Substrate profiling and aldehyde dismutase activity of the Kvβ2 subunit of the mammalian Kv1 potassium channel

Voltage-dependent potassium channels (Kv) are involved in various cellular signalling processes by governing the membrane potential of excitable cells. The cytosolic face of these α subunit-containing channels is associated with β subunits that can modulate channel responses. Surprisingly, the β subunit of the mammalian Kv1 channels, Kvβ2, has a high level of sequence homology with the aldo–keto reductase (AKR) superfamily of proteins. Recent studies have shown that Kvβ2 can catalyze the reduction of aldehydes and, most significantly, that channel function is modulated when Kvβ2-bound NADPH is concomitantly oxidized. As a result, the redox chemistry of this subunit is crucial to understanding its role in K⁺ channel modulation. The present study has extended knowledge of the substrate profile of this subunit using a single turnover fluorimetric assay. Kvβ2 was found to catalyse the reduction of aromatic aldehyde substrates such as 2, 3 and 4-nitrobenzaldehydes, 4-hydroxybenzaldehyde, pyridine 2-aldehyde and benzaldehyde. The presence of an electron withdrawing group at the position para to the aldehyde in aromatic compounds facilitated reduction. Aliphatic aldehydes proved to be poor substrates. We devised a simple HPLC-based assay to identify Kvβ2 reaction products. Using this assay we showed, for the first time, that Kvβ2 can catalyze a slow aldehyde dismutation reaction using 4-nitrobenzaldehyde as substrate and have identified the products of this reaction. The ability of Kvβ2 to carry out both an aldehyde reduction and a dismutation reaction is discussed in the light of current thinking on the role of redox chemistry in channel modulation.     

KCNE1 and KCNE2 provide a checkpoint governing voltage-gated potassium channel α-subunit composition

Voltage-gated potassium (Kv) currents generated by N-type α-subunit homotetramers inactivate rapidly because an N-terminal ball domain blocks the channel pore after activation. Hence, the inactivation rate of heterotetrameric channels comprising both N-type and non-N-type (delayed rectifier) α-subunits depends upon the number of N-type α-subunits in the complex. As Kv channel inactivation and inactivation recovery rates regulate cellular excitability, the composition and expression of these heterotetrameric complexes are expected to be tightly regulated. In a companion article, we showed that the single transmembrane segment ancillary (β) subunits KCNE1 and KCNE2 suppress currents generated by homomeric Kv1.4, Kv3.3, and Kv3.4 channels, by trapping them early in the secretory pathway. Here, we show that this trapping is prevented by coassembly of the N-type α-subunits with intra-subfamily delayed rectifier α-subunits. Extra-subfamily delayed rectifier α-subunits, regardless of their capacity to interact with KCNE1 and KCNE2, cannot rescue Kv1.4 or Kv3.4 surface expression unless engineered to interact with them using N-terminal A and B domain swapping. The KCNE1/2-enforced checkpoint ensures N-type α-subunits only reach the cell surface as part of intra-subfamily mixed-α complexes, thereby governing channel composition, inactivation rate, and—by extension—cellular excitability.     

TGF-β1 enhances Kv2.1 potassium channel protein expression and promotes maturation of cerebellar granule neurons

Members of the transforming growth factor-β (TGF-β) family of cytokines are involved in diverse physiological processes. Although TGF-β is known to play multiple roles in the mammalian central nervous system (CNS), its role in neuronal development has not been explored. We have studied the effects of TGF-β1 on the electrophysiological properties and maturation of rat primary cerebellar granule neurons (CGNs). We report that incubation with TGF-β1 increased delayed rectifier potassium current (I(K) ) amplitudes in a dose- and time-dependent manner, but did not affect the kinetic properties of the channel. Exposure to TGF-β1 (20 ng/ml) for 36 h led to a 37.2% increase in I(K) amplitudes. There was no significant change in mRNA levels for the key Kv2.1 channel protein, but translation blockade abolished the increase in protein levels and channel activity, arguing that TGF-β1 increases I(K) amplitudes by upregulating translation of the Kv2.1 channel protein. Although TGF-β1 treatment did not affect the activity of protein kinase A (PKA), and constitutive activation of PKA with forskolin failed to increase I(K) amplitudes, inhibition of PKA prevented channel upregulation, demonstrating that basal PKA activity is required for TGF-β1 stimulation of I(K) channel activity. TGF-β1 also promoted the expression of the γ-aminobutyric acid (GABA(A) ) receptor α6 subunit, a marker of mature CGNs, and calcium influx during depolarizing stimuli was reduced by TGF-β1. The effects of TGF-β1 were only observed during a narrow developmental time-window, and were lost as CGNs matured. These findings suggest that TGF-β1 upregulates K(+) channel expression and I(K) currents and thereby promotes CGN maturation.     

