Decreased expression of hyperpolarisation-activated cyclic nucleotide-gated channel 3 in Hirschsprung's disease

AIM: To determine if hyperpolarisation-activated nucleotide-gated(HCN) channels exist in human colon, and to investigate the expression of HCN channels in Hirschsprung's disease.METHODS:We investigated HCN1,HCN2,HCN3 and HCN4 protein expression in pull-through specimens from patients with Hirschsprung’s disease(HSCR,n=10)using the proximal-most ganglionic segment and distalmost aganglionic segment,as well as in healthy control specimens obtained at the time of sigmoid colostomy closure in children who had undergone anorectoplasty for imperforate anus(n=10).Fluorescent immunohistochemistry was performed to assess protein distribution,which was then visualized using confocal microscopy.RESULTS:No HCN1 channel expression was observed in any of the tissues studied.Both HCN2 and HCN4proteins were found to be equally expressed in the aganglionic and ganglionic bowel in HSCR and controls.HCN3 channel expression was found to be markedly decreased in the aganglionic colon vs ganglionic colon and controls.HCN2-4 channels were seen to be expressed within neurons of the myenteric and submucosal plexus of the ganglionic bowel and normal controls,and also co-localised to interstitial cells of Cajal in all tissues studied.CONCLUSION:We demonstrate HCN channel expression in human colon for the first time.Reduced HCN3expression in aganglionic bowel suggests its potential role in HSCR pathophysiology.

Dynamic Expression of Hyperpolarization-activated Cyclic Nucleotide-gated Cation Channel 4 Involved in Microwave Induced Pacemaker Cell Injuries

To investigate the mechanisms of microwave induced pacemaker cell injuries, Wistar rats and the primary pacemaker cells of newborn Wistar rats were exposed to microwave at average power density of 50 mW/cm2. Slower spontaneous beating rate, intercellular Ca2＋ aggregation and cell membrane perforation were detected immediately after the exposure. Moreover, hyperpolarizationactivated cyclic nucleotide-gated cation channel 4 （HCN4） was down-regulated immediately after the exposure and up-regulated at 12 h after the exposure. In the sinoatrial node （SAN） of the rats,

The Role of Cyclic Nucleotide-Gated Ion Channels in Plant Immunity

Since the first plant cyclic nucleotide-gated ion channel （CNGC）, HvCBT1, was identified as a calmodulin bind- ing protein, more than a decade has passed and a substantial amount of work has been done to understand the molecular nature and function of these channel proteins. Based on electrophysiological and heterologous expression analyses, plant CNGCs function as non-selective cation channels and, so far, their biological roles have been reported in defense responses, development, and ion homeostasis. Forward genetic approaches identified four AtCNGCs （AtCNGC2, 4, 11, and 12） to be involved in plant immunity, as null mutants for AtCNGC2, 4, 11, and 12 as well as a gain-of- function mutant for AtCNGC11 and 12 exhibited alterations in defense responses. Since ion flux changes have been reported as one of the early events upon pathogen recognition and also are an essential component for the activation of defense responses, the involvement of CNGCs in these ion flux changes has been suggested. However, the recent detailed characterization of null mutants suggested a more complex involvement of this channel family. In this review, we focus on the discoveries and character- ization of these CNGC mutants and discuss possible roles of CNGCs as components in plant immunity.

Electrophysiology of hyperpolarization-activated cyclic nucleotidegated cation channel 2 and hyperpolarization-activated cyclic nucleotide-gated cation channel 4 expressed in HEK293 cells

The action potential of pacemaker cells in the sino-atrial node forms the automatic rhythm of the heart. The automatic depolarization in phase 4 is me DaSlS of the automaticity in pacemaker cells. Many currents are included in phase 4, such as calcium current, TTX-sensitive sodium current, sustained inward current （Isi）, decay of delayed rectifier potassium current, etc. Funny current （If） has long been recognized as important in this phase and is activated at hyperpolarized potentials during cell diastole and in turn activates other currents to form automatic depolarization.

Hyperpolarization-activated cyclic nucleotide-gated 2 contributes to electroacupuncture analgesia on lumbar disc herniation-induced radicular pain through activation of microglia in spinal dorsal horn

