Ifenprodil Reduced Expression of Activated Microglia, BDNF and DREAM Proteins in the Spinal Cord Following Formalin Injection During the Early Stage of Painful Diabetic Neuropathy in Rats.

The pharmacological inhibition of glial activation is one of the new approaches for combating neuropathic pain in which the role of glia in the modulation of neuropathic pain has attracted significant interest and attention. Neuron-glial crosstalk is achieved with N-methyl-D-aspartate-2B receptor (NMDAR-2B) activation. This study aims to determine the effect of ifenprodil, a potent noncompetitive NMDAR-2B antagonist, on activated microglia, brain-derived neurotrophic factors (BDNF) and downstream regulatory element antagonist modulator (DREAM) protein expression in the spinal cord of streptozotocin-induced painful diabetic neuropathy (PDN) rats following formalin injection. In this experimentation, 48 Sprague-Dawley male rats were randomly selected and divided into four groups: (n = 12): control, PDN, and ifenprodil-treated PDN rats at 0.5 µg or 1.0 µg for 7 days. Type I diabetes mellitus was then induced by injecting streptozotocin (60 mg/kg, i.p.) into the rats which were then over a 2-week period allowed to progress into the early phase of PDN. Ifenprodil was administered in PDN rats while saline was administered intrathecally in the control group. A formalin test was conducted during the fourth week to induce inflammatory nerve injury, in which the rats were sacrificed at 72 h post-formalin injection. The lumbar enlargement region (L4-L5) of the spinal cord was dissected for immunohistochemistry and western blot analyses. The results demonstrated a significant increase in formalin-induced flinching and licking behavior with an increased spinal expression of activated microglia, BDNF and DREAM proteins. It was also shown that the ifenprodil-treated rats following both doses reduced the extent of their flinching and duration of licking in PDN in a dose-dependent manner. As such, ifenprodil successfully demonstrated inhibition against microglia activation and suppressed the expression of BDNF and DREAM proteins in the spinal cord of PDN rats. In conclusion, ifenprodil may alleviate PDN by suppressing spinal microglia activation, BDNF and DREAM proteins.

A Soluble Epoxide Hydrolase Inhibitor Upregulated KCNJ12 and KCNIP2 by Downregulating MicroRNA-29 in a Mouse Model of Myocardial Infarction.

BACKGROUND: Soluble epoxide hydrolase inhibitors (sEHi) have anti-arrhythmic effects, and we previously found that the novel sEHi t-AUCB (trans-4[-4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid) significantly inhibited ventricular arrhythmias after myocardial infarction (MI). However, the mechanism is unknown. It's known that microRNA-29 (miR-29) participates in the occurrence of arrhythmias. In this study, we investigated whether sEHi t-AUCB was protective against ischemic arrhythmias by modulating miR-29 and its target genes KCNJ12 and KCNIP2. METHODS: Male 8-week-old C57BL/6 mice were divided into five groups and fed distilled water only or distilled water with t-AUCB of different dosages for seven days. Then, the mice underwent MI or sham surgery. The ischemic region of the myocardium was obtained 24 hours after MI to detect miR-29, KCNJ12, and KCNIP2 mRNA expression levels via real-time PCR and KCNJ12 and KCNIP2 protein expression levels via western blotting. RESULTS: MiR-29 expression levels were significantly increased in the ischemic region of MI mouse hearts and the mRNA and protein expression levels of its target genes KCNJ12 and KCNIP2 were significantly decreased. T-AUCB prevented these changes dose-dependently. CONCLUSION: The sEHi t-AUCB regulates the expression levels of miR-29 and its target genes KCNJ12 and KCNIP2, suggesting a possible mechanism for its potential therapeutic application in ischemic arrhythmia.

Post-treatment with a Hydrogen Sulfide Donor Limits Neuronal Injury and Modulates Potassium Voltage-gated Channel Subfamily D Member 2 (Kv4.2) and Potassium Channel Interacting Protein 3 (KChIP3) During Transient Global Cerebral Ischemia.

