Thalamocortical Circuit Controls Neuropathic Pain via Up-regulation of HCN2 in the Ventral Posterolateral Thalamus / 神经科学通报·英文版

The thalamocortical (TC) circuit is closely associated with pain processing. The hyperpolarization-activated cyclic nucleotide-gated (HCN) 2 channel is predominantly expressed in the ventral posterolateral thalamus (VPL) that has been shown to mediate neuropathic pain. However, the role of VPL HCN2 in modulating TC circuit activity is largely unknown. Here, by using optogenetics, neuronal tracing, electrophysiological recordings, and virus knockdown strategies, we showed that the activation of VPL TC neurons potentiates excitatory synaptic transmission to the hindlimb region of the primary somatosensory cortex (S1HL) as well as mechanical hypersensitivity following spared nerve injury (SNI)-induced neuropathic pain in mice. Either pharmacological blockade or virus knockdown of HCN2 (shRNA-Hcn2) in the VPL was sufficient to alleviate SNI-induced hyperalgesia. Moreover, shRNA-Hcn2 decreased the excitability of TC neurons and synaptic transmission of the VPL-S1HL circuit. Together, our studies provide a novel mechanism by which HCN2 enhances the excitability of the TC circuit to facilitate neuropathic pain.

The effects of PDK1-Akt signaling pathway intervention on cardiomyocyte HCN4 ion channels / 中华心血管病杂志

Objective: To explore the effects of 3-phosphate dependent protein kinase 1-protein kinase B (PDK1-Akt) signaling pathway on the transcription, expression and function of cardiac hyperpolarized activated cyclic nucleotide gated 4 (HCN4) ion channels. Methods: Atrial myocytes were obtained from healthy male wild-type C57 mice and heart-specific PDK1 knockout mice (PDK1-KO) by enzymolysis. Then the atrial myocytes were divided into blank control group and PDK1-KO group. In further studies, the isolated atrial myocytes were cultured and further divided into drug control group (treated with dimethyl sulfoxide (DMSO)) and PDK1 knockdown group (treated with 1 μg/ml PDK1 short hairpin RNA (shRNA) interference plasmid), SC79 group (treated with 8 μmol/ml SC79), GSK2334470 group (treated with 10 nmol/L GSK2334470) and PDK1 knockdown+SC79 group (8 μmol/ml SC79 and 1 μg/ml PDK1 shRNA interference plasmid). Real time quantitative PCR (qRT-PCR) was used to detect the mRNA expression levels of PDK1 and HCN4, Western blot was used to detect the protein expression levels of PDK1, Akt and HCN4, the whole cell patch clamp was used to detecte the current density of HCN, and immunofluorescence was used to detecte the expression of HCN4 protein on atrial cells. Results: (1) the expression levels of HCN4 mRNA (1.46±0.03 vs. 0.99±0.01, P<0.001) and protein (1.14±0.02 vs. 1.00±0.06, P=0.017) in PDK1-KO group were higher than those in blank control group. The HCN current density in PDK1-KO group was higher than that in blank control group((-17.47±2.00) pA/pF vs. (-12.15±2.25) pA/pF, P=0.038). (2) The functions of PDK1 shRNA and specific Akt agonist SC79 were verified by comparing the PDK1 knockdown group and SC79 group with the drug control group. The results showed that the expression levels of PDK1 mRNA and protein in PDK1 knockdown group were lower than those in drug control group, and the expression level of phosphorylated Akt (Thr 308) protein in SC79 group was higher than that in drug control group. (3) The expression levels of HCN4 mRNA (3.61±0.46 vs. 1.00±0.08, P<0.001) and protein (2.33±0.11 vs. 1.00±0.05, P<0.001) in GSK2334470 group were higher than those in drug control group. (4) To reduce the effect of drug-miss target, the cultured atrial myocytes were transfected with shRNA plasmid of PDK1 and intervened with SC79. The results showed that the expression of HCN4 mRNA in PDK1 knockdown group was higher than that in the drug control group (1.76±0.11 vs. 1.00±0.06, P<0.001), and PDK1 knockdown+SC79 group (1.76±0.11 vs. 1.33±0.07, P=0.003). In PDK1 knockdown+SC79 group, the mRNA expression level was also higher than that in the drug control group (1.33±0.07 vs. 1.00±0.06, P<0.001). The expression level of HCN4 protein in PDK1 knockdown group was higher than that in drug control group (1.15±0.04 vs. 1.00±0.05, P=0.003). As for the The expression level of HCN4 protein, there was no significantly statistical difference between the PDK1 knockdown+SC79 group and the drug control group (P>0.05), but PDK1 knockdown+SC79 group was lower than PDK1 knockdown group (0.95±0.01 vs. 1.15±0.04, P<0.001). In patch clamp experiments, the results showed that the HCN current density was (-13.27±1.28) pA/pF in the drug control group, (-18.76±2.03) pA/pF in the PDK1 knockdown group, (-13.50±2.58) pA/pF in the PDK1 knockdown+SC79 group; the HCN current density of PDK1 knockdown group was higher than that of drug control group (P<0.001), but there was no significant difference between PDK1 knockdown+SC79 group and drug control group (P>0.05). (5) The results of immunofluorescence showed that the brightness of green fluorescence of PDK1 knockdown group was higher than that of drug control group, indicating that the expression of HCN4 localized on cell membrane was increased. However, the green fluorescence of PDK1 knockdown+SC79 group was lighter than that of PDK1 knockdown group, suggesting that the expression of HCN4 in PDK1-knockdown cell membrane decreased after further activating Akt. Conclusion: PDK1-Akt signaling pathway is involved in the regulation of HCN4 ion channel transcription, expression and function.