OBJECTIVE:To explore the mechanisms of dorsal root ganglia and spinal microglia cascade cross in electroacupuncture(EA)analgesia in the treatment of lumbar disc herniation.METHODS:A rat model of lumbar disc herniation(LDH)was established,EA was administered at Huantiao(GB30)acupoint 30 min once a day,for 3 d.Before and after modeling,and after EA,mechanical allodynia thresholds were detected.Hyperpolarization-activated cyclic nucleotide-gated 2(HCN2)in dorsal root ganglia was detected by quantitative polymerase chain reaction(qPCR)and Western blot.C-X3-C motif chemokine ligand 1(CX3CL1)and activity of microglia in spinal cord was observed separately via qPCR and immunofluorescence staining.RESULTS:The mechanical allodynia threshold of the right planta of model rats was significantly reduced(P<0.01),EA increased the mechanical pain threshold of rats(P<0.01),and decreased HCN2 mRNA,and protein expression,reduced the expression of CX3CL1 and the activation of microglia.ZD7288(a blocker of HCN channel)reduced the analgesic effect of EA from 1.83±0.84 to 0.74±0.20(P<0.05),and the expression of CX3CL1 in the spinal cord decreased from 0.52±0.11 to 0.15±0.05(P<0.01).CONCLUSION:EA analgesia on the radicular pain of LDH is definite.EA reduced the expression of HCN2 channel in the dorsal root ganglion,thereby decreasing the noxious stimulation entered to microglia in spinal dorsal horn.Our work supports EA is an effective treatment for radicular pain of LDH.

Genome-wide identification and analysis of the CNGC gene family in upland cotton under multiple stress conditions

Background The cyclic nucleotide-gated channel(CNGC)gene family plays a significant role in the uptake of both essential and toxic cations,and has a role in enhancing tolerance to various forms of abiotic stresses as well as the modulation of the heavy metal toxicity to plant through the absorption of heavy metals.Results A complete genome-wide identification and functional characterization of the cotton CNGC genes was carried out,in which 55,28,and 29 CNGC genes were identified in Gossypium hirsutum,G.raimondii,and G.arboreum,respectively.The protein encoded by the CNGC genes exhibited GRAVY value below zero,indicating their hydrophilic property.CNGC genes were unevenly distributed in 19 out of 26 chromosomes,in which the highest density were observed on Ah05,with 8 genes.High gene coverage was observed among the diploid cotton species,with CNGC genes mapped on all A chromosomes and on 11 out of 13 of D chromosomes.The majority of CNGC proteins were localized in the endoplasmic reticulum,nucleus,and plasma membrane.Gene expression analysis revealed the up-regulation of Gh_A01G0520(CNGC4)and Gh_D13G1974(CNGC5)across various forms of abiotic stresses.Moreover,down-regulation of Gh_A01G0520(CNGC4)and Gh_D13G1974(CNGC5)in CNGCs silenced plants caused the significantly reduced ability to tolerate drought and salt stresses.All CNGCs silenced plants were recorded to have significantly low content of antioxidants but relatively higher content of oxidant,including MDA and H_(2)O_(2).Furthermore,SPAD,CMS(cell membrane stability),ELWL(excised leaf water loss),SDW(shoot dry matter weight),and RDW(root dry matter weight)were all lower in CNGCs silenced plants compared with the wild type plants.Conclusion Significant reduction in antioxidant content and negative effects of physiological and morphological characters in CNGCs silenced plants has revealed the novel role of CNGC genes in enhancing cell integrity under abiotic stress conditions.These results provide vital information that will expand our understanding of the CNGC gene family in cotton and other plants,thus promoting the integration of these genes in the development of the environmental resilient plants.

Use of Rats Mesenchymal Stem Cells Modified with mHCN2 Gene to Create Biologic Pacemakers

The possibility of rats mesenchymal stem cells (MSCs) modified with murine hyperpolarization-activated cyclic nucleotide-gated 2 (mHCN2) gene as biological pacemakers in vitro was studied. The cultured MSCs were transfected with pIRES2-EGFP plasmid carrying enhanced green fluorescent protein (EGFP) gene and mHCN2 gene. The identification using restriction enzyme and sequencing indicated that the mHCN2 gene was inserted to the pIRES2-EGFP. Green fluorescence could be seen in MSCs after transfection for 24–48 h. The expression of mHCN2 mRNA and protein in the transfected cells was detected by RT-PCR and Western blot, and the quantity of mHCN2 mRNA and protein expression in transfected MSCs was 5.31 times and 7.55 times higher than that of the non-transfected MSCs respectively (P<0.05, P<0.05). IHCN2 was recorded by whole-cell patch clamp method. The effect of Cs+, a specific blocker of pacemaker current, was measured after perfusion by patch clamp. The results of inward current indicated that there was no inward current recording in non-transfected MSCs and a large voltage-dependent inward and Cs+-sensitive current activated on hyperpolarizations presented in the transfected MSCs. IHCN2 was fully activated around–140 mV with an activation threshold of –60 mV. The midpoint (V50) was –95.1±0.9 mV (n=9). The study demonstrates that mHCN2 mRNA and protein can be expressed and the currents of HCN2 channels can be detected in genetically modified MSCs. The gene-modified MSCs present a novel method for pacemaker genes into the heart or other electrical syncytia.

