【作者】 张敏；

【导师】 徐天乐； 陈林；

【作者基本信息】 中国科学技术大学 ， 神经生物学， 2008， 博士

【摘要】 酸敏感离子通道是可被胞外的H⁺直接激活的配体门控的阳离子通道,在哺乳动物的中枢及外周神经系统中都有广泛的分布。越来越多的证据表明,ASICs在生理及病理的条件下都发挥了重要的功能。在生理条件下,ASICs参与了味觉、嗅觉和视觉的感受;在病理条件下,ASICs在痛觉、癫痫及缺血造成的损伤中扮演了重要的角色。而对于ASICs在听觉通路上的表达及功能研究得不多,仅有的几篇文献主要是研究ASICs在外周的听觉通路上的表达及功能特性。在听觉中枢上ASICs的表达、通道特性及其生理功能目前还不清楚。因此,本研究用RT-PCR、全细胞膜片钳及钙成像等方法,研究了下丘ASICs的表达以及下丘神经元上酸诱导电流的电生理学和药理学特性,鉴定了介导该电流的ASICs的亚基组成,主要结果如下:1.RT-PCR的结果显示在下丘可检测到ASIC1a,ASIC1b,ASIC2a,ASIC2b四种亚基的表达,而ASIC3亚基在下丘上不表达。2.细胞外环境的pH值的下降可在培养的下丘神经元上诱导出快速失活的内向电流;该电流对胞外pH高度敏感,激活阈值约为pH 6.9,半效激活pH为5.92;ASICs通道非选择性拮抗剂amiloride可以阻断下丘神经元上酸诱导的电流(IC₅₀=20.4±0.4μM):该电流的反转电位接近Na⁺的反转电位,且用NMDG取代Na⁺极大的抑制了该电流。这些结果提示酸在下丘神经元上诱导的电流是由ASICs介导的。3.更进一步的证据表明,ASIC1a同聚体通道参与介导酸在下丘神经元上介导的电流:（1）经典的离子替换及钙成像的结果显示,介导下丘神经元上酸诱导电流的通道可以通透Ca²⁺;（2）PcTX1（ASIC1a同聚体通道的选择性拮抗剂）对下丘神经元上酸诱导的电流有明显的抑制作用;（3）胞外的Ca²⁺及pb²⁺可以显著的抑制该电流,进一步验证了ASIC1a亚基的存在。此外,Zn²⁺（300μM）可以增强该电流的幅度,提示ASIC2a亚基的存在。而水杨酸钠对该电流没有影响,进一步验证了在下丘神经元上不表达ASIC3亚基。4.最后,用全细胞电流钳的方法,本研究观察到胞外酸化可以导致下丘神经元膜电位的去极化并诱发动作电位的发放,提示ASICs的激活可以调节神经元的兴奋性。本研究表明,下丘神经元上ASIC1a同聚体通道及包含ASIC2a的异聚体通道的存在。下丘神经元上ASICs在生理及病理条件下的功能,值得进一步的研究探讨。更多还原

【Abstract】 Acid-sensing ion channels （ASICs）, which are widely distributed in the mammalian brain, the spinal cord and the peripheral sensory organs, are ligand-gated cation channels activated by extracellular protons. Abundant experimental evidence shows that ASICs play important roles in physiological/pathological conditions, such as sensory transduction, learning/memory, retinal function, seizure and ischemia. In the auditory system, however, there are only a few studies available describing ASICs in hair cells, the spiral ganglion and the vestibular ganglion. In particular, functional ASICs have not been assessed in the central auditory region, although there is evidence to show their transcription in the inferior colliculus （IC）. In the present study, we have studied the electrophysiological and pharmacological properties of acid-induced current in cultured IC neurons, identified the possible ASIC subunits mediating the current.1. Reverse transcriptase polymerase chain reaction （RT-PCR） experiment demonstrates that four ASIC isoforms, ASIC1a, ASIC1b, ASIC2a and ASIC2b are present in the rat IC, while ASIC3 is absent.2. Our electrophysiological data showed that a rapidly decaying inward current was induced by exogenous application of acidic solution in cultured IC neurons with a response threshold around pH 6.9 and a half activation pH value at 5.92. The current was sensitive to amiloride (IC₅₀ = 20.4±0.4μM), an ASIC blocker, and its reversal potential was close to the theoretical Na⁺ equilibrium potential, indicating that the recorded current was mediated by ASICs.3. Further experiments revealed the presence of homomeric ASIC1a channels in IC neurons: （1） the ASIC-like current was partially carried by Ca²⁺ as demonstrated with an ion-substitution protocol and Ca²⁺ imaging; （2） the current was inhibited by the tarantula venom PcTX1, a specific blocker for homomeric ASIC1a channels; （3） the current could be inhibited by extracellular Ca²⁺ (IC₅₀= 2.31 mM) and Pb²⁺ （10μM）, confirming the presence of ASIC1a subunit. The presence of functional ASIC2a containing channels was revealed by the Zn²⁺（300μM）-induced enhancement of ASIC-like currents and the absence of functional AS1C3 channels was indicated by the insensitivity of ASIC-like currents to salicylate （1mM）, an ASIC3 subunit blocker.4. Finally, we show that activation of ASICs by a pH drop could induce membrane depolarization and evoke neuronal firing in IC neurons. Our study clearly demonstrates that functional homomeric ASIC1a channels and ASIC2a-containing channels, but not ASIC3 channels, are present in the IC. We suggest that ASICs should be taken into consideration for their possible functional roles in information processing and pathological processes in the central auditory system.更多还原