超极化激活环核苷酸门控阳离子通道(HCN)研究进展

HCN是超极化激活环核苷酸门控阳离子通道,其激活后产生If/Ih电流,能被ZD7288和Cs+特异性阻断.该通道有4个亚型,具有稳定细胞膜电位、参与心脏和神经节律调节、参与树突整合,以及调节神经递质释放等生理功能.近期实验中发现豚鼠膀胱ICC上存在Ih电流,其功能特点值得进一步研究和探讨.     

HCN通道:生物学特性及疼痛相关作用

脊椎动物的超极化激活环核苷酸门控通道(hyperpolarization-activated cyclic nucleotide-gated channels,HCN通道)具有反向电压依赖性,其开放依赖细胞表面的超极化。HCN在机体各组织的分布和数量及开放状态存在差异。HCN通道的开放受到cAMP及其它物质或信号传导通路直接或者间接的调控。HCN及其介导的Ih/If电流可以影响细胞膜静息电位,控制神经元兴奋性、突触电位和突触传递并在调节心律等方面起到重要作用,并且参与了疼痛等生理或病理过程的调控。部分药物可以通过对HCN通道的作用治疗疼痛等相关疾病。本文将从HCN通道的结构、分布、调控、在疼痛及其它相关疾病中起到的作用等方面对近年来HCN通道研究的新发现进行回顾和综述。     

HCN 通道在神经系统中的分布及功能

超极化激活的环核苷酸门控的阳离子通道(hyperpolarization activated cyclic nucleotide gated channels,HCN),分为四个亚型:HCN1、HCN2、HCN3和HCN4。关于其在神经系统中作用的研究有很多,但是有些研究的结果似乎是矛盾的,这些矛盾的结果可能与其分布特点有关。在神经系统中,HCN通道的各个亚型的分布具有差异,这决定了其作用的差异性,因此在不同区域有其特定的生理功能。本文从不同脑区、脊髓及外周DRG等方面综述了HCN通道4个亚型在神经系统的分布,并且针对具体组织、核团分析其作用和生理功能。     

人HCN4通道的滞后现象:影响窦房结起搏的潜在决定因素(英文)

超极化活化环核苷酸门控(hyperpolarization-activated cyclic-nucleotide-gated,HCN)通道参与调制心脏跳动的节律和速率。与HCN1和HCN2有所不同,慢通道HCN4可能不存在电压依赖的滞后现象。本研究采用单细胞膜片钳方法,在稳定转染hHCN4的HEK293细胞上进行电生理记录,观察hHCN4通道是否存在滞后现象,以及cAMP对其的调制作用;同时采用实时定量RT-PCR方法检测窦房结和心房组织中HCNs的表达。电压钳实验结果显示hHCN4电流(Ih)激活随着保持电位超极化的变化而向去极化方向移动。三角电位变化钳(triangular ramp)和动作电位钳的结果也显示了hHCN4的滞后现象。cAMP增加Ih电流幅度,且使电流激活向去极化方向移动,从而改变内源性hHCN4滞后行为。RT-PCR结果显示,人窦房结组织主要表达HCN4,占75%,HCN1占21%,HCN2占3%,HCN3占0.7%。以上结果提示,人窦房结组织主要表达HCN4亚型,hHCN4的Ih存在电压依赖性的滞后现象,且受cAMP调制。由此推断,hHCN4通道的滞后现象可能在窦房结起搏活动中起到了关键作用。     