AAV-mediated human CNGB3 restores cone function in an allcone mouse model of CNGB3 achromatopsia

Complete congenital achromatopsia is a devastating hereditary visual disorder. Mutations in the CNGB3 gene account for more than 50% of all known cases of achromatopsia. This work investigated the efficiency of subretinal(SR) delivered AAV8(Y447, 733 F) vector containing a human PR2.1 promoter and a human CNGB3 c DNA in Cngb3-/-/Nrl-/-mice. The Cngb3-/-/Nrl-/- mouse was a cone-dominant model with Cngb3 channel deficiency, which partially mimicked the all-cone foveal structure of human achromatopsia with CNGB3 mutations. Following SR delivery of the vector, AAV-mediated CNGB3 expression restored cone function which was assessed by the restoration of the cone-mediated electroretinogram(ERG) and immunohistochemistry. This therapeutic rescue resulted in long-term improvement of retinal function with the restoration of cone ERG amplitude. This study demonstrated an AAV-mediated gene therapy in a cone-dominant mouse model using a human gene construct and provided the potential to be utilized in clinical trials.

A Calcium-Dependent Protein Kinase Interactswith and Activates A Calcium Channel toRequlate Pollen Tube Growth

ABSTRACT Calcium, as a ubiquitous second messenger, plays essential roles in tip-growing cells, such as animal neu-rons, plant pollen tubes, and root hairs. However, little is known concerning the regulatory mechanisms that code anddecode Ca2＋ signals in plants. The evidence presented here indicates that a calcium-dependent protein kinase, CPK32,controls polar growth of pollen tubes. Overexpression of CPK32 disrupted the polar growth along with excessive Ca2＋accumulation in the tip. A search of downstream effector molecules for CPK32 led to identification of a cyclic nucleotide-gated channel, CNGC18, as an interacting partner for CPK32. Co-expression of CPK32 and CNGC18 resulted in activationof CNGC18 in Xenopus oocytes where expression of CNGC18 alone did not exhibit significant calcium channel activity.Overexpression of CNGC18 produced a growth arrest phenotype coupled with accumulation of calcium in the tip, simi-lar to that induced by CPK32 overexpression. Co-expression of CPK32 and CNGC18 had a synergistic effect leading tomore severe depolarization of pollen tube growth. These results provide a potential feed-forward mechanism in whichcalcium-activated CPK32 activates CNGC18, further promoting calcium entry during the elevation phase of Ca2＋ oscilla-tions in the polar growth of pollen tubes.

Overexpression of connexin 45 in rat mesenchymal stem cells improves the function as cardiac biological pacemakers

Background Extensive research toward creating a biological pacemaker by enhancement of inward depolarizing current has been performed. However, studies have mainly focused on inducing spontaneous activity and have not adequately addressed ways to improve pacemaker function. In this study we attempted to improve pacemaker function by altering connexin expression in rat mesenchymal stem cells （MSCs） to a phenotype similar to native sinus node pacemaker cells. Methods To generate a biological pacemaker, MSCs were transduced with a cardiac pacemaker gene- hyperpolarization-activated cyclic nucleotide-gated channel 4 （HCN4）, via transfection with a lentiviral vector. Funny current （If） in HCN4~ MSCs was recorded by voltage-clamp. Overexpression of connexin 45 （gene Gja7） in MSCs was achieved by transfection with the plasmid pDsRED2-N1-Gja7-RFP. Double-immunolabelling with anti-connexin 43 and anti-connexin 45 antibodies were used to identify the gap junction channels. The effects of the genetically modified MSCs on cardiomyocyte excitability were determined in MSCs cocultured with neonatal rat ventricular myocytes. Spontaneous action potentials of neonatal rat ventricular myocytes were recorded by current-clamp. Results High level time- and voltage-dependent inward hyperpolarization current that was sensitive to 4 mmol/L Cs＋ was detected in HCN4＋ MSCs, confirming that HCN4 acted as Ir channels in MSCs. Connexin 43 and connexin 45 were simultaneously detected in CX45＋ MSCs. Beating frequency was （82±8） beats per minute （n=-5） in myocytes cocultured with non-transfected control MSCs, versus （129±11） beats per minute （n=-5） in myocytes cocultured with HCN4＋ MSCs. Myocytes cocultured with MSCs cotransfected with HCN4 and connexin 45 had the highest beating frequency at （147±9） beats per minute （n=5）. Conclusion These findings demonstrate that overexpression of connexin 45 and subsequent formation of heteromeric connexin 45/connexin 43 gap junction channels in HCN4 expressing MSCs can improve their function as cardiac biological pacemakers in vitro.