A reinterpretation of Na channel gating and permeation in terms of a phase transition between a transmembrane S4 α-helix and a channel-helix

A functional model for the S4/IV -helix of the action potential sodium channel is described by means of a thermodynamic approach. The model is based on a phase transition between the -helix and an ion conducting channel-helix which is similar to the well established helix-coil transition in solution. The right hand channel-helix is a peptide chain with an alternating sequence of torsional angles (¹¹)=(87°, 315°) and (²²)=(22°, 107°) which yields a helix of 13.5 Å per turn. The axial dipole moments of the peptide bonds of this chain of l-amino acids nearly cancel each other out in similar way to those in the gramicidin A channel, which is formed by alternating d-and l-amino acids. The helix, which does not contain any H-bonds, is stabilized by a helical file of water molecules which includes the permeating ion(s). This file turns around the channel-helix to form a relatively stable double helix structure which corresponds to the open channel. Since every third side chain in the S4/IV helix carries a positive charge their environments must be polarized. These polarized regions form a left hand screening-helix around the -helix are broken and the internal -carbon atom is considered as fixed, the outer ten residues leave the membrane while the internal ten residues form the channel-helix. In this configuration every positively charged side chain matches nearly exactly every second polarized region of the screening-helix leaving the three regions in-between exposed to the water file containing the ion(s). This further stabilizes the channel and agrees nicely with the idea of cationic selectivity. An analysis of the energetics of the -helix-channel-helix transition showed that the voltage-independent part of the free energy per helix residue could well be close to 0 kcal/mol and thus be in the range where a transition could occur. Two voltage-dependent contributions were included: the break down of the considerable dipole of the -helix and the outward shift of the positive charges of the side chains upon channel-helix formation. Taking into account the fact that the formation of an -helix is a highly cooperative process the degree of voltage dependence of the probability of formation of a channel-helix proved to be in the same range as experimental values for the open probability of modified Na channels whose inactivation had been removed. With regard to gating currents, the model predicts that 2.74 positive charges are moved in an outward direction. Consequences of the model for other experimental findings are discussed.     

Purification and characterization of a fix ABCX-linked 2[4Fe-4S] ferredoxin from Azotobacter vinelandii

 Ferredoxins that contain 2[4Fe-4S]^2+/+ clusters can be divided into two classes. The "clostridial-type" ferredoxins have two CysXXCysXXCysXXXCysPro motifs. The "photosynthetic bacterial and nif-related" ferredoxins have one motif of that type and one more unusual CysXXCysX_7–9CysXXXCysPro motif. In Azotobacter vinelandii three gene sequences have been reported that contain the latter motif, but until now none of the gene products has been purified. Here we report the purification of a small anionic [Fe-S] protein with yields of ∼3 mg per 500 g cell paste. NH₂-terminal sequence analysis shows that this protein is the product of a previously sequenced A. vinelandii gene that is found upstream of fixA and is cotranscribed with fixABCX. That gene was originally named fixP, but since that gene designation is now commonly used for a very different cb-type cytochrome oxidase we have renamed the gene fixFd and its product Fix Fd. Its sequence places Fix Fd in the class of "photosynthetic bacterial and nif-related" 2[4Fe-4S]^2+/1+ ferredoxins that includes Chromatium vinosum ferredoxin. Studies of the purified protein by Fe analysis, absorption, CD and EPR spectroscopies and electrochemistry confirm this characterization; the reduction potentials of the two clusters are –440 mV vs SHE. The fact that A. vinelandii synthesizes three different proteins with the same sequence motif, each of which is likely to have a different function, shows that although sequence motifs may be used reliably to classify ferredoxins by cluster type they cannot yet be used reliably for classifying ferredoxins by function. Received: 31 January 1997 / Accepted: 9 June 1997     

Probing α-310 Transitions in a Voltage-Sensing S4 Helix

The S4 helix of voltage sensor domains (VSDs) transfers its gating charges across the membrane electrical field in response to changes of the membrane potential. Recent studies suggest that this process may occur via the helical conversion of the entire S4 between α and 3₁₀ conformations. Here, using LRET and FRET, we tested this hypothesis by measuring dynamic changes in the transmembrane length of S4 from engineered VSDs expressed in Xenopus oocytes. Our results suggest that the native S4 from the Ciona intestinalis voltage-sensitive phosphatase (Ci-VSP) does not exhibit extended and long-lived 3₁₀ conformations and remains mostly α-helical. Although the S4 of NavAb displays a fully extended 3₁₀ conformation in x-ray structures, its transplantation in the Ci-VSP VSD scaffold yielded similar results as the native Ci-VSP S4. Taken together, our study does not support the presence of long-lived extended α-to-3₁₀ helical conversions of the S4 in Ci-VSP associated with voltage activation.     