用生物学方法重建心脏起搏点的研究进展

正常人的心脏节律源于右心房的天然起搏点（pacemaker）——窦房结。窦房结的功能异常或者房室传导阻滞会导致心率异常（如心律缓慢）。治疗严重的心动过缓需要植入在技术上已经相当成熟的电子起搏器,但这种治疗存在一些缺陷和不足。近年来,在动物实验模型中应用基因或细胞来重建心脏的生物起搏点已经取得了进展。超极化活化环核苷酸门控（hyperpolarization-activated cyclic-nucleotide-modulated,HCN）通道（起搏通道）通过超极化活化的阳离子电流（hyperpolarization-activated cation current,It）调制心脏的自律性。利用病毒载体或转染HCN基因的细胞将HCN基因导入动物心脏内可重建生物起搏点。也有导入其它基因或植入自律细胞来探索心脏起搏点的重建。本文总结了重建心脏生物起搏点的一些研究进展。一旦稳定性和寿命等关键问题得到相应解决,遗传工程改造的生物起搏点可用于治疗严重的心动过缓。     

HCN通道在焦虑中作用的研究进展

超极化激活环核苷酸门控(hyperpolarization-activated cyclic nucleotide-gated,HCN)通道具有重要的生理功能,尤其是在静息膜电位、树突整合、神经元起搏和动作电位阈值的建立等方面作用明显。研究发现,HCN通道的失调可能会引起焦虑,该通道介导焦虑作用的机制可能受脑源性神经营养因子(brain-derived neurotrophic factor,BDNF)/哺乳动物雷帕霉素靶点(mammalian target of rapamycin,mTOR)、谷氨酸(glutamate,Glu)、γ-氨基丁酸(γ-aminobutyric acid,GABA)、单胺类神经递质、突触可塑性等的调节。现就HCN通道的结构、分布、调节及介导焦虑作用可能的机制进行综述,以期为焦虑症的预防和治疗提供药物治疗靶点。     

HCN 通道在生物胃肠道起搏机制中作用研究现状

HCN通道(hyperpolarization-activated cyclic nucleotide-gated channels)是一种超极化激活的,钠、钾、钙离子混合通透的,直接受cAMP调控的离子通道,在人体内的分布具有一定的组织和细胞特异性,其不仅与神经系统疾病联系紧密,与胃肠道动力障碍疾病也存在一定联系。对HCN通道生理功能、在胃肠起搏电流形成中作用及与疾病的关系的深入了解,必将对今后的研究以及临床治疗有实际意义。     

超极化激活的环核苷酸门控通道（HCN 通道）抑制剂的研究进展

超极化激活的环核苷酸门控通道(HCN通道)有四个亚型,分别为HCN1-4。HCN通道各亚型之间的基本结构相似,在许多组织中均有表达,其中以大脑和心脏组织中表达最为丰富。HCN通道既参与所在组织的正常生理功能,也与所在组织的病理状态密切相关。如神经损伤引起的神经源性疼痛常检测到HCN1通道表达量的增加,肥厚性心肌病和终末期心力衰竭等病理状态下常检测到心室肌细胞HCN4 mRNA及HCN2 mRNA表达增加。鉴于HCN通道与许多疾病密切相关,因此,以其为靶点来治疗相关疾病成为可能,但是由于HCN通道分布广泛,而目前该通道阻滞剂均为非选择性亚型抑制剂,临床应用时不可避免的引起副反应,因此发展选择性HCN通道亚型抑制剂就显得刻不容缓。本文就HCN通道抑制剂的研究发展做进一步探讨。     

HCN通道在爪蟾卵母细胞中的功能表达

目的:建立爪蟾卵母细胞表达的HCN通道的细胞模型,研究其生物学特性,并为药物评价建立细胞模型。方法:将HCN1及HCN2的互补DNA(c DNA)体外转录为互补RNA(c RNA)后,分别注射至去除滤泡膜的非洲爪蟾卵母细胞中,表达1~3 d后采用双电极电压钳技术记录其电流。结果:在爪蟾卵母细胞表达的HCN1及HCN2通道的同聚体细胞模型上,记录到了超极化激活的内向阳离子电流,此电流被称为Ih电流,可被HCN通道特异性阻断剂Cs Cl所阻断;在1 mmol/L的Cs Cl作用下,HCN通道产生的电流幅度显著减小,在-140 m V水平,HCN1电流幅度减少率为83.4%±9.5%(n=5,P0.001),HCN2产生的电流幅度减少率为99.7%±0.6%(n=4,P0.001)。结论:建立了表达HCN1及HCN2通道的爪蟾卵母细胞模型,为研究HCN通道生物学特点以及药物评价奠定了基础。     