BACKGROUND: Although the neuroprotective effect of sodium hydrosulfide (NaHS, a hydrogen sulfide donor) pretreatment has been revealed, the effect of NaHS post-conditioning remains largely unknown. OBJECTIVE: We aimed to investigate the neuroprotective effect of NaHS post-conditioning against transient Global Cerebral Ischemia (tGCI)-induced hippocampal CA1 injury and its underlying molecular mechanism. METHODS: A tGCI rat model was established using the four-vessel occlusion method for 15 min of ischemia. The survival of hippocampal neurons was determined by Nissl staining and NeuN immunostaining. Protein expression of potassium voltage-gated channel subfamily D member 2 (Kv4.2) and potassium channel interacting protein 3 (KChIP3) was assessed by Immunohistochemistry (IHC) and Western blot. RESULTS: Decreased concentrations (12 and 24 µmol/kg) of NaHS post-conditioning significantly increased the numbers of survival neurons and NeuN-positive neurons in the hippocampal CA1 region at 7 days post-tGCI (all P<0.05). NaHS post-conditioning (24 µmol/kg) at 12 and 24 hr posttGCI can achieve the best protective effect (both P<0.05). IHC data demonstrated that NaHS postconditioning (24 µmol/kg) markedly attenuated tGCI-induced down-regulation of Kv4.2 protein in the hippocampal CA1 region at 26 hr post-tGCI. Confocal images showed that Kv4.2 did not express in the neuronal nuclei but predominantly express in the neuronal dendrites. In addition, NaHS post-conditioning significantly up-regulated Kv4.2 and down-regulated KChIP3 in tGCI rats at 26 and 168 hr post- tGCI (all P<0.05). CONCLUSION: Decreased concentrations of NaHS post-conditioning at 12-24 hr post-tGCI effectively protected hippocampal CA1 neurons from tGCI-induced injury, which may be through regulating the expression of Kv4.2 and KChIP3.

Integration of TP53, DREAM, MMB-FOXM1 and RB-E2F target gene analyses identifies cell cycle gene regulatory networks.

Cell cycle (CC) and TP53 regulatory networks are frequently deregulated in cancer. While numerous genome-wide studies of TP53 and CC-regulated genes have been performed, significant variation between studies has made it difficult to assess regulation of any given gene of interest. To overcome the limitation of individual studies, we developed a meta-analysis approach to identify high confidence target genes that reflect their frequency of identification in independent datasets. Gene regulatory networks were generated by comparing differential expression of TP53 and CC-regulated genes with chromatin immunoprecipitation studies for TP53, RB1, E2F, DREAM, B-MYB, FOXM1 and MuvB. RNA-seq data from p21-null cells revealed that gene downregulation by TP53 generally requires p21 (CDKN1A). Genes downregulated by TP53 were also identified as CC genes bound by the DREAM complex. The transcription factors RB, E2F1 and E2F7 bind to a subset of DREAM target genes that function in G1/S of the CC while B-MYB, FOXM1 and MuvB control G2/M gene expression. Our approach yields high confidence ranked target gene maps for TP53, DREAM, MMB-FOXM1 and RB-E2F and enables prediction and distinction of CC regulation. A web-based atlas at www.targetgenereg.org enables assessing the regulation of any human gene of interest.

FMRP Mediates Chronic Ethanol-Induced Changes in NMDA, Kv4.2, and KChIP3 Expression in the Hippocampus.

