【作者】 成秋平；

【导师】 周艳红；

【作者基本信息】 华中科技大学 ， 生物医学工程， 2013， 博士

【摘要】 细胞是生命活动的基本单位。一切有机体都是由细胞构成的，它是有机体生长与发育的基础，也是遗传的基本单位，因此，没有细胞就没有完整的生命。所有的动物细胞都由一层薄膜所包围，这就是细胞膜(plasma membrane)，细胞膜的作用是保持细胞内物质成分的稳定。但是，细胞膜并不是完全封闭的，细胞需要跟外界交换物质和能量，细胞膜上的离子通道就是具备这种功能的蛋白质，离子通道可以分为配体门控通道、电压门控通道、胞内信号门控通道和机械敏感通道四种类型。超极化激活的环核苷酸门控(hyperpolarization-activated cyclic nucleotide-gated，HCN)通道(起搏通道)是一种能被电压和环磷酸腺苷(cAMP)调控的、无选择性阳离子通道，它介导的电流是Ih。Ih广泛的参与了对一系列生理活动的调控，包括心脏和神经元节律，某些可兴奋细胞的静息电位，神经信号的传递以及信号在树突中的整合等。Ih受自主神经系统和神经内分泌系统的调节以及某些病理状态的影响，Ih异常会导致心脏及精神方面的多种疾病。因此，研究Ih有重要的生理学意义。本文着重应用电生理膜片钳、全细胞钙成像、神经疼痛模型等技术对HCN通道的调控机制、在神经疼痛中的作用及其药理学特性进行了研究和探讨。本文主要研究结果和结论如下：（1）PKA信号通路在调控HCN通道活性中的作用。PKA广泛分布于细胞体内，参与了对一些列重要生理活动的调控。然而，抑制PKA对HCN通道活性有何影响，目前仍不清楚。为了弄清这一问题，我们选择了PKA的选择性抑制剂KT5720。在新鲜分离的大鼠DRG神经元上，综合运用全细胞电压钳、电流钳、单通道记录以及钙成像技术。揭示了PKA信号通路在调控HCN通道活性中发挥了极为重要的作用，KT5720抑制HCN通道活性，使其电流密度减小，半数开放电压向超极化方向移动，并使其通道开放的时间常数增加。此外，KT5720抑制PKA使DRG神经元上的动作电位减小、胞内Ca2+浓度降低，从而抑制DRG神经元的兴奋性。（2）HCN通道调控神经疼痛。神经疼痛通常由神经损伤引发，因其具有发病率高、难治愈等特点，已成为当今医学界亟待解决的主要问题之一。另一方面，DRG神经元是感觉传入的第一级神经元，在疼痛信号的传导过程中发挥着不可或缺的重要作用。因此，我们选择DRG神经元为研究对象，利用大鼠幸免神经损伤模型。综合运用多种电生理膜片钳实验方法，研究和探讨了HCN通道在调控神经疼痛中的机制，揭示了HCN通道是触发和维持神经疼痛的关键因素。在幸免神经损伤诱导的疼痛状态下，DRG神经元上的HCN通道活性显著增加，异位放电频率变快，且DRG形态发生变化。因此，HCN通道是有效治疗神经疼痛的潜在靶点。（3）麻黄碱对HCN通道的影响。麻黄碱作为一种肾上腺受体激动药，具有加强心收缩力、增加心输出量、加快心率、升高血压等作用。为了弄清麻黄碱的这些作用是否是通过HCN通道来实现的以及它对HCN通道电流有什么影响。我们采用传统的全细胞膜片钳实验方法，在新生的大鼠海马神经元和HEK细胞上记录到HCN通道电流Ih。实验结果显示，麻黄碱对Ih有抑制作用。经分析，我们认为HCN通道可能具备某种反馈及保护功能，即当血压和心率上升时，HCN通道能部分阻滞这种上升趋势，从而保护机体健康和生命安全。更多还原