植物环核苷酸门控离子通道基因的功能及其调控

环核苷酸(cAMP/cGMP)是生命体重要的信号分子,环核苷酸门控离子通道(CNGC)是环核苷酸主要的受体之一,目前已在植物中克隆并鉴定了多个环核苷酸门控离子通道基因,它们参与调控植物的生长、发育以及抗病等反应.这些通道既可通过一价阳离子,也可通过二价阳离子,其活性受Ca2+/Calmodulin调控.本文概括了近年来植物环核苷酸门控离子通道(CNGC)基因的克隆、植物CNGC对离子的选择特性、CNGC的生物学功能与调控等方面的研究进展.     

TRPM7 is a stretch- and swelling-activated cation channel involved in volume regulation in human epithelial cells

Stretch- and swelling-activated cation (SSAC) channels play essential roles not only in sensing and transducing external mechanical stresses but also in regulating cell volume in living cells. However, the molecular nature of the SSAC channel has not been clarified. In human epithelial HeLa cells, single-channel recordings in cell-attached and inside-out patches revealed expression of a Mg²⁺- and Gd³⁺-sensitive nonselective cation channel that is exquisitely sensitive to membrane stretch. Whole cell recordings revealed that the macroscopic cationic currents exhibit transient receptor potential (TRP) melastatin (TRPM)7-like properties such as outward rectification and sensitivity to Mg²⁺ and Gd³⁺. The whole cell cation current was augmented by osmotic cell swelling. RT-PCR and Western blotting demonstrated molecular expression of TRPM7 in HeLa cells. Treatment with small interfering RNA (siRNA) targeted against TRPM7 led to abolition of single stretch-activated cation channel currents and of swelling-activated, whole cell cation currents in HeLa cells. The silencing of TRPM7 by siRNA reduced the rate of cell volume recovery after osmotic swelling. A similar inhibition of regulatory volume decrease was also observed when extracellular Ca²⁺ was removed or Gd³⁺ was applied. It is thus concluded that TRPM7 represents the SSAC channel endogenously expressed in HeLa cells and that, by serving as a swelling-induced Ca²⁺ influx pathway, it plays an important role in cell volume regulation. regulatory volume decrease     

Hydrogen peroxide and ADP-ribose induce TRPM2-mediated calcium influx and cation currents in microglia

Microglial cells are the host macrophages in the central nervous system and respond to brain injury and various neurological diseases. In this process, microglial cells undergo multiple morphological and functional changes from the resting cell toward a fully activated, phagocyting tissue macrophage. In culture, bacterial lipopolysaccharide (LPS) is a frequently used tool to induce this activation. By using calcium-imaging and patch-clamp techniques, we investigated the effect of hydrogen peroxide (H₂O₂), which is released by macrophagic cells themselves, on the intracellular calcium concentration and ion currents in cultured rat microglia. Application of 0.1–5 mM H₂O₂ for several minutes induced small responses in untreated cells but a large calcium influx and cation current in LPS-treated cells. In both untreated and LPS-treated microglia, internal perfusion of ADP-ribose (ADPR) via the patch pipette elicited large cation currents. Both stimuli, H₂O₂ and ADPR, have been reported to activate the recently cloned nonselective cation channel TRPM2. RT-PCR analysis from cultured rat glial and neuronal cells confirmed a strong expression of TRPM2 in rat microglia but not in astrocytes and cerebellar granule cells. In situ hybridizations from mouse brain showed a distribution of TRPM2, which is compatible with the expression in microglial cells. In conclusion, we describe here a novel calcium influx pathway in microglia coupled to hydrogen peroxide and ADPR and provide evidence that this pathway involves TRPM2. The increased sensitivity to H₂O₂ in LPS-stimulated cells suggests a role for TRPM2 in the calcium signaling of activated microglia. nonselective cation channel; transient receptor potential channel; H₂O₂; activated microglia     