Novel estrone mimetics with high 17β-HSD1 inhibitory activity

17β-Hydroxysteroid dehydrogenase type 1 (17β-HSD1) catalyzes the reduction of estrone into estradiol, which is the most potent estrogen in humans. Lowering intracellular estradiol concentration by inhibition of this enzyme is a promising new option for the treatment of estrogen-dependent diseases like breast cancer and endometriosis. Combination of ligand- and structure-based design resulted in heterocyclic substituted biphenylols and their aza-analogs as new 17β-HSD1 inhibitors. The design was based on mimicking estrone, especially focusing on the imitation of the D-ring keto group with (substituted) heterocycles. Molecular docking provided insights into plausible protein–ligand interactions for this class of compounds. The most promising compound 12 showed an inhibitory activity in the high nanomolar range and very low affinity for the estrogen receptors α and β. Thus, compound 12 is a novel tool for the elucidation of the pharmacological relevance of 17β-HSD1 and might be a lead for the treatment of estrogen-dependent diseases.     

Integrin α9β1-mediated cell migration in glioblastoma via SSAT and Kir4.2 potassium channel pathway

The α9β1 integrin accelerates cell migration through binding of the α9 cytoplasmic domain to SSAT, which catalyzes the catabolism of higher order polyamines, spermidine and spermine, to the lower order polyamine, putrescine. SSAT levels were downregulated at both the mRNA and protein levels by shRNA-mediated simultaneous knockdown of MMP-9 and uPAR/cathepsin B. In addition, we noted a prominent reduction in the expression of SSAT with MMP-9 and uPAR/cathepsin B knockdown in the tumor regions of 5310 injected nude mice brains. Further, SSAT knockdown in glioma xenograft cells significantly reduced their migration potential. Interestingly, MMP-9, uPAR and cathepsin B overexpression in these xenograft cells significantly elevated SSAT mRNA and protein levels. The migratory potential of MMP-9/uPAR/cathepsin B-overexpressed 4910 and 5310 cells was not affected by either glybenclamide (Kir 6.x inhibitor) or tertiapin-Q (Kir 1.1 and 3.x inhibitor) but instead was significantly inhibited by either barium or Kir4.2 siRNA treatments. Co-localization of α9 integrin with Kir4.2 was observed in both 4910 and 5310 xenograft cells. However, MMP-9 and uPAR/cathepsin B knockdown in these cells prominently reduced the co-localization of α9 with Kir4.2. Taken together, our results clearly demonstrate that α9β1 integrin-mediated cell migration utilizes SSAT and the Kir4.2 potassium channel pathway, and inhibition of the migratory potential of these glioma xenograft cells by simultaneous knockdown of MMP-9 and uPAR/cathepsin B could be attributed to the reduced SSAT levels and co-localization of α9 integrin with Kir4.2 inward rectifier potassium channels.     

Molecular simulation of the interaction of κ-conotoxin-PVIIA with the Shaker potassium channel pore

Molecular simulation techniques were appplied to predict the interaction of the voltage-dependent Shaker potassium channel with the channel-blocking toxin kappa-conotoxin-PVIIA (PVIIA). A structural thee-dimensional model of the extracellular vestibule of the potassium channel was constructed based on structural homologies with the bacterial potassium channel Kcsa, whose structure has been solved by X-ray crystallography. The docking of the PVIIA molecule was obtained by a geometric recognition algorithm, yielding 100 possible conformations. A series of residue-residue distance restraints, predicted from mutation-cycle experiments, were used to select a small set of a plausible channel-toxin complex models among the resulting possible conformations. The four final conformations, with similar characteristics, can explain most of the single-point mutation experiments done with this system. The models of the Shaker-PVIIA interaction predict two clusters of amino acids, critical for the binding of the toxin to the channel. The first cluster is the amino acids R2, I3, Q6 and K7 that form the plug of the toxin that interacts with the entrance to the selectivity filter of the channel. The second cluster of residues, R22, F23, N24 and K25, interacts with a channel region near to the external entrance of the pore vestibule. The consistency of the obtained models and the experimental data indicate that the Shaker-PVIIA complex model is reasonable and can be used in further biological studies such as the rational design of blocking agents of potassium channels and the mutagenesis of both toxins and potassium channels.     