【Abstract】 Cell is the basic unit of life. All organisms composed of cells, it is the basis oforganism growth and development. It’s also the basic unit of heredity. Overall, cell is thefundamental element of life. All animal cells are surrounded by a layer of film, which iscell membrane. It serves as a fence to keep intracellular materious and circumstancesstable. However, cell membrane is not absolutly sealed. Cells need to exchange materialsand energies with outside. Ion channels located in cell membrane perform this function.Ion channels can be classified into four types: ligand-gated channels, voltage-gatedchannels, intracellular signal-gated channels and mechanical sensitive channels.hyperpolarization-activated cyclic nucleotide-gated channel (pacemaker channel) is anon-selective cation channel which can be regulated by both voltage and cyclic adenosinemonophosphate (cAMP), the associated underlying current is Ih, Ihinvolved in regulatinga series of physiological processes, such as controling pacemaker activities in both heartand neurons, governing the resting membrane potential of some excitable cells, transferingnerve signals and signals in dendritic integration. Ihis regulated by the autonomic nervoussystem and neuroendocrine system and is affected by some pathological conditions. Itsdysfunction may induce many heart and mental diseases. Therefore, study Ihhas importantphysiological significances.At this present study, by using electrophysiological patch-clamp recording, whole cellCa2+imaging as well as a rat spared-nerve injury model, its regulation mechanisms, itsrole in neuropathic pain and its pharmacological properties are investigated.The main results are as follows:(1) The role of PKA signal pathway on regulating HCN channel activity. ProteinKinase A (PKA) is widely expressed in almost all types of cells, it involved in controllingand regulating a serial of pivotal physiological processes, while the effect of PKAinhibition on HCN channel activity is yet to be decided. To address this issue, we chosedKT5720, a selective PKA inhibitor. With using whole-cell voltage clamp, current clamp,single channel patch-clamp recording as well as Ca2+imaging, on freshly isolated DRGneurons. Our investigations revealed that PKA signal pathway plays a crucial role on regulating HCN channel activity, KT5720attenuated HCN channel activity. In presence of3μM KT5720, HCN channel currents density decreased. Its V1/2shifted tohyperpolarization direction, and the channel activation time constant significantlyincreased. Moreover, PKA inhibition with KT5720attenuated the action potentials (APs)and decreased intracellular Ca2+level, which therefore dampened DRG neuronsexcitability.(2) The role of HCN channel on regulating neuropathic pain. Neuropathic pain wascharacterized with high incidence and refractory, which often associated with nerve injury.It’s a major medical problem yet to be resolved. On the other hand, DRG neuron is theprimary sensory afferent, it’s essential for pain signal transportation. Hence, we choseDRG neuron as our study subject, on spared-nerve injury model, with using multipleelectrophysiological patch-clamp recording methods. We studied the underlyingmechanisms of HCN channel regulating neuropathic pain, unveiled that HCN channel is akey mediator on triggering and maintaining neuropathic pain. Under spared nerve injuryinduced pain condition, HCN channel activity in DRG neuron significantly increased, theectopic discharge frequency potentiated, and DRG morphology changed. Therefore, HCNchannel represents a novel potential target for the treatment of this disease.(3) The impact of ephedrine on HCN channel activity. Ephedrine is an adrenalagonist, which has the functions of strengthening heart contraction, enhancing cardiacoutput, potentiating heart rate and improving blood pressure as well. To investigate ifHCN channel involved in these regulations, we carried out experiments with conventionalwhole-cell patch-clamp recording method. We obtained HCN channel current Ihonneonatal rat hippocampal neurons and HEK cells. Our experimental results indicated thatIhattenuated by ephedrine. The likelihood mechanism is that HCN channel has somefeedback functions. When blood pressure or heart rate increases abnormally, HCN channelbeing able to partly block these momentums, and therefor maintains organisms’ health andlife safety.更多还原