Ca²⁺ could bind to two action sites located on ASIC1a, a blocking site and a putative modulating site, to modulate ASIC1a currents. We modified and employed this model to explain the modulation of ASICs by Pb²⁺. When binding to the blocking site, Pb²⁺ blocked ASIC currents in the open state of the channel, whereas binding to the modulating site, Pb²⁺ induced a conformational change of the channel protein, resulting in an inhibition of ASIC currents. When binding to either blocking site or modulating site, Pb²⁺ influences the effects on ASICs by H⁺.2. The modulation of ASICs by Cu²⁺Cu²⁺ is the essential element in human body and plays important roles in maintaining normal functions of the brain. Abnormal amount of Cu²⁺ are associated with some neurological diseases. In the cultured hypothalamic neurons, we found that this divalent cation reversibly and concentration-dependently inhibited the amplitude of ASIC currents, and slowed down the desensitization of ASIC channels. Similar results were obtained from hippocampal and cortical neurons of the rat. These results identified Cu²⁺ as an endogenous modulator of ASIC activity. Further study showed that the Cu²⁺ modulation of ASICs was independent of change in membrane potentials suggesting that the action site（s） of Cu²⁺ on ASIC channel is located on extracellular domain. In addition, the effects of Cu²⁺ on activation curve of ASICs indicate a non-competitive mechanism, which implies an allosteric modulation. The prolonged desensitization of ASICs by Cu²⁺ supports this speculation. Employed three different modes of drug application, we found that the inhibitory effect in pre-treatment protocol was statistically identical to that of the liner summation from co-application protocol and sequential application protocol. Based on these results, we speculate that two separate action sites, a blocking site and a modulating site, are located on the extracellular domain of ASICs. Cu²⁺ reduced sensitivity of ASICs to H⁺ via these action sites, which accounts for the reduced amplitude of ASIC currents, and the prolonged desensitization of these channels. Since the two sites are separate, we thus observed a cumulative inhibition caused by Cu²⁺ binding to both sites.Another finding in this study was that Cu²⁺ reversibly attenuated the increased membrane excitability mediated by activation of ASICs. This negative modulation may be helpful to maintain normal neuronal excitation during synaptic transmission. From the results from CHO cells expressing ASIC1a subunit, we found this subunit is inhibited by Cu²⁺. Since ASIC la-mediated acidotoxicity is the important mechanism of ischemic brain injury, Cu²⁺-induced inhibition of ASIC1a may be protective in the conditions of stroke.3. The ASICs in the hypothalamusWe recorded proton-induced cation currents in the cultured hypothalamic neurons of the rat. These transient inward currents with an activation threshold of around pH 6.8, were mainly carried by Na⁺, and reversibly blocked by amiloride, the known ASIC antagonist. Based on these typical electrophysiological and pharmacological properties, we attribute these currents in the hypothalamic neurons to ASICs. Further study exhibited that homomeric ASIC1a channels and heteromeric ASIC1a+2a channels may mediate the ASIC currents in the hypothalamic neurons. We can not exclude the presence of heteromeric ASIC1a+2b responsible for ASIC currents. A novel finding in this study is that functional ASIC3 channels might be present in the central neurons of the rat. These conclusions have been supported by the preliminary results from RT-PCR. Another finding in this study is that membrane depolarization in the hypothalamic neurons could be induced by a mild and moderate drop in extracellular pH that is within the range locally reached by pH fluctuations during normal synaptic transmission or due to tissue acidosis occurring in the pathological conditions. Therefore, it is conceivable that the hypothalamic ASICs may regulate hormone release from hypothalamic neurons via ASIC-triggered membrane depolarization. Considering that different ASIC subtypes are involved in many physiological and pathological processes, and the hypothalamus mediates distinct functions, the presence of multiple ASIC subunits suggests their potential roles in this important brain region.更多还原