Structural basis of gating modulation of Kv4 channel complexes.

Modulation of voltage-gated potassium (Kv) channels by auxiliary subunits is central to the physiological function of channels in the brain and heart1,2. Native Kv4 tetrameric channels form macromolecular ternary complexes with two auxiliary ß-subunits-intracellular Kv channel-interacting proteins (KChIPs) and transmembrane dipeptidyl peptidase-related proteins (DPPs)-to evoke rapidly activating and inactivating A-type currents, which prevent the backpropagation of action potentials1-5. However, the modulatory mechanisms of Kv4 channel complexes remain largely unknown. Here we report cryo-electron microscopy structures of the Kv4.2-DPP6S-KChIP1 dodecamer complex, the Kv4.2-KChIP1 and Kv4.2-DPP6S octamer complexes, and Kv4.2 alone. The structure of the Kv4.2-KChIP1 complex reveals that the intracellular N terminus of Kv4.2 interacts with its C terminus that extends from the S6 gating helix of the neighbouring Kv4.2 subunit. KChIP1 captures both the N and the C terminus of Kv4.2. In consequence, KChIP1 would prevent N-type inactivation and stabilize the S6 conformation to modulate gating of the S6 helices within the tetramer. By contrast, unlike the reported auxiliary subunits of voltage-gated channel complexes, DPP6S interacts with the S1 and S2 helices of the Kv4.2 voltage-sensing domain, which suggests that DPP6S stabilizes the conformation of the S1-S2 helices. DPP6S may therefore accelerate the voltage-dependent movement of the S4 helices. KChIP1 and DPP6S do not directly interact with each other in the Kv4.2-KChIP1-DPP6S ternary complex. Thus, our data suggest that two distinct modes of modulation contribute in an additive manner to evoke A-type currents from the native Kv4 macromolecular complex.

The transcription factor DREAM represses the deubiquitinase A20 and mediates inflammation.

Here we found that the transcription repressor DREAM bound to the promoter of the gene encoding A20 to repress expression of this deubiquitinase that suppresses inflammatory NF-κB signaling. DREAM-deficient mice displayed persistent and unchecked A20 expression in response to endotoxin. DREAM functioned by transcriptionally repressing A20 through binding to downstream regulatory elements (DREs). In contrast, binding of the transcription factor USF1 to the DRE-associated E-box domain in the gene encoding A20 activated its expression in response to inflammatory stimuli. Our studies define the critical opposing functions of DREAM and USF1 in inhibiting and inducing A20 expression, respectively, and thereby the strength of NF-κB signaling. Targeting of DREAM to induce USF1-mediated A20 expression is therefore a potential anti-inflammatory strategy for the treatment of diseases associated with unconstrained NF-κB activity, such as acute lung injury.

The p21CIP1-CDK4-DREAM axis is a master regulator of genotoxic stress-induced cellular senescence.

Cellular senescence, a major driver of aging, can be stimulated by DNA damage, and is counteracted by the DNA repair machinery. Here we show that in p16INK4a-deficient cells, senescence induction by the environmental genotoxin B[a]P or ionizing radiation (IR) completely depends on p21CIP1. Immunoprecipitation-based mass spectrometry interactomics data revealed that during senescence induction and maintenance, p21CIP1 specifically inhibits CDK4 and thereby activates the DREAM complex. Genome-wide transcriptomics revealed striking similarities in the response induced by B[a]P and IR. Among the top 100 repressed genes 78 were identical between B[a]P and IR and 76 were DREAM targets. The DREAM complex transcriptionally silences the main proliferation-associated transcription factors E2F1, FOXM1 and B-Myb as well as multiple DNA repair factors. Knockdown of p21CIP1, E2F4 or E2F5 diminished both, repression of these factors and senescence. The transcriptional profiles evoked by B[a]P and IR largely overlapped with the profile induced by pharmacological CDK4 inhibition, further illustrating the role of CDK4 inhibition in genotoxic stress-induced senescence. Moreover, data obtained by live-cell time-lapse microscopy suggest the inhibition of CDK4 by p21CIP1 is especially important for arresting cells which slip through mitosis. Overall, we identified the p21CIP1/CDK4/DREAM axis as a master regulator of genotoxic stress-induced senescence.