BACKGROUND: Exposure to chronic ethanol (EtOH) results in changes in the expression of proteins that regulate neuronal excitability. This study examined whether chronic EtOH alters the hippocampal expression and function of fragile X mental retardation protein (FMRP) and the role of FMRP in the modulation of chronic EtOH-induced changes in the expression of NMDA receptors and Kv4.2 channels. METHODS: For in vivo studies, C57BL/6J mice underwent a chronic intermittent EtOH (CIE) vapor exposure procedure. After CIE, hippocampal tissue was collected and subjected to immunoblot blot analysis of NMDA receptor subunits (GluN1, GluN2B), Kv4.2, and its accessory protein KChIP3. For in vitro studies, hippocampal slice cultures were exposed to 75 mM EtOH for 8 days. Following EtOH exposure, mRNAs bound to FMRP was measured. In a separate set of studies, cultures were exposed to an inhibitor of S6K1 (PF-4708671 [PF], 6 µM) in order to assess whether EtOH-induced homeostatic changes in protein expression depend upon changes in FMRP activity. RESULTS: Immunoblot blot analysis revealed increases in GluN1 and GluN2B but reductions in Kv4.2 and KChIP3. Analysis of mRNAs bound to FMRP revealed a similar bidirectional change observed as reduction of GluN2B and increase in Kv4.2 and KChIP3 mRNA transcripts. Analysis of FMRP further revealed that while chronic EtOH did not alter the expression of FMRP, it significantly increased phosphorylation of FMRP at the S499 residue that is known to critically regulate its activity. Inhibition of S6K1 prevented the chronic EtOH-induced increase in phospho-FMRP and changes in NMDA subunits, Kv4.2, and KChIP3. In contrast, PF had no effect in the absence of alcohol, indicating it was specific for the chronic EtOH-induced changes. CONCLUSIONS: These findings demonstrate that chronic EtOH exposure enhances translational control of plasticity-related proteins by FMRP, and that S6K1 and FMRP activities are required for expression of chronic EtOH-induced homeostatic plasticity at glutamatergic synapses in the hippocampus.

Evaluation of Downstream Regulatory Element Antagonistic Modulator Gene in Human Multinodular Goiter.

BACKGROUND DREAM (Downstream Regulatory Element Antagonistic Modulator) is a neuronal calcium sensor that was suggested to modulate TSH receptor activity and whose overexpression provokes an enlargement of the thyroid gland in transgenic mice. The aim of this study was to investigate somatic mutations and DREAM gene expression in human multinodular goiter (MNG). MATERIAL AND METHODS DNA and RNA samples were obtained from hyperplastic thyroid glands of 60 patients (54 females) with benign MNG. DREAM mutations were evaluated by PCR and direct automatic sequencing, whereas relative quantification of mRNA was performed by real-time PCR. Over- and under-expression were defined as a 2-fold increase and decrease in comparison to normal thyroid tissue, respectively. RQ M (relative quantification mean); SD (standard deviation). RESULTS DREAM expression was detected in all nodules evaluated. DREAM mRNA was overexpressed in 31.7% of MNG (RQ M=6.26; SD=5.08), whereas 53.3% and 15% had either normal (RQ M=1.16; SD=0.46) or underexpression (RQ M=0.30; SD=0.10), respectively. Regarding DREAM mutations analysis, only previously described intronic polymorphisms were observed. CONCLUSIONS We report DREAM gene expression in the hyperplastic thyroid gland of MNG patients. However, DREAM expression did not vary significantly, and was somewhat underexpressed in most patients, suggesting that DREAM upregulation does not significantly affect nodular development in human goiter.

Decreased Expression of DREAM Promotes the Degeneration of Retinal Neurons.

