Design and characterization of a highly selective peptide inhibitor of the small conductance calcium-activated K+ channel, SkCa2

Apamin-sensitive small conductance calcium-activated potassium channels (SKCa1-3) mediate the slow afterhyperpolarization in neurons, but the molecular identity of the channel has not been defined because of the lack of specific inhibitors. Here we describe the structure-based design of a selective inhibitor of SKCa2. Leiurotoxin I (Lei) and PO5, peptide toxins that share the RXCQ motif, potently blocked human SKCa2 and SKCa3 but not SKCa1, whereas maurotoxin, Pi1, Tskappa, and PO1 were ineffective. Lei blocked these channels more potently than PO5 because of the presence of Ala(1), Phe(2), and Met(7). By replacing Met(7) in the RXCQ motif of Lei with the shorter, unnatural, positively charged diaminobutanoic acid (Dab), we generated Lei-Dab(7), a selective SKCa2 inhibitor (K(d) = 3.8 nm) that interacts with residues in the external vestibule of the channel. SKCa3 was rendered sensitive to Lei-Dab(7) by replacing His(521) with the corresponding SKCa2 residue (Asn(367)). Intracerebroventricular injection of Lei-Dab(7) into mice resulted in no gross central nervous system toxicity at concentrations that specifically blocked SKCa2 homotetramers. Lei-Dab(7) will be a useful tool to investigate the functional role of SKCa2 in mammalian tissues.     

Bradykinin activates calcium-dependent potassium channels in cultured human airway smooth muscle cells

Bradykinin (BK) is an inflammatory mediator that can cause bronchoconstriction. In this study, we investigated the membrane currents induced by BK in cultured human airway smooth muscle (ASM) cells. Depolarization of the cells induced outward currents, which were inhibited by tetraethylammonium (TEA) in a concentration-dependent manner with an IC50 of 0.33 microM. The currents were increased by elevating intracellular free Ca2+ concentration, suggesting they are calcium-activated potassium channels [I(K(Ca))]. Preexposure to inhibitor of I(K(Ca)) of large conductance (BKCa), iberiotoxin, and small conductance (SKCa), apamin, inhibited the increase of outward current induced by BK. The relative contribution of BKCa was greatest in early passage cells. Both nickel and SKF-96365 (10 microM) inhibited the increase of the I(K(Ca)) induced by BK; however, the l-type Ca2+ channel blocker, nifedipine, had no effect. Activation of the BK-induced current was inhibited by heparin, indicating dependence on intact inositol 1,4,5-triphosphate (IP3)-sensitive intracellular Ca2+ stores. BK also increased inositol phosphate accumulation and induced a transient Ca2+-activated chloride current (CACC) and a sustained nonselective cation current (I(CAT)). In summary, BK activates BKCa, SKCa, CACC, and I(CAT) via IP3-sensitive stores in human ASM.     

Subcellular localisation of human inositol 1,4,5-trisphosphate 3-kinase C: species-specific use of alternative export sites for nucleo-cytoplasmic shuttling indicates divergent roles of the catalytic and N-terminal domains

The three isoforms of human Ins(1,4,5)P3 3-kinase (IP3K) show remarkable differences in their intracellular targeting. Whereas predominant targeting to the cytoskeleton and endoplasmic reticulum has been shown for IP3K-A and IP3K-B, rat IP3K-C shuttles actively between the nucleus and cytoplasm. In the present study we examined the expression and intracellular localisation of endogenous IP3K-C in different mammalian cell lines using an isoform-specific antibody. In addition, human IP3K-C, showing remarkable differences to its rat homologue in the N-terminal targeting domain, was tagged with EGFP and used to examine active transport mechanisms into and out of the nucleus. We found both a nuclear import activity residing in its N-terminal domain and a nuclear export activity sensitive to treatment with leptomycin B. Different from the rat isoform, an exportin 1-dependent nuclear export site of the human enzyme resides outside the N-terminal targeting domain in the catalytic enzyme domain. A phylogenetic survey of vertebrate IP3K sequences indicates that in each of the three isoforms a nuclear export signal has evolved in the catalytic domain either de novo (IP3K-A) or as a substitute for an earlier evolved corresponding N-terminal signal (IP3K-B and IP3K-C). In higher vertebrates, and in particular in primates, re-export of nuclear IP3K activity may be guaranteed by the mechanism discovered.     