Characterisation of canine KCNIP4: A novel gene for cerebellar ataxia identified by whole-genome sequencing two affected Norwegian Buhund dogs.

A form of hereditary cerebellar ataxia has recently been described in the Norwegian Buhund dog breed. This study aimed to identify the genetic cause of the disease. Whole-genome sequencing of two Norwegian Buhund siblings diagnosed with progressive cerebellar ataxia was carried out, and sequences compared with 405 whole genome sequences of dogs of other breeds to filter benign common variants. Nine variants predicted to be deleterious segregated among the genomes in concordance with an autosomal recessive mode of inheritance, only one of which segregated within the breed when genotyped in additional Norwegian Buhunds. In total this variant was assessed in 802 whole genome sequences, and genotyped in an additional 505 unaffected dogs (including 146 Buhunds), and only four affected Norwegian Buhunds were homozygous for the variant. The variant identified, a T to C single nucleotide polymorphism (SNP) (NC_006585.3:g.88890674T>C), is predicted to cause a tryptophan to arginine substitution in a highly conserved region of the potassium voltage-gated channel interacting protein KCNIP4. This gene has not been implicated previously in hereditary ataxia in any species. Evaluation of KCNIP4 protein expression through western blot and immunohistochemical analysis using cerebellum tissue of affected and control dogs demonstrated that the mutation causes a dramatic reduction of KCNIP4 protein expression. The expression of alternative KCNIP4 transcripts within the canine cerebellum, and regional differences in KCNIP4 protein expression, were characterised through RT-PCR and immunohistochemistry respectively. The voltage-gated potassium channel protein KCND3 has previously been implicated in spinocerebellar ataxia, and our findings suggest that the Kv4 channel complex KCNIP accessory subunits also have an essential role in voltage-gated potassium channel function in the cerebellum and should be investigated as potential candidate genes for cerebellar ataxia in future studies in other species.

Emerging Role of DREAM in Healthy Brain and Neurological Diseases.

The downstream regulatory element antagonist modulator (DREAM) is a multifunctional Ca2+-sensitive protein exerting a dual mechanism of action to regulate several Ca2+-dependent processes. Upon sumoylation, DREAM enters in nucleus where it downregulates the expression of several genes provided with a consensus sequence named dream regulatory element (DRE). On the other hand, DREAM could also directly modulate the activity or the localization of several cytosolic and plasma membrane proteins. In this review, we summarize recent advances in the knowledge of DREAM dysregulation and DREAM-dependent epigenetic remodeling as a central mechanism in the progression of several diseases affecting central nervous system, including stroke, Alzheimer's and Huntington's diseases, amyotrophic lateral sclerosis, and neuropathic pain. Interestingly, DREAM seems to exert a common detrimental role in these diseases by inhibiting the transcription of several neuroprotective genes, including the sodium/calcium exchanger isoform 3 (NCX3), brain-derived neurotrophic factor (BDNF), pro-dynorphin, and c-fos. These findings lead to the concept that DREAM might represent a pharmacological target to ameliorate symptoms and reduce neurodegenerative processes in several pathological conditions affecting central nervous system.

Repaglinide Induces ATF6 Processing and Neuroprotection in Transgenic SOD1G93A Mice.