Blockade of HCN2 Channels Provides Neuroprotection Against Ischemic Injury via Accelerating Autophagic Degradation in Hippocampal Neurons / 神经科学通报·英文版

In the central nervous system, hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are essential to maintain normal neuronal function. Recent studies have shown that HCN channels may be involved in the pathological process of ischemic brain injury, but the mechanisms remain unclear. Autophagy is activated in cerebral ischemia, but its role in cell death/survival remains controversial. In this study, our results showed that the HCN channel blocker ZD7288 remarkably decreased the percentage of apoptotic neurons and corrected the excessive autophagy induced by oxygen-glucose deprivation followed by reperfusion (OGD/R) in hippocampal HT22 neurons. Furthermore, in the OGD/R group, p-mTOR, p-ULK1 (Ser), and p62 were significantly decreased, while p-ULK1 (Ser), atg5, and beclin1 were remarkably increased. ZD7288 did not change the expression of p-ULK1 (Ser), ULK1 (Ser), p62, Beclin1, and atg5, which are involved in regulating autophagosome formation. Besides, we found that OGD/R induced a significant increase in Cathepsin D expression, but not LAMP-1. Treatment with ZD7288 at 10 μmol/L in the OGD/R group did not change the expression of cathepsin D and LAMP-1. However, chloroquine (CQ), which decreases autophagosome-lysosome fusion, eliminated the correction of excessive autophagy and neuroprotection by ZD7288. Besides, shRNA knockdown of HCN2 channels significantly reduced the accumulation of LC3-II and increased neuron survival in the OGD/R and transient global cerebral ischemia (TGCI) models, and CQ also eliminated the effects of HCN2-shRNA. Furthermore, we found that the percentage of LC3-positive puncta that co-localized with LAMP-1-positive lysosomes decreased in Con-shRNA-transfected HT22 neurons exposed to OGD/R or CQ. In HCN2-shRNA-transfected HT22 neurons, the percentage of LC3-positive puncta that co-localized with LAMP-1-positive lysosomes increased under OGD/R; however, the percentage was significantly decreased by the addition of CQ to HCN2-shRNA-transfected HT22 neurons. The present results demonstrated that blockade of HCN2 channels provides neuroprotection against OGD/R and TGCI by accelerating autophagic degradation attributable to the promotion of autophagosome and lysosome fusion.