Dissecting the structural and functional roles of the S3-S4 linker of pacemaker (hyperpolarization-activated cyclic nucleotide-modulated) channels by systematic length alterations

If or Ih, a key player in neuronal and cardiac pacing, is encoded by the hyperpolarization-activated cyclic nucleotide-modulated (HCN) channel gene family. We have recently reported that the S3-S4 linker (i.e. residues 229EKGMDSEVY237 of HCN1) prominently influences the activation phenotypes of HCN channels and that part of the linker may conform a secondary helical structure. Here we further dissected the structural and functional roles of this linker by systematic alterations of its length. In contrast to voltage-gated K+ channels, complete deletion of the S3-S4 linker (Delta229-237) did not produce functional channels. Similarly, the deletions Delta229-234, Delta232-234, and Delta232-237 also abolished normal current activity. Interestingly, Delta229-231, Delta233-237, Delta234-237, Delta235-237, Delta229-231/Delta233-237, Delta229-231/Delta234-237, and Delta229-231/Delta235-237 all yielded robust hyperpolarization-activated inward currents, indicating that loss-of-function caused by deletion could be rescued by keeping the single functionally important residue Met232 alone. Whereas shortening the linker by deletion generally shifted steady-state activation in the depolarizing direction (e.g. DeltaV1/2 of Delta229-231, Delta233-237, Delta235-237 > +10 mV relative to wild type), linker prolongation by duplicating the entire linker (Dup229-237) or by glutamine insertion (InsQ233Q, InsQQ233QQ and InsQQQ233QQQ, or Ins237QQQ) produced length-dependent progressive hyperpolarizing activation shifts (-35 mV < DeltaV1/2 < -4 mV). Based on these results, we conclude that only Met232 is prerequisite for channels to function, but the length and other constituents of the S3-S4 linker shape the ultimate activation phenotype. Our results also highlight several evolutionary similarities and differences between HCN and voltage-gated K+ channels. Manipulations of the S3-S4 linker length may provide a flexible approach to customize HCN gating for engineering electrically active cells (such as stem cell-derived neuronal and cardiac pacemakers) for gene- and cell-based therapies.     

The localization of histone H3K27me3 demethylase Jmjd3 is dynamically regulated

Jmjd3 is required for cellular differentiation and senescence, and inhibits the induction of pluripotent stem cells by demethylating histone 3 lysine 27 trimethylation (H3K27me3). Although recent studies reveal crucial biological roles for Jmjd3, it is unclear how its demethylase activity is controlled. Here, we show that nuclear localization of Jmjd3 is required for effective demethylation of H3K27me3. Our subcellular localization analysis of Jmjd3 shows that the N-terminal region of the protein is responsible for its nuclear placement, whereas the C-terminal region harboring the catalytic Jumonji C (JmjC) domain cannot situate into the nucleus. We identify two classical nuclear localization signals (cNLSs) in the N-terminal domain of Jmjd3. Forced nuclear emplacement of the catalytic domain of Jmjd3 by fusion with a heterologous cNLS significantly enhances its H3K27me3 demethylation activity. A dynamic nucleocytoplasmic shuttling of endogenous Jmjd3 occurs in mouse embryonic fibroblasts. Jmjd3 is localized both into the cytoplasm and the nucleus, and its nuclear export is dependent on Exportin-1, as treatment with leptomycin B triggers nuclear accumulation of Jmjd3. These results suggest that the subcellular localization of Jmjd3 is dynamically regulated and has pivotal roles for H3K27me3 status.     