The interaction of the activating transcription factor 6 (ATF6), a key effector of the unfolded protein response (UPR) in the endoplasmic reticulum, with the neuronal calcium sensor Downstream Regulatory Element Antagonist Modulator (DREAM) is a potential therapeutic target in neurodegeneration. Modulation of the ATF6-DREAM interaction with repaglinide (RP) induced neuroprotection in a model of Huntington's disease. Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder with no cure, characterized by the progressive loss of motoneurons resulting in muscle denervation, atrophy, paralysis, and death. The aim of this work was to investigate the potential therapeutic significance of DREAM as a target for intervention in ALS. We found that the expression of the DREAM protein was reduced in the spinal cord of SOD1G93A mice compared to wild-type littermates. RP treatment improved motor strength and reduced the expression of the ALS progression marker collagen type XIXα1 (Col19α1 mRNA) in the quadriceps muscle in SOD1G93A mice. Moreover, treated SOD1G93A mice showed reduced motoneuron loss and glial activation and increased ATF6 processing in the spinal cord. These results indicate that the modulation of the DREAM-ATF6 interaction ameliorates ALS symptoms in SOD1G93A mice.

Kv4.2 phosphorylation by PKA drives Kv4.2-KChIP2 dissociation, leading to Kv4.2 out of lipid rafts and internalization.

Sympathetic regulation of the Kv4.2 transient outward potassium current (Ito) is critical for the acute electrical and contractile response of the myocardium under physiological and pathological conditions. Previous studies have suggested that KChIP2, the key auxiliary subunit of Kv4 channels, is required for the sympathetic regulation of Kv4.2 current densities. Of interest, Kv4.2 and KChIP2, and key components mediating acute sympathetic signaling transduction are present in lipid rafts, which are profoundly involved in regulation of Ito densities in rat ventricular myocytes. However, little is known about the mechanisms of Kv4.2-raft association and its connection with acute sympathetic regulation. With the aid of high-resolution fluorescent microscope, we demonstrated that KChIP2 assisted Kv4.2 localization in lipid rafts in HEK293 cells. Moreover, PKA-mediated Kv4.2 phosphorylation, the downstream signaling event of acute sympathetic stimulation, induced dissociation between Kv4.2 and KChIP2, resulting in Kv4.2 shifting out of lipid rafts in KChIP2-expressed HEK293. The mutation that mimics Kv4.2 phosphorylation by PKA (K4.2-S552D) similarly disrupted Kv4.2 interaction with KChIP2 and also decreased the surface stability of Kv4.2. The attenuated Kv4.2-KChIP2 interaction was also observed in native neonatal rat ventricular myocytes (NRVMs) upon acute adrenergic stimulation with phenylephrine (PE). Furthermore, PE stimulation decreased Kv4.2 location at lipid rafts and induced internalization of Kv4.2 as well as the effect of lipid rafts disruption. In conclusion, KChIP2 contributes to targeting Kv4.2 to lipid rafts. Acute adrenergic stimulation induces Kv4.2-KChIP2 dissociation, leading to Kv4.2 out of lipid rafts and internalization, reinforcing the critical role of Kv4.2-lipid raft association in the essential physiological response of Ito to acute sympathetic regulation.

Benzo[a]pyrene represses DNA repair through altered E2F1/E2F4 function marking an early event in DNA damage-induced cellular senescence.

Transcriptional regulation of DNA repair is of outmost importance for the restoration of DNA integrity upon genotoxic stress. Here we report that the potent environmental carcinogen benzo[a]pyrene (B[a]P) activates a cellular DNA damage response resulting in transcriptional repression of mismatch repair (MMR) genes (MSH2, MSH6, EXO1) and of RAD51, the central homologous recombination repair (HR) component, ultimately leading to downregulation of MMR and HR. B[a]P-induced gene repression is caused by abrogated E2F1 signalling. This occurs through proteasomal degradation of E2F1 in G2-arrested cells and downregulation of E2F1 mRNA expression in G1-arrested cells. Repression of E2F1-mediated transcription and silencing of repair genes is further mediated by the p21-dependent E2F4/DREAM complex. Notably, repression of DNA repair is also observed following exposure to the active B[a]P metabolite BPDE and upon ionizing radiation and occurs in response to a p53/p21-triggered, irreversible cell cycle arrest marking the onset of cellular senescence. Overall, our results suggest that repression of MMR and HR is an early event during genotoxic-stress induced senescence. We propose that persistent downregulation of DNA repair might play a role in the maintenance of the senescence phenotype, which is associated with an accumulation of unrepairable DNA lesions.