Hyperpolarization activated cyclic nucleotide-gated cation channels involved in central nervous system diseases:research advances / 国际药学研究杂志

Hyperpolarization activated cyclic nucleotide-gated cation (HCN) channels are involved in neuronal rhythm regulation, synaptic activity, membrane resistance and dendritic integration due to their voltage-gated physiological properties. Recent studies have shown that HCN channels play an important role in central nervous system diseases, such as temporal lobe epilepsy, neuropathic pain, learning and memory disorder, substance abuse addiction and other related diseases. In this paper, we summarize the research progress of HCN channels in central nervous system diseases in recent years.

HCN and Kv4 channel in epilepsy / 国际儿科学杂志

Voltage-gated ion channels play an important role in cell's excitability.HCN is short for hyperpolarization-activated cyclic nucleotide-gated channels, and Kv4 family is the main ingredients of transient outward potassium current (IA) produced by hippocampal neuron, both of which belong to voltage-gated ion channels.HCN channel and its mediated Ih current could influence the resting potential of cell membrane, control the excitability of neurons, synaptic potential and synaptic transmission.They also participate in cardiac pacing and rhythmic activity of some neurons.IA is the main ingredients of outward potassium current in the early period of action potential repolarization, which mainly takes part in regulating the synaptic input and counterpropagation of action potential.Kv4 channel could reduce the excitability of neurons.Studies indicate that mutations of these two channels lead to epilepsy.This article focuses on the structure、 distribution and function of HCN and Kv4 in epilepsy.

Effect of XinBao pill on electrophysiological properties of human HCN4 channel / 中国生化药物杂志

Objective To explore the basic medical mechanism of XinBao pill on electrophysiological characteristics of hyperpolarization-activated cyclic nucleotide-gated channel 4 (HCN4), and illustrate the mechanism of its therapeutical effect on bradycardia.Methods Human HCN4 mRNA was injected into the Xenopus laevis oocytes, after incubated for 2 ~3 days, channel current properties of HCN4 perfused with 40 mg/L XinBao Pill were observed by double electrode voltage clamp technique.Results At-90mV test potential, compared with control group (no XinBao pill), HCN4 channel peak current and tail current in 40 mg/L XinBao pill group had obvious changes, and V1/2 from ( -103.61 ±3.57)mV to ( -106.42 ±5.33)mV in XinBao pill group, from( -81.11 ±4.26)mV to( -86.36 ±7.44)mV in control group.The values of k from (15.15 ±2.23)mV to (17.33 ±3.58) mV in XinBao pill group, from(11.78 ±0.85)mV to(12.39 ±1.51)mV in control group(n=10).At test potential -90 mV, 40 mg/L XinBao pill perfusion fluid decreased the instantaneous current of(0.15 ±0.24)%, the EC50 was (30.8 ±4.8)mg/L (n=8).At test potential-140 mV~-100 mV level, 40 mg/L XinBao pill group increased the channel activation time constant compared with control group[(226.73 ±31.36)ms vs(143.67 ± 21.44)ms;-140 mV,n=10,P<0.05].40 mg/L XinBao pill group increased the channel deactivation time constant compared with control group [(1293.53 ±95.02)ms vs (647.12 ±61.35)ms;-140 mV,n=10,P<0.05].Conclusion The XinBao pill enhances the instantaneous current of HCN4 in a concentration-dependent manner, and extents channel activation and deactivation processes.