Regulation of nucleo-cytoplasmic shuttling of human annexin A2—a proposed mechanism

Studies have long been focused on the functions of annexin A2 in the cytoplasm. However, the involvement of annexin A2 in DNA replication as a part of primer recognition protein complex and the presence of nuclear export signal (NES) suggest that annexin A2 is also functional in the nucleus, and its localization in the nucleus is under regulation by interaction with other nuclear factors through its N-terminus. During the study of the mechanism of annexin A2 sequestering in the nucleus and the regulation of its export from the nucleus, in this study, we show that endogenous annexin A2 is present in both the cytoplasm and the nucleus in HeLa, PC-3 and DU-145 cells. While exogenously expressed annexin A2 is excluded from nuclei of annexin A2-null LNCaP cells in a CRM1 (Chromosome Maintenance Region 1) mediated nuclear export, endogenous annexin A2 in HeLa, PC-3 and DU-145 cell lines does not undergo the CRM1 mediated nuclear export. While investigating the mechanism of the nuclear retention of annexin A2, we found that an anti-annexin A2 antibody that recognizes the C-terminus of annexin A2 (D1/274.5) cannot recognize nuclear annexin A2, suggesting that the domain recognized by this antibody may be masked in the nuclei. In order to find out the role of annexin A2 C-terminus in the nuclear retention of annexin A2, we transiently transfected green fluorescence protein (GFP)-fused N-terminal 29 amino acids of annexin A2 to LNCaP, PC-3 and DU-145 cells, and determined that the C-terminus is not required for the nuclear retention of annexin A2. Based on the finding described above, we propose a model for nuclear retention of annexin A2 where the regulation sites reside in the N-terminus and are adjacent to the NES, and upon modification, the NES is exposed and annexin A2 is exported from the nucleus. Electronic Supplementary Material The online version of this article (doi) contains supplementary material, which is available to authorized users.     

The localization of histone H3K27me3 demethylase Jmjd3 is dynamically regulated

Jmjd3 is required for cellular differentiation and senescence, and inhibits the induction of pluripotent stem cells by demethylating histone 3 lysine 27 trimethylation (H3K27me3). Although recent studies reveal crucial biological roles for Jmjd3, it is unclear how its demethylase activity is controlled. Here, we show that nuclear localization of Jmjd3 is required for effective demethylation of H3K27me3. Our subcellular localization analysis of Jmjd3 shows that the N-terminal region of the protein is responsible for its nuclear placement, whereas the C-terminal region harboring the catalytic Jumonji C (JmjC) domain cannot situate into the nucleus. We identify two classical nuclear localization signals (cNLSs) in the N-terminal domain of Jmjd3. Forced nuclear emplacement of the catalytic domain of Jmjd3 by fusion with a heterologous cNLS significantly enhances its H3K27me3 demethylation activity. A dynamic nucleocytoplasmic shuttling of endogenous Jmjd3 occurs in mouse embryonic fibroblasts. Jmjd3 is localized both into the cytoplasm and the nucleus, and its nuclear export is dependent on Exportin-1, as treatment with leptomycin B triggers nuclear accumulation of Jmjd3. These results suggest that the subcellular localization of Jmjd3 is dynamically regulated and has pivotal roles for H3K27me3 status.     

Distinct molecular regulation of glycogen synthase kinase-3alpha isozyme controlled by its N-terminal region: functional role in calcium/calpain signaling