Modulation of KV4.3-KChIP2 Channels by IQM-266: Role of DPP6 and KCNE2.

The transient outward potassium current (Itof) is generated by the activation of KV4 channels assembled with KChIP2 and other accessory subunits (DPP6 and KCNE2). To test the hypothesis that these subunits modify the channel pharmacology, we analyzed the electrophysiological effects of (3-(2-(3-phenoxyphenyl)acetamido)-2-naphthoic acid) (IQM-266), a new KChIP2 ligand, on the currents generated by KV4.3/KChIP2, KV4.3/KChIP2/DPP6 and KV4.3/KChIP2/KCNE2 channels. CHO cells were transiently transfected with cDNAs codifying for different proteins (KV4.3/KChIP2, KV4.3/KChIP2/DPP6 or KV4.3/KChIP2/KCNE2), and the potassium currents were recorded using the whole-cell patch-clamp technique. IQM-266 decreased the maximum peak of KV4.3/KChIP2, KV4.3/KChIP2/DPP6 and KV4.3/KChIP2/KCNE2 currents, slowing their time course of inactivation in a concentration-, voltage-, time- and use-dependent manner. IQM-266 produced an increase in the charge in KV4.3/KChIP2 channels that was intensified when DPP6 was present and abolished in the presence of KCNE2. IQM-266 induced an activation unblocking effect during the application of trains of pulses to cells expressing KV4.3/KChIP2 and KV4.3/KChIP2/KCNE2, but not in KV4.3/KChIP2/DPP6 channels. Overall, all these results are consistent with a preferential IQM-266 binding to an active closed state of Kv4.3/KChIP2 and Kv4.3/KChIP2/KCNE2 channels, whereas in the presence of DPP6, IQM-266 binds preferentially to an inactivated state. In conclusion, DPP6 and KCNE2 modify the pharmacological response of KV4.3/KChIP2 channels to IQM-266.

Neuronal Roles of the Multifunctional Protein Dipeptidyl Peptidase-like 6 (DPP6).

The concerted action of voltage-gated ion channels in the brain is fundamental in controlling neuronal physiology and circuit function. Ion channels often associate in multi-protein complexes together with auxiliary subunits, which can strongly influence channel expression and function and, therefore, neuronal computation. One such auxiliary subunit that displays prominent expression in multiple brain regions is the Dipeptidyl aminopeptidase-like protein 6 (DPP6). This protein associates with A-type K+ channels to control their cellular distribution and gating properties. Intriguingly, DPP6 has been found to be multifunctional with an additional, independent role in synapse formation and maintenance. Here, we feature the role of DPP6 in regulating neuronal function in the context of its modulation of A-type K+ channels as well as its independent involvement in synaptic development. The prevalence of DPP6 in these processes underscores its importance in brain function, and recent work has identified that its dysfunction is associated with host of neurological disorders. We provide a brief overview of these and discuss research directions currently underway to advance our understanding of the contribution of DPP6 to their etiology.

IQM-PC332, a Novel DREAM Ligand with Antinociceptive Effect on Peripheral Nerve Injury-Induced Pain.

Neuropathic pain is a form of chronic pain arising from damage of the neural cells that sense, transmit or process sensory information. Given its growing prevalence and common refractoriness to conventional analgesics, the development of new drugs with pain relief effects constitutes a prominent clinical need. In this respect, drugs that reduce activity of sensory neurons by modulating ion channels hold the promise to become effective analgesics. Here, we evaluated the mechanical antinociceptive effect of IQM-PC332, a novel ligand of the multifunctional protein downstream regulatory element antagonist modulator (DREAM) in rats subjected to chronic constriction injury of the sciatic nerve as a model of neuropathic pain. IQM-PC332 administered by intraplantar (0.01-10 µg) or intraperitoneal (0.02-1 µg/kg) injection reduced mechanical sensitivity by ≈100% of the maximum possible effect, with ED50 of 0.27 ± 0.05 µg and 0.09 ± 0.01 µg/kg, respectively. Perforated-patch whole-cell recordings in isolated dorsal root ganglion (DRG) neurons showed that IQM-PC332 (1 and 10 µM) reduced ionic currents through voltage-gated K+ channels responsible for A-type potassium currents, low, T-type, and high voltage-activated Ca2+ channels, and transient receptor potential vanilloid-1 (TRPV1) channels. Furthermore, IQM-PC332 (1 µM) reduced electrically evoked action potentials in DRG neurons from neuropathic animals. It is suggested that by modulating multiple DREAM-ion channel signaling complexes, IQM-PC332 may serve a lead compound of novel multimodal analgesics.