3₁₀-helix conformation facilitates the transition of a voltage sensor S4 segment toward the down state

The activation of voltage-gated ion channels is controlled by the S4 helix, with arginines every third residue. The x-ray structures are believed to reflect an open-inactivated state, and models propose combinations of translation, rotation, and tilt to reach the resting state. Recently, experiments and simulations have independently observed occurrence of 3₁₀-helix in S4. This suggests S4 might make a transition from α- to 3₁₀-helix in the gating process. Here, we show 3₁₀-helix structure between Q1 and R3 in the S4 segment of a voltage sensor appears to facilitate the early stage of the motion toward a down state. We use multiple microsecond-steered molecular simulations to calculate the work required for translating S4 both as α-helix and transformed to 3₁₀-helix. The barrier appears to be caused by salt-bridge reformation simultaneous to R4 passing the F233 hydrophobic lock, and it is almost a factor-two lower with 3₁₀-helix. The latter facilitates translation because R2/R3 line up to face E183/E226, which reduces the requirement to rotate S4. This is also reflected in a lower root mean-square deviation distortion of the rest of the voltage sensor. This supports the 3₁₀ hypothesis, and could explain some of the differences between the open-inactivated- versus activated-states.     

Structural determination of a directly mutagenic amino-nitrobiphenyl as the S9 metabolite of 2,4,2′,4′-tetranitrobiphenyl in Salmonella typhimuriumTA98

In order to elucidate the mechanisms of mutagenic activation of nitrobiphenyls by mammalian activation systems, 2,4,2′,4′-tetranitrobiphenyl was incubated with S9 and its mutagenic metabolites were separated by SiO₂ and Al₂O₃ column chromatography. The most mutagenic diamino-dinitrobiphenyl was isolated from the reaction mixture of 2,4,2′ ,4′-tetranitrobiphenyl with S9 mix at 37°C for 48 h, and its mutability was 4646 revertants/50 ng in Salmonella typhimurium TA98 without S9 mix. The deamination product of this most mutagenic metabolite was identical to 2,4′-dinitrobiphenyl by gas chromatography-mass spectrometry. Therefore, the structure of the metabolite was determined as 2,4′-diamino-2′,4-dinitrobiphenyl by its chemical and physico-chemical properties.     

Detection of a 16S rRNA · initiator-tRNA complex by a new selective labelling method

Cupric-ion induced hydrolysis of [³⁵S]Met-tRNA but not of N-formyl-Met-tRNA _inff ^supMet permitted the specific terminal labelling of initiator tRNA. Initiator tRNA, labeled in this way, was suitable for sequence analysis without the need for further purification. By probing labeled initiator tRNA with specific RNases, changes in this molecule during its interaction with the 30S particle or with 16S rRNA were investigated. Initiation complexes were resistant to the action of single-strand, base-specific nucleases Bc and Phy M and, except for one base of the anticodon stem, were also resistant to digestion by the double-strand-specific V₁ nuclease of Naja venom. In contrast, T₁ RNase digestion of the initiator tRNA in the presence of 16S rRNA enhanced cleavage of bases in the T stem of the molecule.     

Exendin-4 upregulates the expression of Wnt-4, a novel regulator of pancreatic β-cell proliferation

The Wnt-signaling pathway regulates β-cell functions. It is not known how the expression of endogenous Wnt-signaling molecules is regulated in β-cells. Therefore, we investigated the effect of antidiabetic drugs and glucose on the expression of Wnt-signaling molecules in β-cells. Primary islets were isolated and cultured. The expression of Wnt-signaling molecules (Wnt-4, Wnt-10b, Frizzled-4, LRP5, TCF7L2) and TNFα was analyzed by semiquantitative PCR and Western blotting. Transient transfections were carried out and proliferation assays of INS-1 β-cells performed using [(3)H]thymidine uptake and BrdU ELISA. Insulin secretion was quantified. A knockdown (siRNA) of Wnt-4 in β-cells was carried out. Exendin-4 significantly increased the expression of Wnt-4 in β-cells on the mRNA level (2.8-fold) and the protein level (3-fold) (P < 0.001). The effect was dose dependent, with strongest stimulation at 10 nM, and it was maintained after long-term stimulation over 4 wk. Addition of exd-(9-39), a GLP-1 receptor antagonist, abolished the effect of exendin-4. Treatment with glucose, insulin, or other antidiabetic drugs had no effect on the expression of any of the examined Wnt-signaling molecules. Functionally, Wnt-4 antagonized the activation of canonical Wnt-signaling in β-cells. Wnt-4 had no effect on glucose-stimulated insulin secretion or insulin gene expression. Knocking down Wnt-4 decreased β-cell proliferation to 45% of controls (P < 0.05). In addition, Wnt-4 and exendin-4 treatment decreased the expression of TNFaα mRNA in primary β-cells. These data demonstrate that stimulation with exendin-4 increases the expression of Wnt-4 in β-cells. Wnt-4 modulates canonical Wnt signaling and acts as regulator of β-cell proliferation and inflammatory cytokine release. This suggests a novel mechanism through which GLP-1 can regulate β-cell proliferation.     