Role of hyperpolarization-activated cyclic nucleotide-gated cation nonselective channel in spontaneous contrac-tion of smooth muscles in human isolated ureters / 局解手术学杂志

Objective To investigate effects of hyperpolarization-activated cyclic nucleotide-gated cation nonselective channel in the hu-man ureter on the spontaneous contraction of smooth muscles. Methods Four HCN subtypes were detected in human ureteral tissue using reverse transcription polymerase chain reaction,Western blotting and immunohistochemical. ZD7288,the HCN blocker, was used to observe the changes of ureteral muscle contraction amplitude and frequency by applying the ureteral smooth muscle strip test in vitro. Results HCN1-4 isoforms were all identified in human ureter using reverse transcription-polymerase chain reaction and Western blotting. Through the immunohistochemical,HCN channel was found mostly in the urothelium layer and muscular layer of human ureteral wall. ZD7288 significantly decreased the bladder excitation. Conclusion All 4 HCN channel hypotypes exist in the human ureter, and affect the ureteral excitation.

The role of hyperpolarization-activated cyclic nucleotide-gated channels in learning and memory / 国际儿科学杂志

Hyperpolarization-activated cyclic nucleotide (HCN)-gated channels belong to a nonselective ion channel family,which are common in neurocyte and cardiac muscle cell.These channels play important roles in the nervous system because of their characterstics of hyperpolarization activation.Many studies have shown that HCN channels play key point in excitatory synaptic transmission and integration of output signal.With the development of research,more evidences have been found to prove that the motor learning is damaged and spatial learning ability is enhanced in HCN1 knockout mice.There are two potential mechanisms leading to these changes,one is the lack of HCN1 in cerebellum cell,and the other is the lack of HCN1 in trisynaptic circuits of hippocampus.

Production of biocontrol traits by banana field fluorescent Pseudomonads and comparison with chemical fungicide.

Pseudomonas aeruginosa isolated from banana field rhizosphere produced different antifungal metabolites like bactriocin, hydrogen cyanide and siderophore. Bacteriocinogenic, siderophoregenic, and HCN rich broth of isolate inhibited the growth of phytopathogen like Aspergilus niger, Aspergilus flavus, Fusarium oxysporum and Alternaria alternata. The isolate exhibited more antifungal activity and comparatively low MIC vis-a-vis commonly used copper based systemic chemical fungicide;bil cop.

Histology of canine pulmonary veins and expression of HCN channel / 重庆医学

Objective To observe the anisotropy of myocardial fiber orientation and conduction cells in canine pulmonary vein , and to investigate the expression of funny current (If ) channel subunit HCN1 ,HCN2 ,HCN4 mRNA in cadiocytes of pulmonary veins and left atrial in acute atrial fibrillation (AF) modle of caine .Methods Fourteen adult mongrel canines were randomly divided into atrial fibrillation group(n=7) and control group(n=7) .A model of acute AF has been developed in rapid atrial pacing (RAP) . The cardiocytes in control group were detect using hematoxylin hematoxylin and eosin (HE) and Periodic acid-Schiff(PAS) stain in the pulmonary vein .The mRNA expressions of HCN1 ,HCN2 ,HCN4 in two group were quantified by semiquantitative reverse transcription transcription-polymerase chain reaction(RT-PCR) .Results There were complex myocardial fiber orientation in pul-monary veins especially in PV-LA junction .PAS-positive cells could be seen in the endomembrane of pulmonary veins muscle sleeve .There were mRNA expression of HCN2 and HCN4 in pulmonary veins and left arial in two group ,but there was no mRNA expression of HCN1 .The mRNA levels of HCN4 from high to low were:pulmonary veins of atrial fibrillation group ,left atrial of atrial fibrillation group ,pulmonary veins of control group ,left atrial of control group(P0 .05) .Conclusion There are anisotro-py ,conduction cells in pulmonary veins .Up-regulation of pulmonary veins and left atrial HCN 2 ,HCN4 mRNA in atrial fibrillation caine ,may be correlated with the atrial fibrillation and maintenance .