Modulation of Kv4.2/KChIP3 interaction by the ceroid lipofuscinosis neuronal 3 protein CLN3.

Voltage-gated potassium (Kv) channels of the Kv4 subfamily associate with Kv channel-interacting proteins (KChIPs), which leads to enhanced surface expression and shapes the inactivation gating of these channels. KChIP3 has been reported to also interact with the late endosomal/lysosomal membrane glycoprotein CLN3 (ceroid lipofuscinosis neuronal 3), which is modified because of gene mutation in juvenile neuronal ceroid lipofuscinosis (JNCL). The present study was undertaken to find out whether and how CLN3, by its interaction with KChIP3, may indirectly modulate Kv4.2 channel expression and function. To this end, we expressed KChIP3 and CLN3, either individually or simultaneously, together with Kv4.2 in HEK 293 cells. We performed co-immunoprecipitation experiments and found a lower amount of KChIP3 bound to Kv4.2 in the presence of CLN3. In whole-cell patch-clamp experiments, we examined the effects of CLN3 co-expression on the KChIP3-mediated modulation of Kv4.2 channels. Simultaneous co-expression of CLN3 and KChIP3 with Kv4.2 resulted in a suppression of the typical KChIP3-mediated modulation; i.e. we observed less increase in current density, less slowing of macroscopic current decay, less acceleration of recovery from inactivation, and a less positively shifted voltage dependence of steady-state inactivation. The suppression of the KChIP3-mediated modulation of Kv4.2 channels was weaker for the JNCL-related missense mutant CLN3R334C and for a JNCL-related C-terminal deletion mutant (CLN3ΔC). Our data support the notion that CLN3 is involved in Kv4.2/KChIP3 somatodendritic A-type channel formation, trafficking, and function, a feature that may be lost in JNCL.

Electrical remodeling and cardiotoxicity precedes structural and functional remodeling of mouse hearts under hyperoxia treatment.

Clinical reports suggest a high incidence of ICU mortality with the use of hyperoxia during mechanical ventilation in patients. Our laboratory is pioneer in studying effect of hyperoxia on cardiac pathophysiology. In this study for the first time, we are reporting the sequence of cardiac pathophysiological events in mice under hyperoxic conditions in time-dependent manner. C57BL/6J male mice, aged 8-10 weeks, were treated with either normal air or >90% oxygen for 24, 48, and 72 h. Following normal air or hyperoxia treatment, physical, biochemical, functional, electrical, and molecular parameters were analyzed. Our data showed that significant reduction of body weight observed as early as 24 h hyperoxia treatment, whereas, no significant changes in heart weight until 72 h. Although we do not see any fibrosis in these hearts, but observed significant increase in cardiomyocyte size with hyperoxia treatment in time-dependent manner. Our data also demonstrated that arrhythmias were present in mice at 24 h hyperoxia, and worsened comparatively after 48 and 72 h. Echocardiogram data confirmed cardiac dysfunction in time-dependent manner. Dysregulation of ion channels such as Kv4.2 and KChIP2; and serum cardiac markers confirmed that hyperoxia-induced effects worsen with each time point. From these observations, it is evident that electrical remodeling precedes structural remodeling, both of which gets worse with length of hyperoxia exposure, therefore shorter periods of hyperoxia exposure is always beneficial for better outcome in ICU/critical care units.