Glycogen synthase kinase-3 (GSK-3) is expressed as two isozymes α and β. They share high similarity in their catalytic domains but differ in their N- and C-terminal regions, with GSK-3α having an extended glycine-rich N terminus. Here, we undertook live cell imaging combined with molecular and bioinformatic studies to understand the distinct functions of the GSK-3 isozymes focusing on GSK-3α N-terminal region. We found that unlike GSK-3β, which shuttles between the nucleus and cytoplasm, GSK-3α was excluded from the nucleus. Deletion of the N-terminal region of GSK-3α resulted in nuclear localization, and treatment with leptomycin B resulted in GSK-3α accumulation in the nucleus. GSK-3α rapidly accumulated in the nucleus in response to calcium or serum deprivation, and accumulation was strongly inhibited by the calpain inhibitor calpeptin. This nuclear accumulation was not mediated by cleavage of the N-terminal region or phosphorylation of GSK-3α. Rather, we show that calcium-induced GSK-3α nuclear accumulation was governed by GSK-3α binding with as yet unknown calpain-sensitive protein or proteins; this binding was mediated by the N-terminal region. Bioinformatic and experimental analyses indicated that nuclear exclusion of GSK-3α was likely an exclusive characteristic of mammalian GSK-3α. Finally, we show that nuclear localization of GSK-3α reduced the nuclear pool of β-catenin and its target cyclin D1. Taken together, these data suggest that the N-terminal region of GSK-3α is responsible for its nuclear exclusion and that binding with a calcium/calpain-sensitive product enables GSK-3α nuclear retention. We further uncovered a novel link between calcium and nuclear GSK-3α-mediated inhibition of the canonical Wnt/β-catenin pathway.     

Intracellular blockade of GABAA receptors in the rat hippocampal neurons

The intracellular blockade of GABA_A-receptor-mediated currents is a useful approach to suppress the GABAergic conductance in a single cell and to isolate the glutamatergic component of network-driven activities. Previously an approach has been described allowing intracellular blockade of GABA_A receptors by means of intracellular dialysis of a neuron with the pipette-filling solution, in which fluoride ions that hardly pass through the GABA_A receptor channels substitute for Cl^? and in which Mg²⁺ and ATP are omitted to induce rundown of the GABA_A receptors during whole-cell patch-clamp recordings. However, the kinetics of suppression of GABAergic conductance and the effect on the currents mediated by glutamate receptors remain unknown. Here, using whole-cell recordings with fluoride-based, Mg²⁺- and ATP-free solution on CA3 hippocampal neurons of neonatal rats, we show that after 1 h of such dialysis, both spontaneous and evoked GABA_A-receptor-mediated synaptic currents and responses induced by the GABA_A receptor agonist isoguvacine were completely suppressed. Inward GABAergic postsynaptic currents were suppressed prior to outward currents. Synaptic responses mediated by AMPA receptors were not affected by the dialysis, whereas the NMDA-receptor-mediated postsynaptic currents were reduced by approximately 20%. Dialysis with fluoride-based Mg²⁺, ATP-free solution either fully blocked giant depolarizing potentials (GDPs) in CA3 pyramidal cells (n = 2) or reduced the charge crossing the membrane during GDPs and shifted the GDP reversal potential to more positive values (n = 5). The dialysis-resistant component of GDPs was mediated by glutamate receptors, since: (i) it reversed around 0 mV; (ii) it demonstrated a negative slope conductance at negative membrane voltages, which is characteristic of NMDA receptor-mediated responses; (iii) kinetics of the individual events composing the dialysis-resistant component of GDPs at negative voltages were very similar to those of AMPA receptor-mediated synaptic currents. Thus, this procedure can be useful to isolate the glutamate receptor-mediated component of neuronal network-driven activities.     

八肋游仆虫第二类肽链释放因子核定位信号分析

蛋白质合成终止过程中肽链释放因子负责终止密码子的识别.真核生物第二类肽链释放因子（eRF3）是一类GTP酶,协助第一类肽链释放因子（eRF1）识别终止密码子和水解肽酰 tRNA酯键.之前的研究表明,两类肽链释放因子在细胞核中发挥功能,参与蛋白质合成和纺锤体的组装.本研究根据软件预测结果,构建了一系列八肋游仆虫eRF3的截短型突变体,分析在其N端是否存在引导eRF3的核定位信号.结果表明,在eRF3的N端有两个区域（NLS1:23-36 aa 和 NLS2: 236-272 aa）可以引导eRF3进入细胞核中,而且这两个区域具有典型的核定位信号的氨基酸序列特征. eRF3的核定位与其作为一种穿梭蛋白的功能相一致,即参与细胞有丝分裂纺锤体的形成和无义介导的mRNA降解途径.     