The intrinsic mechanisms that promote the degeneration of retinal ganglion cells (RGCs) following the activation of N-Methyl-D-aspartic acid-type glutamate receptors (NMDARs) are unclear. In this study, we have investigated the role of downstream regulatory element antagonist modulator (DREAM) in NMDA-mediated degeneration of the retina. NMDA, phosphate-buffered saline (PBS), and MK801 were injected into the vitreous humor of C57BL/6 mice. At 12, 24, and 48 hours after injection, expression of DREAM in the retina was determined by immunohistochemistry, western blot analysis, and electrophoretic mobility-shift assay (EMSA). Apoptotic death of cells in the retina was determined by terminal deoxynucleotidyl transferace dUTP nick end labeling (TUNEL) assays. Degeneration of RGCs in cross sections and in whole mount retinas was determined by using antibodies against Tuj1 and Brn3a respectively. Degeneration of amacrine cells and bipolar cells was determined by using antibodies against calretinin and protein kinase C (PKC)-alpha respectively. DREAM was expressed constitutively in RGCs, amacrine cells, bipolar cells, as well as in the inner plexiform layer (IPL). NMDA promoted a progressive decrease in DREAM levels in all three cell types over time, and at 48 h after NMDA-treatment very low DREAM levels were evident in the IPL only. DREAM expression in retinal nuclear proteins was decreased progressively after NMDA-treatment, and correlated with its decreased binding to the c-fos-DRE oligonucleotides. A decrease in DREAM expression correlated significantly with apoptotic death of RGCs, amacrine cells and bipolar cells. Treatment of eyes with NMDA antagonist MK801, restored DREAM expression to almost normal levels in the retina, and significantly decreased NMDA-mediated apoptotic death of RGCs, amacrine cells, and bipolar cells. Results presented in this study show for the first time that down-regulation of DREAM promotes the degeneration of RGCs, amacrine cells, and bipolar cells.

Cardiomyocyte-derived mitochondrial superoxide causes myocardial electrical remodeling by downregulating potassium channels and related molecules.

BACKGROUND: This study was designed to investigate the role of a primary hyperoxidative stress in myocardial electrical remodeling using heterozygous heart/muscle-specific manganese superoxide dismutase-deficient (H/M-Sod2(+/-)) mice treated with L-buthionine-sulfoximine (BSO). METHODS AND RESULTS: Both H/M-Sod2(+/-)and wild-type (WT) mice were treated with intra-peritoneal BSO or saline for 7 days, and divided into 4 groups: H/M-Sod2(+/-)+BSO, WT+BSO, H/M-Sod2(+/-)control, and WT control. The ventricular effective refractory period (ERP) and the monophasic action potential duration (MAPD) were determined. Levels of oxidative stress, potassium channel-related molecules, and K(+)channel-interacting protein-2 (KChIP2) were also evaluated. The H/M-Sod2(+/-)+BSO group exhibited markedly prolonged MAPD20, MAPD90 and ERP in comparison with the other groups (MAPD20: 14 ± 1 vs. 11 ± 1 ms, MAPD90: 77 ± 7 vs. 58 ± 4 ms, ERP: 61 ± 6 vs. 41 ± 3 ms, H/M-Sod2(+/-)+BSO vs. WT control; P<0.05). Mitochondrial superoxide and hydrogen peroxide formation in the myocardium increased in the H/M-Sod2(+/-)+BSO group in comparison with the WT+BSO group (P<0.05). Real-time RT-PCR and Western blotting revealed that Kv4.2 expression was downregulated in both BSO-treated groups, whereas KChIP2 expression was downregulated only in the H/M-Sod2(+/-)+BSO group (P<0.05). CONCLUSIONS: BSO treatment caused hyperoxidative stress in the myocardium of H/M-Sod2(+/-)mice. Changes in the expression and function of potassium channels were considered to be involved in the mechanism of electrical remodeling in this model.

Circadian rhythms govern cardiac repolarization and arrhythmogenesis.

Sudden cardiac death exhibits diurnal variation in both acquired and hereditary forms of heart disease, but the molecular basis of this variation is unknown. A common mechanism that underlies susceptibility to ventricular arrhythmias is abnormalities in the duration (for example, short or long QT syndromes and heart failure) or pattern (for example, Brugada's syndrome) of myocardial repolarization. Here we provide molecular evidence that links circadian rhythms to vulnerability in ventricular arrhythmias in mice. Specifically, we show that cardiac ion-channel expression and QT-interval duration (an index of myocardial repolarization) exhibit endogenous circadian rhythmicity under the control of a clock-dependent oscillator, krüppel-like factor 15 (Klf15). Klf15 transcriptionally controls rhythmic expression of Kv channel-interacting protein 2 (KChIP2), a critical subunit required for generating the transient outward potassium current. Deficiency or excess of Klf15 causes loss of rhythmic QT variation, abnormal repolarization and enhanced susceptibility to ventricular arrhythmias. These findings identify circadian transcription of ion channels as a mechanism for cardiac arrhythmogenesis.