DREAM and RB cooperate to induce gene repression and cell-cycle arrest in response to p53 activation.

Most human cancers acquire mutations causing defects in the p53 signaling pathway. The tumor suppressor p53 becomes activated in response to genotoxic stress and is essential for arresting the cell cycle to facilitate DNA repair or to initiate apoptosis. p53-induced cell cycle-arrest is mediated by expression of the CDK inhibitor p21WAF1/Cip1, which prevents phosphorylation and inactivation of the pocket proteins RB, p130, and p107. In a hypophosphorylated state, pocket proteins bind to E2F factors forming RB-E2F and DREAM transcriptional repressor complexes. Here, we analyze the influence of RB and DREAM on p53-induced gene repression and cell-cycle arrest. We show that abrogation of DREAM function by knockout of the DREAM component LIN37 results in a reduced repression of cell-cycle genes. We identify the genes repressed by the p53-DREAM pathway and describe a set of genes that is downregulated by p53 independent of LIN37/DREAM. Most strikingly, p53-dependent repression of cell-cycle genes is completely abrogated in LIN37-/-;RB-/- cells leading to a loss of the G1/S checkpoint. Taken together, we show that DREAM and RB are key factors in the p53 signaling pathway to downregulate a large number of cell-cycle genes and to arrest the cell cycle at the G1/S transition.

Kv4.2 autism and epilepsy mutation enhances inactivation of closed channels but impairs access to inactivated state after opening.

A de novo mutation in the KCND2 gene, which encodes the Kv4.2 K+ channel, was identified in twin boys with intractable, infant-onset epilepsy and autism. Kv4.2 channels undergo closed-state inactivation (CSI), a mechanism by which channels inactivate without opening during subthreshold depolarizations. CSI dynamically modulates neuronal excitability and action potential back propagation in response to excitatory synaptic input, controlling Ca2+ influx into dendrites and regulating spike timing-dependent plasticity. Here, we show that the V404M mutation specifically affects the mechanism of CSI, enhancing the inactivation of channels that have not opened while dramatically impairing the inactivation of channels that have opened. The mutation gives rise to these opposing effects by increasing the stability of the inactivated state and in parallel, profoundly slowing the closure of open channels, which according to our data, is required for CSI. The larger volume of methionine compared with valine is a major factor underlying altered inactivation gating. Our results suggest that V404M increases the strength of the physical interaction between the pore gate and the voltage sensor regardless of whether the gate is open or closed. Furthermore, in contrast to previous proposals, our data strongly suggest that physical coupling between the voltage sensor and the pore gate is maintained in the inactivated state. The state-dependent effects of V404M on CSI are expected to disturb the regulation of neuronal excitability and the induction of spike timing-dependent plasticity. Our results strongly support a role for altered CSI gating in the etiology of epilepsy and autism in the affected twins.

Pharmacological Approaches for the Modulation of the Potassium Channel KV4.x and KChIPs.

Ion channels are macromolecular complexes present in the plasma membrane and intracellular organelles of cells. Dysfunction of ion channels results in a group of disorders named channelopathies, which represent an extraordinary challenge for study and treatment. In this review, we will focus on voltage-gated potassium channels (KV), specifically on the KV4-family. The activation of these channels generates outward currents operating at subthreshold membrane potentials as recorded from myocardial cells (ITO, transient outward current) and from the somata of hippocampal neurons (ISA). In the heart, KV4 dysfunctions are related to Brugada syndrome, atrial fibrillation, hypertrophy, and heart failure. In hippocampus, KV4.x channelopathies are linked to schizophrenia, epilepsy, and Alzheimer's disease. KV4.x channels need to assemble with other accessory subunits (ß) to fully reproduce the ITO and ISA currents. ß Subunits affect channel gating and/or the traffic to the plasma membrane, and their dysfunctions may influence channel pharmacology. Among KV4 regulatory subunits, this review aims to analyze the KV4/KChIPs interaction and the effect of small molecule KChIP ligands in the A-type currents generated by the modulation of the KV4/KChIP channel complex. Knowledge gained from structural and functional studies using activators or inhibitors of the potassium current mediated by KV4/KChIPs will better help understand the underlying mechanism involving KV4-mediated-channelopathies, establishing the foundations for drug discovery, and hence their treatments.