Inhibition of transient receptor potential melastatin 7 (TRPM7) protects against Schwann cell trans-dedifferentiation and proliferation during Wallerian degeneration

ABSTRACT

Irreversible peripheral neurodegenerative diseases such as diabetic peripheral neuropathy are becoming increasingly common due to rising rates of diabetes mellitus; however, no effective therapeutic treatments have been developed. One of main causes of irreversible peripheral neurodegenerative diseases is dysfunction in Schwann cells, which are neuroglia unique to the peripheral nervous system (PNS). Because homeostasis of calcium (Ca²⁺) and magnesium (Mg²⁺) is essential for Schwann cell dynamics, the regulation of these cations is important for controlling peripheral nerve degeneration and regeneration. Transient receptor potential melastatin 7 (TRPM7) is a non-selective ion (Ca²⁺ and Mg²⁺) channel that is expressed in Schwann cells. In the present study, we demonstrated in an ex vivo culture system that inhibition of TRPM7 during peripheral nerve degeneration (Wallerian degeneration) suppressed dedifferentiable or degenerative features (trans-dedifferentiation and proliferation) and conserved a differentiable feature of Schwann cells. Our results indicate that TRPM7 could be very useful as a molecular target for irreversible peripheral neurodegenerative diseases, facilitating discovery of new therapeutic methods for improving human health.     

Functional transient receptor potential melastatin 7 channels are critical for human mast cell survival

Mast cells play a significant role in the pathophysiology of many diverse diseases such as asthma and pulmonary fibrosis. Ca2+ influx is essential for mast cell degranulation and release of proinflammatory mediators, while Mg2+ plays an important role in cellular homeostasis. The channels supporting divalent cation influx in human mast cells have not been identified, but candidate channels include the transient receptor potential melastatin (TRPM) family. In this study, we have investigated TRPM7 expression and function in primary human lung mast cells (HLMCs) and in the human mast cell lines LAD2 and HMC-1, using RT-PCR, patch clamp electrophysiology, and RNA interference. Whole cell voltage-clamp recordings revealed a nonselective cation current that activated spontaneously following loss of intracellular Mg2+. The current had a nonlinear current-voltage relationship with the characteristic steep outward rectification associated with TRPM7 channels. Reducing external divalent concentration from 3 to 0.3 mM dramatically increased the size of the outward current, whereas the current was markedly inhibited by elevated intracellular Mg2+ (6 mM). Ion substitution experiments revealed cation selectivity and Ca2+ permeability. RT-PCR confirmed the presence of mRNA for TRPM7 in HLMC, LAD2, and HMC-1 cells. Adenoviral-mediated knockdown of TRPM7 in HLMC with short hairpin RNA and in HMC-1 with short interfering RNA markedly reduced TRPM7 currents and induced cell death, an effect that was not rescued by raising extracellular Mg2+. In summary, HLMC and human mast cell lines express the nonselective cation channel TRPM7 whose presence is essential for cell survival.     