Hyperglycemia induces early upregulation of the calcium sensor KChIP3/DREAM/calsenilin in the rat retina.

Hyperglycemia alters the tight control of intracellular calcium dynamics in retinal cells and may lead to the development of diabetic retinopathy. The potassium channel interacting protein 3 (KChIP3) also known as DREAM (Downstream Regulatory Element Antagonist Modulator) or calsenilin (KChIP3/DREAM/calsenilin), a member of the neuronal calcium sensor protein family, is expressed in Müller glial cells and upregulated under high glucose experimental culture conditions. Here, we analyzed the expression and function of KChIP3 in the retina of streptozotocin induced diabetic Long Evans rats by immunofluorescence confocal microscopy, western blot, co-immunoprecipitation, whole cell patch clamp recording on isolated cells and KChIP3 gene silencing by RNA interference. Three weeks after streptozotocin application, KChIP3 was increased throughout the different retinal layers and this process was not linked to augmented apoptosis. KChIP3 co-immunoprecipitated with voltage gated K(+) channels of the K(V)4.2-4.3 subtype in retinal extracts from control and hyperglycemic rats. Electrophysiological analysis showed that control cells did not express A type (K(V)4-mediated) K(+) currents but most of the cells from streptozotocin treated retinas displayed macroscopic currents with an inactivating component sensitive to 4-AP, suggesting the persistence of the A type currents at early times after treatment. siRNA analysis in Müller cells cultures grown under high glucose experimental conditions corroborated that, when the expression of KChIP3 is 50% reduced, the number of cells expressing A type currents decreases significantly. Together these data suggest an altered expression and function of KChIP3 after streptozotocin induced hyperglycemia that might help explain some pathological alterations in early diabetic retinopathy.

Expression and high glucose-mediated regulation of K+ channel interacting protein 3 (KChIP3) and KV4 channels in retinal Müller glial cells.

Normal vision depends on the correct function of retinal neurons and glia and it is impaired in the course of diabetic retinopathy. Müller cells, the main glial cells of the retina, suffer morphological and functional alterations during diabetes participating in the pathological retinal dysfunction. Recently, we showed that Müller cells express the pleiotropic protein potassium channel interacting protein 3 (KChIP3), an integral component of the voltage-gated K(+) channels K(V)4. Here, we sought to analyze the role of KChIP3 in the molecular mechanisms underlying hyperglycemia-induced phenotypic changes in the glial elements of the retina. The expression and function of KChIp3 was analyzed in vitro in rat Müller primary cultures grown under control (5.6 mM) or high glucose (25 mM) (diabetic-like) conditions. We show the up-regulation of KChIP3 expression in Müller cell cultures under high glucose conditions and demonstrate a previously unknown interaction between the K(V)4 channel and KChIP3 in Müller cells. We show evidence for the expression of a 4-AP-sensitive transient outward voltage-gated K(+) current and an alteration in the inactivation of the macroscopic outward K(+) currents expressed in high glucose-cultured Müller cells. Our data support the notion that induction of KChIP3 and functional changes of K(V)4 channels in Müller cells could exert a physiological role in the onset of diabetic retinopathy.

Expression and distribution of Kv4 potassium channel subunits and potassium channel interacting proteins in subpopulations of interneurons in the basolateral amygdala.