Distinct Regional and Cellular Localization of Hyperpolarization-activated Cyclic Nucleotide-gated Channel 1 in Cerebellar Cortex of Rat

OBJECTIVE: Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels mediate the hyperpolarizationactivated currents (Ih) that participate in regulating neuronal membrane potential and contribute critically to pacemaker activity, promoting synchronization of neuronal networks. However, distinct regional and cellular localization of HCN channels in the brain have not been precisely defined. Aim of this study was to verify the precise cellular location of HCN1 channels in rat cerebellum to better understand the physiological role these channels play in synaptic transmission between CNS neurons. METHODS: HCN1 expression in rat brain was analyzed using immunohistochemistry and electron-microscopic observations. Postsynaptic density-95 (PSD-95), otherwise known as locating and clustering protein, was also examined to clarify its role in the subcellular location of HCN1 channels. In addition, to presume the binding of HCN1 channels with PSD-95, putative binding motifs in these channels were investigated using softwaresearching method. RESULTS: HCN1 channels were locally distributed at the presynaptic terminal of basket cell and exactly corresponded with the location of PSD-95. Moreover, nine putative SH3 domain of PSD-95 binding motifs were discovered in HCN1 channels from motif analysis. CONCLUSION: Distinct localization of HCN1 channels in rat cerebellum is possible, especially when analyzed in conjunction with the SH3 domain of PSD-95. Considering that HCN1 channels contribute to spontaneous rhythmic action potentials, it is suggested that HCN1 channels located at the presynaptic terminal of neurons may play an important role in synaptic plasticity.

Effect of Non-specific HCN1 Blocker CsCl on Spatial Learning and Memory in Mouse / 华中科技大学学报（医学）（英德文版）

It has been suggested that HCN1 is primarily expressed in hippocampus, however little is known about its effects on spatial learning and memory. In the present study, we investigated the effects of non-specific HCN1 blocker CsCl on spatial learning and memory by using Morris water maze and in situ hybridization in mice. The results showed CsCl 160 mg/kg ip for 4 days, and the mean escape latency was 34 s longer than that of normal control (P＜0.01). In hippocampal tissues, staining for the HCN1 mRNA was stronger in the DG and CA1 region of the hippocampus (P ＜0.05, P＜0.05, when CsCl-administration group was compared with normal group). Our results suggested that CsCl could significantly affect the spatial learning and memory in mice, and HCN channel is involved in the process of learning and memory.

Differentiation of rats bone marrow mesynchymal stem cells into cardiomyogenic cells with pacemaking function / 基础医学与临床

Objective To find the way of inducing the bone marrow mesynchymal stem cells(MSCs)into cardiac cells with pacemaking function in vitro.Methods Dissociate the rat MSCs and induce them with 5AZA,bFGF+EGF,HGF,SCF and lysate of the sinoatrial cells respectively.The morphological changes were observed,and the expressing of protein cTnT,connexin 43 and HCN2/4 were analyse by immunohistologic and flowcytometry techniques.The pacmaking current If were evaluted by patch clamp techniques.Results All the methods can induce the bone marrow MSCs to differentiate into cardiac cells,which expressing cardiac cell specific protein and HCN2.Cells induced by 5AZA,bFGF+EGF and SAN CMs show higher rate of HCN2 expressing(22.9%,22.3%,11%).The cells of these groups have the pacmaking current If.Conclusion Lysate of the sinoatrial cells are ideal methods of inducing the bone marrow MSCs to differentiate into cardiac cells with pacemaking function in vitro.HCN is a promising marker protein to select pacmaking cells out of the differentiated cells.

Effects of DMAP, NaNO_2 and Co_2EDTA on Hemodynamics of Severely HCN-poisoned Dogs / 第三军医大学学报