Death and survival of heterozygous Lurcher Purkinje cells In vitro

The differentiation and survival of heterozygous Lurcher (+/Lc) Purkinje cells in vitro was examined as a model system for studying how chronic ionic stress affects neuronal differentiation and survival. The Lurcher mutation in the δ2 glutamate receptor (GluRδ2) converts an orphan receptor into a membrane channel that constitutively passes an inward cation current. In the GluRδ2^+/Lc mutant, Purkinje cell dendritic differentiation is disrupted and the cells degenerate following the first week of postnatal development. To determine if the GluRδ2^+/Lc Purkinje cell phenotype is recapitulated in vitro, +/+, and +/Lc Purkinje cells from postnatal Day 0 pups were grown in either isolated cell or cerebellar slice cultures. GluRδ2^+/+ and GluRδ2^+/Lc Purkinje cells appeared to develop normally through the first 7 days in vitro (DIV), but by 11 DIV GluRδ2^+/Lc Purkinje cells exhibited a significantly higher cation leak current. By 14 DIV, GluRδ2^+/Lc Purkinje cell dendrites were stunted and the number of surviving GluRδ2^+/Lc Purkinje cells was reduced by 75% compared to controls. However, treatment of +/Lc cerebellar cultures with 1‐naphthyl acetyl spermine increased +/Lc Purkinje cell survival to wild type levels. These results support the conclusion that the Lurcher mutation in GluRδ2 induces cell autonomous defects in differentiation and survival. The establishment of a tissue culture system for studying cell injury and death mechanisms in a relatively simple system like GluRδ2^+/Lc Purkinje cells will provide a valuable model for studying how the induction of a chronic inward cation current in a single cell type affects neuronal differentiation and survival. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2009     

内皮细胞电压门控钙离子通道及其功能研究进展

内皮细胞（endothelial cell,EC）作为不可兴奋细胞,早前通常被认为缺乏功能性电压门控钙离子通道（voltagegated calcium channel,VGCC）,如人脐静脉内皮细胞、牛肺动脉内皮细胞、牛主动脉内皮细胞等。随着膜片钳技术、荧光显微技术、聚合酶链式反应（PCR）技术的发展,越来越多的VGCC在各种内皮细胞中被发现,如人主动脉内皮细胞、大鼠主动脉内皮细胞、大鼠肺微血管内皮细胞等。目前对于VGCC存在与否主要有3种检测方法:利用膜片钳技术对离子通道电流的检测、利用荧光显微技术对胞内钙离子浓度变化的检测、利用PCR技术对离子通道基因或蛋白质表达的检测。内皮细胞不单单是血液和其他相邻组织细胞及基质蛋白间的物理屏障,更重要的是通过细胞膜上VGCC的开放和关闭对细胞和血管组织的生理变化产生显著的影响。一方面,VGCC对胞内钙离子浓度变化的影响,控制着一氧化氮（NO）等血管舒张因子的释放,调节血管张力的平衡。另一方面,作为钙离子内流重要途经的VGCC,经过Ras和MEK通路的诱导、磷酸化PI3K和Akt通路,影响内皮细胞迁移和增殖。此外,部分生理现象,如血管内压力产生...     

Voltage-dependent L-type Ca channels and a novel type of non-selective cation channel activated by cAMP-dependent phosphorylation in mesoderm-like (MES-1) cells

Undifferentiated P19 embryonal carcinoma cells (ECC P19), the P19-derived clonal cell lines END-2 (visceral endoderm-like), EPI-7 (epithelioid ectoderm-like), MES-1 (mesoderm-like) and a parietal yolk sac cell line (PYS-2) were used as cellular models to examine the functional expression of voltage-dependent Ca channels and other Ca-permeable cation channels at various stages of early embryonic development. Whole-cell currents were recorded by means of the patch clamp technique. Whereas more than 75% of MES-1 cells possessed Ca channel currents, neither P19, END-2, EPI-7 nor PYS-2 cells had detectable voltage-dependent inward currents. Ca channel currents of MES-1 cells were highly sensitive towards 1,4-dihydropyridines and blocked by cadmium. Adrenaline (10 μM) caused Ca channel stimulation in only 14% of MES-1 cells examined. However, in 62% of the cells adrenaline activated a linear current component which under physiological conditions reversed close to 0 mV. Removal of extracellular Na⁺ suppressed the adrenaline-induced inward current, while reducing extracellular Cl⁻ had no significant effect. These findings suggest that the adrenaline-induced current is carried through non-selective cation channels which were found to be permeable for Na⁺, K⁺, Cs⁺ å Ca²⁺. Remarkably, the intracellular signalling pathway for activation of the non-selective cation current involved the cascade of reactions leading to cAMP-dependent phosphorylation, a regulatory pathway well known for cardiac Ca channels. A possible functional role of adrenaline-induced non-selective cation currents and Ca channels in embryonal development is discussed.     