Design,recombinant expression,and antibacterial activity of a novel hybrid magainin–thanatin antimicrobial peptide

Antimicrobial peptides are small molecule polypeptides with biological activity, which can avoid the drug resistance. Magainin and thanatin are antimicrobial peptides with a broad spectrum of inhibitory microbes, and the core sequence of magainin is linked to a core sequence of thanatin. Here, the hybrid magainin–thanatin (MT) antimicrobial peptide was designed through bioinformatics analysis. The recombinant MT antimicrobial peptide was successfully expressed and purified in Escherichia coli BL21 (DE3). The molecular weight of the hybrid MT antimicrobial peptide was about 3.35?kDa. Moreover, the target protein indeed has an inhibitory effect on Staphylococcus aureus, E. coli DH5α, and Bacillus subtilis, with the minimum inhibitory concentrations 16.5, 20, and 9?μM, respectively. The rational designed hybrid MT antimicrobial peptide will hopefully provide large-scale fermentable antimicrobial peptides in the industrial production in the future.     

Anticonvulsant effect of cytoskeletal depolymerizers in combination with potassium channel opener and adenylate cyclase activator; a causative link with nerve growth factor?

Anticonvulsant effect of cytoskeletal depolymerizing drugs in combination with potassium channel (KATP) opener and adenylate cyclase activator was evaluated in animal models of epilepsy. Seizures were induced in the animals by subjecting them to maximal electroshock (MES) or by injecting a chemical convulsant, pentylenetetrazole (PTZ). Moreover a correlation with the nerve growth factor (NGF) was also investigated. The anticonvulsant effect of minoxidil (1200 micrograms/kg i.p.) and Deacetylforskolin (600 micrograms/kg i.p.) was significantly enhanced in the mice pre-treated with cytoskeletal depolymerizing drugs. On the other hand nerve growth factor potentiated the convulsive phenomenon and decreased the seizure threshold in both the electroshock and chemically induced convulsions. Another interesting feature was the interaction of cytochalasin B, a microfilament disrupter in preventing the action of mNGF and PTZ. This study demonstrates the importance of interaction between cytoskeletal structures and signalling molecules in determining the convulsive threshold. This study clearly points to the importance of the nerve growth factor in convulsive phenomenon.     

Spatial association of the Cav1.2 calcium channel with α5β1-integrin

Engagement of α(5)β(1)-integrin by fibronectin (FN) acutely enhances Cav1.2 channel (Ca(L)) current in rat arteriolar smooth muscle and human embryonic kidney cells (HEK293-T) expressing Ca(L). Using coimmunoprecipitation strategies, we show that coassociation of Ca(L) with α(5)- or β(1)-integrin in HEK293-T cells is specific and depends on cell adhesion to FN. In rat arteriolar smooth muscle, coassociations between Ca(L) and α(5)β(1)-integrin and between Ca(L) and phosphorylated c-Src are also revealed and enhanced by FN treatment. Using site-directed mutagenesis of Ca(L) heterologously expressed in HEK293-T cells, we identified two regions of Ca(L) required for these interactions: 1) COOH-terminal residues Ser(1901) and Tyr(2122), known to be phosphorylated by protein kinase A (PKA) and c-Src, respectively; and 2) two proline-rich domains (PRDs) near the middle of the COOH terminus. Immunofluorescence confocal imaging revealed a moderate degree of wild-type Ca(L) colocalization with β(1)-integrin on the plasma membrane. Collectively, our results strongly suggest that 1) upon ligation by FN, Ca(L) associates with α(5)β(1)-integrin in a macromolecular complex including PKA, c-Src, and potentially other protein kinases; 2) phosphorylation of Ca(L) at Y(2122) and/or S(1901) is required for association of Ca(L) with α(5)β(1)-integrin; and 3) c-Src, via binding to PRDs that reside in the II-III linker region and/or the COOH terminus of Ca(L), mediates current potentiation following α(5)β(1)-integrin engagement. These findings provide new evidence for how interactions between α(5)β(1)-integrin and FN can modulate Ca(L) entry and consequently alter the physiological function of multiple types of excitable cells.     

Hyptenolide,a new α-pyrone with spasmolytic activity from Hyptis macrostachys

A new α-pyrone was isolated from aerial parts of Hyptis macrostachys Benth. Its structure was determined as 6R-[(5′S,6′S-diacetoxy)-1′Z,3′E-heptenyl]-5,6-dihydro-2H-pyran-2-one, named hyptenolide based on a combination of 1D and 2D NMR techniques and CD data. Hyptenolide inhibited the contractions induced by CCh (IC₅₀ = 1.7 ± 0.3 × 10⁻⁴ M) or histamine (IC₅₀ = 0.9 ± 0.05 × 10⁻⁴ M) in guinea pig ileum, demonstrating for the first time a pharmacological activity for the pyrone.     