G protein-coupled receptor kinase 5 contains a DNA-binding nuclear localization sequence

G protein-coupled receptor kinases (GRKs) mediate desensitization of agonist-occupied G protein-coupled receptors (GPCRs). Here we report that GRK5 contains a DNA-binding nuclear localization sequence (NLS) and that its nuclear localization is regulated by GPCR activation, results that suggest potential nuclear functions for GRK5. As assessed by fluorescence confocal microscopy, transfected and endogenous GRK5 is present in the nuclei of HEp2 cells. Mutation of basic residues in the catalytic domain of GRK5 (between amino acids 388 and 395) results in the nuclear exclusion of the mutant enzyme (GRK5(Delta)(NLS)), demonstrating that GRK5 contains a functional NLS. The nuclear localization of GRK5 is subject to dynamic regulation. Calcium ionophore treatment or activation of Gq-coupled muscarinic-M3 receptors promotes the nuclear export of the kinase in a Ca(2+)/calmodulin (Ca(2+)/CaM)-dependent fashion. Ca(2+)/CaM binding to the N-terminal CaM binding site of GRK5 mediates this effect. Furthermore, GRK5, but not GRK5(Delta)(NLS) or GRK2, binds specifically and directly to DNA in vitro. Consistent with their presence in the nuclei of transfected cells, all the GRK4, but not GRK2, subfamily members contain putative NLSs. These results suggest that the GRK4 subfamily of GRKs may play a signaling role in the nucleus and that GRK4 and GRK2 subfamily members perform divergent cellular functions.     

Primary structure of a dynamin-related mouse mitochondrial GTPase and its distribution in brain, subcellular localization, and effect on mitochondrial morphology

A new member of the dynamin GTPase family (OPA1) was recently identified in humans and shown to be mutated in patients with dominant optic atrophy. To understand better the function of mammalian OPA1, we isolated a mouse ortholog (mOPA1) from brain and raised a specific antibody against its C terminus. The subcellular distribution of mOPA1 overexpressed in COS-7 cells largely overlapped that of endogenous cytochrome c, a well known mitochondrial marker, and dramatically affected mitochondrial morphology, altering it from tubular to vesicular. Mitochondrial targeting was mediated by the N-terminal region of mOPA1 as follows: deletion of the 124 N-terminal amino acids eliminated mitochondrial targeting, although fusion of the N-terminal 60 or 90 amino acids of mOPA1 with green fluorescent protein resulted in its mitochondrial targeting. mOPA1 was expressed widely in the mouse brain, especially in neurons of olfactory bulb, cerebral cortex, piriform cortex, hypothalamus, hippocampus, red nucleus, cochlear nucleus, motor trigeminal nucleus, facial nucleus, cerebellar nucleus, and Purkinje cells. Within dissociated cerebellar cells, mOPA1 protein was clearly observed in the dendrites and somas of neuronal cells, as well as in astrocytes and meningeal cells. In each case, it was distributed in the vesicular pattern seen in other cell types.     

14,15-EET Suppresses Neuronal Apoptosis in Ischemia–Reperfusion Through the Mitochondrial Pathway