The Kv4 potassium channel α subunits, Kv4.1, Kv4.2, and Kv4.3, determine some of the fundamental physiological properties of neurons in the CNS. Kv4 subunits are associated with auxiliary ß-subunits, such as the potassium channel interacting proteins (KChIP1 - 4), which are thought to regulate the trafficking and gating of native Kv4 potassium channels. Intriguingly, KChIP1 is thought to show cell type-selective expression in GABA-ergic inhibitory interneurons, while other ß-subunits (KChIP2-4) are associated with principal glutamatergic neurons. However, nothing is known about the expression of Kv4 family α- and ß-subunits in specific interneurons populations in the BLA. Here, we have used immunofluorescence, co-immunoprecipitation, and Western Blotting to determine the relative expression of KChIP1 in the different interneuron subtypes within the BLA, and its co-localization with one or more of the Kv4 α subunits. We show that all three α-subunits of Kv4 potassium channel are found in rat BLA neurons, and that the immunoreactivity of KChIP1 closely resembles that of Kv4.3. Indeed, Kv4.3 showed almost complete co-localization with KChIP1 in the soma and dendrites of a distinct subpopulation of BLA neurons. Dual-immunofluorescence studies revealed this to be in BLA interneurons immunoreactive for parvalbumin, cholecystokin-8, and somatostatin. Finally, co-immunoprecipitation studies showed that KChIP1 was associated with all three Kv4 α subunits. Together our results suggest that KChIP1 is selectively expressed in BLA interneurons where it may function to regulate the activity of A-type potassium channels. Hence, KChIP1 might be considered as a cell type-specific regulator of GABAergic inhibitory circuits in the BLA.

MicroRNA-133a protects against myocardial fibrosis and modulates electrical repolarization without affecting hypertrophy in pressure-overloaded adult hearts.

RATIONALE: MicroRNA (miR)-133a regulates cardiac and skeletal muscle differentiation and plays an important role in cardiac development. Because miR-133a levels decrease during reactive cardiac hypertrophy, some have considered that restoring miR-133a levels could suppress hypertrophic remodeling. OBJECTIVE: To prevent the "normal" downregulation of miR-133a induced by an acute hypertrophic stimulus in the adult heart. METHODS AND RESULTS: miR-133a is downregulated in transverse aortic constriction (TAC) and isoproterenol-induced hypertrophy, but not in 2 genetic hypertrophy models. Using MYH6 promoter-directed expression of a miR-133a genomic precursor, increased cardiomyocyte miR-133a had no effect on postnatal cardiac development assessed by measures of structure, function, and mRNA profile. However, increased miR-133a levels increased QT intervals in surface electrocardiographic recordings and action potential durations in isolated ventricular myocytes, with a decrease in the fast component of the transient outward K+ current, I(to,f), at baseline. Transgenic expression of miR-133a prevented TAC-associated miR-133a downregulation and improved myocardial fibrosis and diastolic function without affecting the extent of hypertrophy. I(to,f) downregulation normally observed post-TAC was prevented in miR-133a transgenic mice, although action potential duration and QT intervals did not reflect this benefit. miR-133a transgenic hearts had no significant alterations of basal or post-TAC mRNA expression profiles, although decreased mRNA and protein levels were observed for the I(to,f) auxiliary KChIP2 subunit, which is not a predicted target. CONCLUSIONS: These results reveal striking differences between in vitro and in vivo phenotypes of miR expression, and further suggest that mRNA signatures do not reliably predict either direct miR targets or major miR effects.

[Construction of shRNA lentivirus vector on rat DREAM gene and its analgesic effect on CCI rats].