Activation of a nonselective cation channel by swelling in atrial cells

Cell swelling has been shown to increase the permeability of the plasma membrane to ions such as K⁺, Na⁺, Ca²⁺ or Cl^– in many types of cells. In cardiac cells, swelling has been reported to increase Cl^– conductance, but whether cation-selective currents are activated by swelling is not known. Low Cl^– or Cl^–-free solutions were used to study the presence of such currents. Lowering the osmolarity of the extracellular medium from 299 to 219 mOsm resulted in cell swelling and concurrent activation of a cation-selective whole-cell current. When cell-attached patches were formed on swollen cells, opening of bursting single channel currents were observed in 18% of the patches studied. Ion substitution experiments indicated that the channel discriminated poorly among monovalent cations, and was impermeable to Cl^–. The channel was permeable to Ca²⁺. In symmetrical 140 mM K⁺, the current-voltage relation was linear with a single channel conductance of 36 ± 3 pS. Depolarization increased channel open probability. Interestingly, depending on the membrane patch studied, application of negative pressure to the pipette caused either an increase or a decrease in the open probability of the channel already activated by swelling. Thus, the sensitivity to tension of the swelling-activated channel was different from those of previously reported stretch-activated channels. These findings suggest that nonselective cation channels exist in rat atrial cells and may be involved in swelling-induced changes in cell function.Dr. Kim is an Established Investigator of the American Heart Association.     

TRP channels in disease

“Transient receptor potential” cation channels (TRP channels) play a unique role as cell sensors, are involved in a plethora of Ca²⁺-mediated cell functions, and play a role as “gate-keepers” in many homeostatic processes such as Ca²⁺ and Mg²⁺ reabsorption. The variety of functions to which TRP channels contribute and the polymodal character of their activation predict that failures in correct channel gating or permeation will likely contribute to complex pathophysiological mechanisms. Dysfunctions of TRPs cause human diseases but are also involved in a complex manner to contribute and determine the progress of several diseases. Contributions to this special issue discuss channelopathias for which mutations in TRP channels that induce “loss-“ or “gain-of-function” of the channel and can be considered “disease-causing” have been identified. The role of TRPs will be further elucidated in complex diseases of the intestinal, renal, urogenital, respiratory, and cardiovascular systems. Finally, the role of TRPs will be discussed in neuronal diseases and neurodegenerative disorders.     

The vanilloid transient receptor potential channel TRPV4: From structure to disease

The Transient Receptor Potential Vanilloid 4 channel, TRPV4, is a Ca²⁺ and Mg²⁺ permeable non-selective cation channel involved in many different cellular functions. It is activated by a variety of physical and chemical stimuli, including heat, mechano-stimuli, endogenous substances such as arachidonic acid and its cytochrome P450-derived metabolites (epoxyeicosatrienoic acids), endocannabinoids (anandamide and 2-arachidonoylglycerol), as well as synthetic α-phorbol derivatives. Recently, TRPV4 has been characterized as an important player modulating osteoclast differentiation in bone remodelling and as a urothelial mechanosensor that controls normal voiding. Several TRPV4 gain-of-function mutations are shown to cause autosomal-dominant bone dysplasias such as brachyolmia and Koszlowski disease. In this review we comprehensively describe the structural, biophysical and (patho)physiological properties of the TRPV4 channel and we summarize the current knowledge about the role of TRPV4 in the pathogenesis of several diseases.