Neuronal apoptosis mediated by the mitochondrial apoptosis pathway is an important pathological process in cerebral ischemia–reperfusion injury. 14,15-EET, an intermediate metabolite of arachidonic acid, can promote cell survival during ischemia/reperfusion. However, whether the mitochondrial apoptotic pathway is involved this survival mechanism is not fully understood. In this study, we observed that infarct size in ischemia–reperfusion injury was reduced in sEH gene knockout mice. In addition, Caspase 3 activation, cytochrome C release and AIF nuclear translocation were also inhibited. In this study, 14,15-EET pretreatment reduced neuronal apoptosis in the oxygen–glucose deprivation and re-oxygenation group in vitro. The mitochondrial apoptosis pathway was also inhibited, as evidenced by AIF translocation from the mitochondria to nucleus and the reduction in the expressions of cleaved-caspase 3 and cytochrome C in the cytoplasm. 14,15-EET could reduce neuronal apoptosis through upregulation of the ratio of Bcl-2 (anti-apoptotic protein) to Bax (apoptosis protein) and inhibition of Bax aggregation onto mitochondria. PI3K/AKT pathway is also probably involved in the reduction of neuronal apoptosis by EET. Our study suggests that 14,15-EET could suppress neuronal apoptosis and reduce infarct volume through the mitochondrial apoptotic pathway. Furthermore, the PI3K/AKT pathway also appears to be involved in the neuroprotection against ischemia–reperfusion by 14,15-EET.     

The cytoplasmic C-terminal fragment of polycystin-1 regulates a Ca2+-permeable cation channel

The cytoplasmic C-terminal portion of the polycystin-1 polypeptide (PKD1(1-226)) regulates several important cell signaling pathways, and its deletion suffices to cause autosomal dominant polycystic kidney disease. However, a functional link between PKD1 and the ion transport processes required to drive renal cyst enlargement has remained elusive. We report here that expression at the Xenopus oocyte surface of a transmembrane fusion protein encoding the C-terminal portion of the PKD1 cytoplasmic tail, PKD1(115-226), but not the N-terminal portion, induced a large, Ca(2+)-permeable cation current, which shifted oocyte reversal potential (E(rev)) by +33 mV. Whole cell currents were sensitive to inhibition by La(3+), Gd(3+), and Zn(2+), and partially inhibited by SKF96365 and amiloride. Currents were not activated by bath hypertonicity, but were inhibited by acid pH. Outside-out patches pulled from PKD1(115-226)-expressing oocytes exhibited a 5.1-fold increased NP(o) of endogenous 20-picosiemens cation channels of linear conductance. PKD1(115-226)-injected oocytes also exhibited elevated NP(o) of unitary calcium currents in outside-out and cell-attached patches, and elevated calcium permeability documented by fluorescence ratio and (45)Ca(2+) flux experiments. Both Ca(2+) conductance and influx were inhibited by La(3+). Mutation of candidate phosphorylation sites within PKD1(115-226) abolished the cation current. We conclude that the C-terminal cytoplasmic tail of PKD1 up-regulates inward current that includes a major contribution from Ca(2+)-permeable nonspecific cation channels. Dysregulation of these or similar channels in autosomal dominant polycystic kidney disease may contribute to cyst formation or expansion.     

Induction of K-channel expression in a neuroblastoma cell line

Whole-cell currents were examined in mouse neuroblastoma cells of the N2AB-1 line. In standard culture medium, N2AB-1 cells exhibited large voltage-dependent Na currents but no discernible K currents. Treatment of N2AB-1 cells with either dimethylsulfoxide (DMSO) in low-serum medium or with retinoic acid (RA) caused the expression of delayed rectifier K currents. Currents from two types of K channel with single channel slope conductances of 15.0 pS and 6.4 pS were observed in outside-out patches from cells of both treatment groups. Thus, while N2AB-1 cells did not exhibit K currents under standard culture conditions, they did possess the gene(s) encoding K channels. The treatments caused other changes that were not directly linked to K-channel expression. RA treatment caused neurite extension in most, but not all, N2AB-1 cells; however, all RA-treated cells, including those without neurites, expressed K currents. RA treatment did not suppress cell division or cause hypertrophy. In contrast, treatment with DMSO/low serum suppressed cell division and caused cellular hypertrophy, but did not cause long neurites to form. Thus, the regulation of K channels was not coupled in a simple fashion to properties that have been associated with a differentiated neuronal phenotype: neurite elaboration, changes in cell size, and inhibition of cell division. These results suggest that N2AB-1 cells may be a good model system for investigating the processes regulating K-channel expression.