Structure–activity relationship study on polyglutamine binding peptide QBP1

Aggregation and deposition of expanded polyglutamine proteins in the brain cause neurodegenerative diseases including Huntington disease. This pathogenic process is suppressed and delayed in the presence of polyglutamine binding peptide 1 (QBP1), which we previously identified as an undecapeptide binding to pathogenic polyglutamine proteins from phage display peptide libraries. In this paper, a structure–activity relationship study on QBP1 was conducted to determine the pharmacophores for inhibition of polyglutamine aggregation. Furthermore, a truncation study identified an octapeptide as the minimum structure for suppressing aggregation of polyglutamine proteins, which is equipotent to the parent undecapeptide QBP1.     

Reactivity of δ-substituted α,β-unsaturated cyclic lactones with antileishmanial activity

The present study examines a set of 43 compounds for their antileishmanial activities and cytotoxicities. Negative lowest unoccupied molecular orbitas and similar values for the electrophilic Fukui function condensed at the β-position for a subset of δ-substituted α,β-unsaturated cyclic lactones classify them as strong Michael acceptors. There was a well-defined trend of increasing antileishmanial activity with increasing cytotoxicity and large selectivity indices for the most active compounds. Softer compounds were more active than harder ones as observed from the experimental data and rationalised by calculated reactivity indices.     

Ultracytochemical demonstration and probable localization of 3β-hydroxysteroid dehydrogenase activity with a ferricyanide technique

Summary In order to localize 3-hydroxysteroid dehydrogenase activity on the ultrastructural level, sections of Newt and Rat adrenocortical tissues, fixed in a mixture of glutaraldehyde (0.25%) and formaldehyde (1%), were incubated in a medium containing namely a 3-hydroxysteroid as substrate, NAD, potassium ferricyanide as final electron acceptor, and copper sulfate. In some experiments, phenazine methosulfate (PMS), an electron carrier which can substitute for the activity of the endogenous NADH-diaphorase, is added at various concentrations to the incubation medium.A final precipitate of copper ferrocyanide is observed in the immediate vicinity of the tubules of the smooth endoplasmic reticulum, or in contact with their external faces. The reaction product can also be seen in mitochondrial cristae. The reaction does not take place in incubation media lacking substrate or containing cyanoketone, a specific inhibitor of 3-hydroxysteroid dehydrogenase. The addition of PMS to the incubation medium increases the intensity of the reaction, but does not modify the localization of the precipitate.     

Synthesis of Nα,Nδ-dicarbobenzoxy-L-ornithyl-β-alanine benzyl ester,a derivative of salty peptide,in 1,1,1-trichloroethane with polyethylene glycol-modified papain

Summary N,N-Dicarbobenzoxy-L-ornithyl--alanine benzyl ester, a derivative of salty peptide, was synthesized from N,N-dicarbobenzoxy-L-ornithine ethyl ester and -alanine benzyl ester in 1,1,1-trichloroethane using papain modified with polyethylene glycol. The peptide bond formation proceeded in a transparent organic solvent at room temperature and the product was obtained as precipitates from the reaction system.     

Synthesis,antitumor activity evaluation of some new N-aroyl-α,β-unsaturated piperidones with fluorescence

Novel N-aroyl-α,β-unsaturated piperidones, series 1, series 2 and series 3 (featuring 2-bromo-4,5-dimethoxybenzylidene, 4-dimethylaminobenzylidene and 4-trifluoromethylbenzylidene, respectively), were synthesized as candidate cytotoxins. Most of the compounds displayed potent cytotoxicity against the human neoplastic cell lines SK-BR-3, PG-BE1, NCI-H460, MIA PaCa-2 and SW1990 in vitro, and approximately 64% of the IC₅₀ values were lower than 5?μM. Among those tested, compound 1b of series 1, 3a, 3d and 3e of series 3 proved to be the most active. Importantly, 1b displayed marked inhibitory effects on tumor growth in vivo and had no apparent toxicity to mice; this was evaluated by a nude mouse PG-BE1 xenograft model. In addition, the fluorescent properties of compounds series 1–3 were investigated. The interesting fluorescence exhibited by these compounds could be useful for their visualization in tumor cells, permitting further studies on these α,β-unsaturated piperidones as candidates for novel fluorescent antitumor agents.     