A polybasic motif in alternatively spliced KChIP2 isoforms prevents Ca2+ regulation of Kv4 channels.

The Kv4 family of A-type voltage-gated K+ channels regulates the excitability in hippocampal pyramidal neuron dendrites and are key determinants of dendritic integration, spike timing-dependent plasticity, long-term potentiation, and learning. Kv4.2 channel expression is down-regulated following hippocampal seizures and in epilepsy, suggesting A-type currents as therapeutic targets. In addition to pore-forming Kv4 subunits, modulatory auxiliary subunits called K+ channel-interacting proteins (KChIPs) modulate Kv4 expression and activity and are required to recapitulate native hippocampal A-type currents in heterologous expression systems. KChIP mRNAs contain multiple start sites and alternative exons that generate considerable N-terminal variation and functional diversity in shaping Kv4 currents. As members of the EF-hand domain-containing neuronal Ca2+ sensor protein family, KChIP auxiliary proteins may convey Ca2+ sensitivity upon Kv4 channels; however, to what degree intracellular Ca2+ regulates KChIP-Kv4.2 complexes is unclear. To answer this question, we expressed KChIP2 with Kv4.2 in HEK293T cells, and, with whole-cell patch-clamp electrophysiology, measured an ∼1.5-fold increase in Kv4.2 current density in the presence of elevated intracellular Ca2+ Intriguingly, the Ca2+ regulation of Kv4 current was specific to KChIP2b and KChIP2c splice isoforms that lack a putative polybasic domain that is present in longer KChIP2a1 and KChIP2a isoforms. Site-directed acidification of the basic residues within the polybasic motif of KChIP2a1 rescued Ca2+-mediated regulation of Kv4 current density. These results support divergent Ca2+ regulation of Kv4 channels mediated by alternative splicing of KChIP2 isoforms. They suggest that distinct KChIP-Kv4 interactions may differentially control excitability and function of hippocampal dendrites.

MG53, A Novel Regulator of KChIP2 and Ito,f, Plays a Critical Role in Electrophysiological Remodeling in Cardiac Hypertrophy.

BACKGROUND: KChIP2 (K+ channel interacting protein) is the auxiliary subunit of the fast transient outward K+ current ( Ito,f) in the heart, and insufficient KChIP2 expression induces Ito,f downregulation and arrhythmogenesis in cardiac hypertrophy. Studies have shown muscle-specific mitsugumin 53 (MG53) has promiscuity of function in the context of normal and diseased heart. This study investigates the possible roles of cardiac MG53 in regulation of KChIP2 expression and Ito,f, and the arrhythmogenic potential in hypertrophy. METHODS: MG53 expression is manipulated by genetic ablation of MG53 in mice and adenoviral overexpression or knockdown of MG53 by RNA interference in cultured neonatal rat ventricular myocytes. Cardiomyocyte hypertrophy is produced by phenylephrine stimulation in neonatal rat ventricular myocytes, and pressure overload-induced mouse cardiac hypertrophy is produced by transverse aortic constriction. RESULTS: KChIP2 expression and Ito,f density are downregulated in hearts from MG53-knockout mice and MG53-knockdown neonatal rat ventricular myocytes, but upregulated in MG53-overexpressing cells. In phenylephrine-induced cardiomyocyte hypertrophy, MG53 expression is reduced with concomitant downregulation of KChIP2 and Ito,f, which can be reversed by MG53 overexpression, but exaggerated by MG53 knockdown. MG53 knockout enhances Ito,f remodeling and action potential duration prolongation and increases susceptibility to ventricular arrhythmia in mouse cardiac hypertrophy. Mechanistically, MG53 regulates NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) activity and subsequently controls KChIP2 transcription. Chromatin immunoprecipitation demonstrates NF-κB protein has interaction with KChIP2 gene. MG53 overexpression decreases, whereas MG53 knockdown increases NF-κB enrichment at the 5' regulatory region of KChIP2 gene. Normalizing NF-κB activity reverses the alterations in KChIP2 in MG53-overexpressing or knockdown cells. Coimmunoprecipitation and Western blotting assays demonstrate MG53 has physical interaction with TAK1 (transforming growth factor-b [TGFb]-activated kinase 1) and IκBα (nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha), critical components of the NF-κB pathway. CONCLUSIONS: These findings establish MG53 as a novel regulator of KChIP2 and Ito,f by modulating NF-κB activity and reveal its critical role in electrophysiological remodeling in cardiac hypertrophy.