OBJECTIVE: To construct the recombinant lentivirus vector containing short hairpin RNA (shRNA) inhibited DREAM expression and to investigate the gene therapy of neuropathic pain by inhibiting the expression of DREAM gene by RNA interference. METHODS: An effective short hairpin RNA targeting to rat DREAM was cloned into the plasmids on the base of Lentivirous vectors, pKCSHR-Puro/GFP, and both of the pKCSHR-Puro/GFP-DREAM and Lentivector package plasmids mix were transferred into the 293T cells. The culture supernatant was harvested, and the virus titer was detected 48 hours after transferring. Thirty-six sheer breed pathogen free adult Sprague Dawley rats were randomly divided into 6 groups (6 in each group): normal control group (N); sham-operated group (S); CCI group (C0 group):CCI model without any intervention; Saline control group (C1 group); empty vector control group (C2 group); and LV-shRNADREAM lentiviral vector treatment group (C3 group). The rats in the last 3 groups respectively accepted injection of normal saline, blank vector, LV-shRNADREAM lentiviral vector in the subarachnoid on the 7th day after CCI, and the pain behavior was observed after 3, 7, 10, 14, 21 d after CCI. Green fluorescent protein (GFP) expression was detected by fluorescence microscope and the contents of DREAM mRNA and DREAM protein were detected by Realtime PCR and Western blot respectively in the rat lumbar spinal cord. RESULTS: The short hairpin RNA sequences targeting at rat DREAM were cloned into the vectors, and an entry clone and an expression clone were constructed successfully confirmed by sequence analysis. Lentiviral packaging was successful in 293 T cell line and the transfection titer of the lentivirus was 1 x 10(8) IFU/mL. LV-shRNADREAM lentivirus vector was transfected successfully in the rat spine with Intrathecal injection of LV-shRNADREAM. Compared with the other 3 groups, heat pain threshold and mechanical pain threshold in Group C3 improved significantly (P<0.01), and the expression of DREAM mRNA and DREAM protein in the lumbar spinal cord in Group C3 were lowered significantly (P<0.01). CONCLUSION: Lentivirus vectors containing rat DREAM gene are constructed successfully, and lentivirus mediated shRNA can inhibit the DREAM expression in the rat spine, which may prove to be an effective method for neuropathic pain.

Estrogen contributes to gender differences in mouse ventricular repolarization.

RATIONALE: Fast-transient outward K(+) (I(to,f)) and ultrarapid delayed rectifier K(+) currents (I(K,slow), also known as I(Kur)) contribute to mouse cardiac repolarization. Gender studies on these currents have reported conflicting results. OBJECTIVE: Key missing information in these studies is the estral stage of the animals. We revisited gender-related differences in K(+) currents, taking into consideration the females' estral stage. We hypothesized that changes in estrogen levels during the estral cycle could play a role in determining the densities of K(+) currents underlying ventricular repolarization. METHODS AND RESULTS: Peak total K(+) current (I(K,total)) densities (pA/pF, at +40 mV) were much higher in males (48.6+/-3.0) versus females at estrus (27.2+/-2.3) but not at diestrus-2 (39.1+/-3.4). Underlying this change, I(to,f) and I(K,slow) were lower in females at estrus versus males and diestrus-2 (I(K,slow): male 21.9+/-1.8, estrus 14.6+/-0.6, diestrus-2 20.3+/-1.4; I(to,f): male 26.8+/-1.9, estrus 14.9+/-1.6, diestrus-2 22.1+/-2.1). Lower I(K,slow) in estrus was attributable to only I(K,slow)(1) reduction, without changes in I(K,slow)(2). Estrogen treatment of ovariectomized mice decreased I(K,total) (46.4+/-3.0 to 28.4+/-1.6), I(to,f) (26.6+/-1.6 to 12.8+/-1.0) and I(K,slow) (22.2+/-1.6 to 17.2+/-1.4). Transcript levels of Kv4.3 and Kv1.5 (underlying I(to,f) and I(K,slow), respectively) were lower in estrus versus diestrus-2 and male. In ovariectomized mice, estrogen treatment resulted in downregulation of Kv4.3 and Kv1.5 but not Kv4.2, KChIP2, or Kv2.1 transcripts. K(+) current reduction in high estrogenic conditions were associated with prolongation of the action potential duration and corrected QT interval. CONCLUSION: Downregulation of Kv4.3 and Kv1.5 transcripts by estrogen are one mechanism defining gender-related differences in mouse ventricular repolarization.

Chemical sympathetic denervation, suppression of myocardial transient outward potassium current, and ventricular fibrillation in the rat.