Under anesthesia and artificial respiration, all the dogs were injected with HCN 8 mg/kg intravenously. 2 minutes after HCN injection, 4-dimethylaminophenol( DM -AP) 3.2mg/kg,sodium nitrite(NaNO2)20 mg/kg,or dicobalt edetate(Co2EDTA) 15 mg/kg was given intravenously to different animals respectively. The changes of hemodynamics of the dogs were observed before and after the administration of the 3 HCN antidotes.It was found that intravenous injection of HCN rapidly induced a progressive decrease of heart rate(HR),stroke index(SI), cardiac index (CI),mean aortic pressure (MAP),left ventricular pressure(LVP) and ?dp/dtmax and a persistent increase of total peripheral vascular resistance (TPVR) and mean pulmonary arterial pressure (MPAP). If not treated,the dogs would died subsquently 4-9.5 minutes after HCN injection because of cardiac failure.2 to 8 minutes after DMAP administration, CI, MAP, LVP and?dp/dtmax increased markedly,and TPVR and MPAP would recover a little later. 4-6minutes after NaNO2 administration, ?dp/dtmax increased markedly, and CI, MAP, LVP, TPVR and MPAP returned to the normal. 6 minutes after Co2EDTA administration, MAP elevated markedly, LVP and?dp/dtmax returued to the normal, and C1, TPVR and MPAP would recover a little later. When observation was made again one hour after treatment, there was a remarkable decrease of blood pressure in NaNO2 and Co2EDTA treated animals and a slight decrease of blood pressure in DMAP treated ones.The conclusions based on the results are as follows:1. The ability of DMAP to restore the circulatory functions after HCN poisoning is more rapid and powerful than that of Co2EDTA or NaNO2.2. The cardiovascular functions restored by DMAP are more stable than those restored by the other two agents.3. DMAP and NaNO2 can rapidly reverse the HCN-iuduced damages on the cardiovascular functions, which may be a part of the anticyanide ability of these 2 drugs.

A Comparison of Antidotal Potency of DMAP & Am NO_2 against Inhaled HCN Intoxication in Dogs / 第三军医大学学报

The toxicity of HCN inhaled via the respiratory tract in dogs and the therapeutic effects of DMAP and AmNO2 for such form of HCN intoxication were studied. The LD50 of HCN when inhaled was 850.4?80.4?g/kg.When the dosage of 2?LD50 of HCN was given to the experimental animals, the signs of intoxication developed rapidly. Uneasiness, struggling, strident barking, and respiratory excitation were observed several seconds after the exposure. Subsequently there was spasm of the extensors and rigidity of the extremities. Finally general inhibition and paralysis ensued. Respiration and heart beat stopped 6 and 7 minutes after exposure. If DMAP (3.25 mg/kg intramuscularly) or AmNO2 (2 ampules through inhalation) was administered 15 seconds after exposure, the survival rate of the animals was 90% or 100% respectively.When the dosage of 4?LD50 of HCN was inhaled and the two drugs were administered 45 seconds after exposure, the survival rate of the animals treated with DMAP remained as high as 90% (9/10) .But the survival rate of those treated with two ampules of AmNO2 or even three ampules reduced to 20% or 40% respectively.The amount of HCN inhaled during respiratory intoxication, the problems of early diagnosis and the administration of DMAP at the site of accident were analyzed and discussed.

Study on morphology and surface antigen of primary cultured neonatal rats' sinoatrial node cells / 西安交通大学学报(医学版)

Objective To understand morphological characteristics of sinoatrial node cells(SNCs) by taking purified isolation and culture of neonatal rat's SNCs in vitro and to understand the expression characteristics of surface antigens hyperpolarization activated cyclic nucleotide gated cation channel 4(HCN4) and connexin 45(Cx45) on SNCs.Methods The SNCs were isolated from sinus node tissues removed from neonatal rats by trypsin and purified cultured with differential attachment and 5'-bromodeoxyuridine(BrdU) treatment.Results Three different types of cells were observed among the purified cultured SNCs: spindle,triangular and irregular cells.The spindle cells took up the greatest proportion,had the fastest beats,and had less developed organelle and scarce muscular fibrils.Immunofluorescence staining was positive against HCN4 and Cx45 on the spindle cells.Small amounts of HCN4 was observed on triangular cells.Conclusion ① Isolation by trypsin combined with differential attachment and BrdU treatment is a reliable approach to culturing sinoatrial node cells.② The spindle cells with the fastest beats and positive expressions of HCN4 and Cx45 may be the sinus node pacemaker cells.