Down-regulation of miR-3068-3p enhances kcnip4-regulated A-type potassium current to protect against glutamate-induced excitotoxicity.

The main cause of excitotoxic neuronal death in ischemic stroke is the massive release of glutamate. Recently, microRNAs (miRNAs) have been found to play an essential role in stroke pathology, although the molecular mechanisms remain to be investigated. Here, to identify potential candidate miRNAs involved in excitotoxicity, we treated rat primary cortical neurons with glutamate and found that miR-3068-3p, a novel miRNA, was up-regulated. We hypothesized that restoring miR-3068-3p expression might influence the neuronal injury outcomes. The inhibition of miR-3068-3p, using tough decoy lentiviruses, significantly attenuated the effects of glutamate on neuronal viability and intracellular calcium overload. To unravel the mechanisms, we employed bioinformatics analysis and RNA sequencing to identify downstream target genes. Additional luciferase assays and western blots validated kcnip4, a Kv4-mediated A-type potassium current (IA ) regulator, as a direct target of miR-3068-3p. The inhibition of miR-3068-3p increased kcnip4 expression and vice versa. In addition, the knockdown of kcnip4 by shRNA abolished the protective effect of miR-3068-3p, and over-expressing kcnip4 alone was sufficient to play a neuroprotective role in excitotoxicity. Moreover the inhibition of miR-3068-3p enhanced the IA density, and the pharmacological inhibition of IA abrogated the protective role of miR-3068-3p inhibition and kcnip4 over-expression. Therefore, we conclude that inhibition of miR-3068-3p protects against excitotoxicity via its target gene, kcnip4, and kcnip4-regulated IA . Our data suggest that the miR-3068-3p/kcnip4 axis may serve as a novel target for the treatment of ischemic stroke.

Effect and Molecular Mechanisms of Collateral Vessel Growth Mediated by Activation of Transient Receptor Potential Vanilloid Type 1.

Information on the function of transient receptor potential vanilloid 1 (TRPV1) in arteriogenesis is limited. We aimed to verify whether TRPV1 is involved in collateral vessel growth in rat hind limbs and elucidate the possible subcellular action mechanisms. Adult Sprague Dawley rats were chosen to establish the hind limb ischemic model and treatment with capsaicin. Angiographies were performed, and tissue was isolated for immunohistochemistry. In vitro, rat aortic endothelial cells (RAECs) were treated with capsaicin and antagonist capsazepine. The RAEC proliferation was determined, and the protein and mRNA levels of Ca2+-dependent transcription factors were assessed. In vivo, the collateral vessels exhibited positive outward remodeling characterized by enhanced inflammatory cell/macrophage accumulation in the adventitia and activated cell proliferation in all layers of the vascular wall and elevated endothelial NO synthetase expression in the rats with hind limb ligation. In RAECs, TRPV1 activation-induced Ca2+-dependent transcriptional factors, nuclear factor of activated T cells 1, calsenilin and myocyte enhancer factor 2C increase, and augmented RAEC proliferation could be a subcellular mechanism for TRPV1 in endothelial cells and ultimately contribute to collateral vessel growth. TRPV1, a novel candidate, positively regulates arteriogenesis, meriting further studies to unravel the potential therapeutic target leading to improved collateral vessel growth for treating ischemic diseases.