Sympathetic denervation is frequently observed in heart disease. To investigate the linkage of sympathetic denervation and cardiac arrhythmia, we developed a rat model of chemical sympathectomy by subcutaneous injections of 6-hydroxydopamine (6-OHDA). Cardiac sympathetic innervation was visualized by means of a glyoxylic catecholaminergic histofluorescence method. Transient outward current (Ito) of ventricular myocytes was recorded with the whole-cell configuration of the patch clamp technique. We observed that sympathectomy (i) decreased cardiac sympathetic nerve density and norepinephrine level, (ii) reduced the protein expression of Kv4.2, Kv1.4, and Kv channel-interacting protein 2 (KChIP2), (iii) decreased current densities and delayed activation of Ito channels, (iv) reduced the phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) and cAMP response element-binding protein (CREB), and (v) increased the severity of ventricular fibrillation induced by rapid pacing. Three weeks after 6-OHDA injections, which allowed time for sympathetic regeneration, we found cardiac sympathetic nerve density, norepinephrine levels, expression levels of Kv4.2 and KChIP2 proteins, and I(to) densities were partially normalized and ventricular fibrillation severity was decreased. We conclude that chemical sympathectomy downregulates the expression of selective Kv channel subunits and decreases myocardial I(to) channel activities, contributing to the elevated susceptibility to ventricular fibrillation.

Dendritic A-type potassium channel subunit expression in CA1 hippocampal interneurons.

Voltage-gated potassium (Kv) channels are important and diverse determinants of neuronal excitability and exhibit specific expression patterns throughout the brain. Among Kv channels, Kv4 channels are major determinants of somatodendritic A-type current and are essential in controlling the amplitude of backpropagating action potentials (BAPs) into neuronal dendrites. BAPs have been well studied in a variety of neurons, and have been recently described in hippocampal and cortical interneurons, a heterogeneous population of GABAergic inhibitory cells that regulate activity of principal cells and neuronal networks. We used well-characterized mouse monoclonal antibodies against the Kv4.3 and potassium channel interacting protein (KChIP) 1 subunits of A-type Kv channels, and antibodies against different interneuron markers in single- and double-label immunohistochemistry experiments to analyze the expression patterns of Kv4.3 and KChIP1 in hippocampal Ammon's horn (CA1) neurons. Immunohistochemistry was performed on 40 mum rat brain sections using nickel-enhanced diaminobenzidine staining or multiple-label immunofluorescence. Our results show that Kv4.3 and KChIP1 component subunits of A-type channels are co-localized in the soma and dendrites of a large number of GABAergic hippocampal interneurons. These subunits co-localize extensively but not completely with markers defining the four major interneuron subpopulations tested (parvalbumin, calbindin, calretinin, and somatostatin). These results suggest that CA1 hippocampal interneurons can be divided in two groups according to the expression of Kv4.3/KChIP1 channel subunits. Antibodies against Kv4.3 and KChIP1 represent an important new tool for identifying a subpopulation of hippocampal interneurons with a unique dendritic A-type channel complement and ability to control BAPs.

NMR analysis of KChIP4a reveals structural basis for control of surface expression of Kv4 channel complexes.

Potassium channel-interacting proteins (KChIPs) are EF-hand calcium-binding proteins of the recoverin/neuronal calcium sensor 1 family that co-assemble with the pore-forming Kv4 alpha-subunits and thus control surface trafficking of the voltage-gated potassium channels mediating the neuronal I(A) and cardiac I(to) currents. Different from the other KChIPs, KChIP4a largely reduces surface expression of the Kv4 channel complexes. Using solution NMR we show that the unique N terminus of KChIP4a forms a 6-turn alpha-helix that is connected to the highly conserved core of the KChIP protein via a solvent-exposed linker. As identified by chemical shift changes, N-terminal alpha-helix and core domain of KChIP4a interact with each other through the same hydrophobic surface pocket that is involved in intermolecular interaction between the N-terminal helix of Kv4alpha and KChIP in Kv4-KChIP complexes. Electrophysiological recordings and biochemical interaction assays of complexes formed by wild-type and mutant Kv4alpha and KChIP4a proteins suggest that competition of these two helical domains for the surface groove is responsible for the reduced trafficking of Kv4-KChIP4a complexes to the plasma membrane. Surface expression of Kv4 complexes may thus be controlled by an auto-inhibitory domain in the KChIP subunit.