Structure–activity relationships of the antimicrobial peptide gramicidin S and its analogs: Aqueous solubility,self-association,conformation, antimicrobial activity and interaction with model lipid membranes

GS10 [cyclo-(VKLdYPVKLdYP)] is a synthetic analog of the naturally occurring antimicrobial peptide gramicidin (GS) in which the two positively charged ornithine (Orn) residues are replaced by two positively charged lysine (Lys) residues and the two less polar aromatic phenylalanine (Phe) residues are replaced by the more polar tyrosine (Tyr) residues. In this study, we examine the effects of these seemingly conservative modifications to the parent GS molecule on the physical properties of the peptide, and on its interactions with lipid bilayer model and biological membranes, by a variety of biophysical techniques. We show that although GS10 retains the largely β-sheet conformation characteristic of GS, it is less structured in both water and membrane-mimetic solvents. GS10 is also more water soluble and less hydrophobic than GS, as predicted, and also exhibits a reduced tendency for self-association in aqueous solution. Surprisingly, GS10 associates more strongly with zwitterionic and anionic phospholipid bilayer model membranes than does GS, despite its greater water solubility, and the presence of anionic phospholipids and cholesterol (Chol) modestly reduces the association of both GS10 and GS to these model membranes. The strong partitioning of both peptides into lipid bilayers is driven by a large favorable entropy change opposed by a much smaller unfavorable enthalpy change. However, GS10 is also less potent than GS at inducing inverted cubic phases in phospholipid bilayer model membranes and at inhibiting the growth of the cell wall-less bacterium Acholeplasma laidlawii B. These results are discussed in terms of the comparative antibiotic and hemolytic activities of these peptides.     

Enhancement of mycobactericidal activity of glutaraldehyde with α,β-unsaturated and aromatic aldehydes

Summary Several ,-unsaturated and aromatic aldehydes were evaluated for antimicrobial activity usingMycobacterium bovis as the test strain. Activity of most of the compounds was determined in the presence and absence of 2% glutaraldehyde. Several compounds highly active against this organism, e.g. 2-pentenal, benzaldehyde, ando-phthalaldehyde showed rapid kill of >10⁵ CFU ml^–1 in 5 min. Activity of ,-unsaturated compounds substituted in the ₁ position showed increasing activity with increasing chain length. Of the aromatic aldehydes tested, benzaldehyde andp-dimethylamino benzaldehyde showed little activity alone, but when combined with 2% glutaraldehyde showed increased activity. Substituents added to the benzaldehyde ring (nitro, chloro, methyl, and methoxy) all detracted from the synergism, but still showed increased activity over the activity of 2% glutaraldehyde. The same affect was noted with disubstituted benzaldehyde compounds but not with substitutedo-phthaladehyde (2-formylformaldehyde).     

Interaction of thymic peptide thymosinα 1 with vasoactive intestinal peptide (VIP) receptors

Thymic peptide thymosin ₁ (10^–9 to 3 x 1010^–7 M) is shown to inhibit the specific binding of [¹²⁵I]VIP to rat blood mononuclear cells and liver plasma membranes. Thymosin ₁ was 160 and 6250 times less potent that VIP at inhibiting [¹²⁵I]VIP binding to blood mononuclear cells and liver plasma membranes, respectively. Thymosin ₁ (10^–10 to 1010^–7 M) was weak in stimulating adenylate cyclase activity. Its efficacy is about 25 % and 27 % that of native VIP in blood mononuclear cells and liver plasma membranes, respectively. Thymosin ₁ may behave as a partial VIP agonist in rat.Abbreviations GRF growth hormone releasing factor - PHI porcine intestinal peptide having N-terminal histidine and C-terminal isoleucine amide - GIP gastric inhibitory polypeptide - VIP vasoactive intestinal peptide     

Modulation of interleukin-1β-induced inflammatory responses by a synthetic cationic innate defence regulator peptide, IDR-1002, in synovial fibroblasts

Introduction  

Innate defence regulator (IDR) peptides are synthetic cationic peptides, variants of naturally occurring innate immune effector molecules known as host defence peptides. IDR peptides were recently demonstrated to limit infection-associated inflammation selectively without compromising host innate immune functions. This study examined the impact of a 12-amino acid IDR peptide, IDR-1002, in pro-inflammatory cytokine interleukin (IL)-1β-induced responses in synovial fibroblasts, a critical cell type in the pathogenesis of inflammatory arthritis.