EPR analysis of multiple forms of [4Fe–4S]3+ clusters in HiPIPs

The electron paramagnetic resonance (EPR) spectrum from the [4Fe–4S]³⁺ cluster in several high-potential iron–sulfur proteins (HiPIPs) is complex: it is not the pattern of a single, isolated S=1/2 system. Multifrequency EPR from 9 to 130 GHz reveals that the apparent peak positions (g values) are frequency-independent: the spectrum is dominated by the Zeeman interaction plus g-strain broadening. The spectra taken at frequencies above the X-band are increasingly sensitive to rapid-passage effects; therefore, the X-band data, which are slightly additionally broadened by dipolar interaction, were used for quantitative spectral analysis. For a single geometrical [4Fe–4S]³⁺ structure the (Fe–Fe)⁵⁺ mixed-valence dimer can be assigned in six different ways to a pair of iron ions, and this defines six valence isomers. Systematic multicomponent g-strain simulation shows that the [4Fe–4S]³⁺ paramagnets in seven HiPIPs from different bacteria each consist of three to four discernible species, and these are assigned to valence isomers of the clusters. This interpretation builds on previous EPR analyzes of [4Fe–4S]³⁺ model compounds, and it constitutes a high-resolution extension of the current literature model, proposed from paramagnetic NMR studies.     

DNA repair glycosylases with a [4Fe-4S] cluster: a redox cofactor for DNA-mediated charge transport?

The [4Fe-4S] cluster is ubiquitous to a class of base excision repair enzymes in organisms ranging from bacteria to man and was first considered as a structural element, owing to its redox stability under physiological conditions. When studied bound to DNA, two of these repair proteins (MutY and Endonuclease III from Escherichia coli) display DNA-dependent reversible electron transfer with characteristics typical of high potential iron proteins. These results have inspired a reexamination of the role of the [4Fe-4S] cluster in this class of enzymes. Might the [4Fe-4S] cluster be used as a redox cofactor to search for damaged sites using DNA-mediated charge transport, a process well known to be highly sensitive to lesions and mismatched bases? Described here are experiments demonstrating the utility of DNA-mediated charge transport in characterizing these DNA-binding metalloproteins, as well as efforts to elucidate this new function for DNA as an electronic signaling medium among the proteins.     

The CCG-domain-containing subunit SdhE of succinate:quinone oxidoreductase from Sulfolobus solfataricus P2 binds a [4Fe–4S] cluster

In type E succinate:quinone reductase (SQR), subunit SdhE (formerly SdhC) is thought to function as monotopic membrane anchor of the enzyme. SdhE contains two copies of a cysteine-rich sequence motif (CX _n CCGX _m CXXC), designated as the CCG domain in the Pfam database and conserved in many proteins. On the basis of the spectroscopic characterization of heterologously produced SdhE from Sulfolobus tokodaii, the protein was proposed in a previous study to contain a labile [2Fe–2S] cluster ligated by cysteine residues of the CCG domains. Using UV/vis, electron paramagnetic resonance (EPR), ⁵⁷Fe electron–nuclear double resonance (ENDOR) and Mössbauer spectroscopies, we show that after an in vitro cluster reconstitution, SdhE from S. solfataricus P2 contains a [4Fe–4S] cluster in reduced (2+) and oxidized (3+) states. The reduced form of the [4Fe–4S]²⁺ cluster is diamagnetic. The individual iron sites of the reduced cluster are noticeably heterogeneous and show partial valence localization, which is particularly strong for one unique ferrous site. In contrast, the paramagnetic form of the cluster exhibits a characteristic rhombic EPR signal with g _zyx  = 2.015, 2.008, and 1.947. This EPR signal is reminiscent of a signal observed previously in intact SQR from S. tokodaii with g _zyx  = 2.016, 2.00, and 1.957. In addition, zinc K-edge X-ray absorption spectroscopy indicated the presence of an isolated zinc site with an S₃(O/N)₁ coordination in reconstituted SdhE. Since cysteine residues in SdhE are restricted to the two CCG domains, we conclude that these domains provide the ligands to both the iron–sulfur cluster and the zinc site.     

Preliminary X-ray diffraction studies on a [4Fe4S] ferredoxin from Bacillus thermoproteolyticus

A [4Fe4S] ferredoxin from Bacillus thermoproteolyticus has been crystallized. The space group is P1 with two molecules in the unit cell, with the dimensions $\text{a = 32.96}\text{A}\text{?}\text{, b = 37.83}\text{A}\text{?}\text{, c = 39.82}\text{A}\text{?}\text{, α = 118.1 °, β = 104.2 °}\text{and}\text{γ = 89.7 °}$. The Bijvoet-difference Patterson map of the native crystal shows up a prominent peak of [4Fe4S] cluster.     

Synthesis and structure–activity relationships of a series of 3-aryl-4-isoxazolecarboxamides as a new class of TGR5 agonists

A series of 3-aryl-4-isoxazolecarboxamides identified from a high-throughput screening campaign as novel, potent agonists of the human TGR5 G-protein-coupled receptor is described. Many analogues were readily accessible via solution-phase synthesis which resulted in the rapid identification of key structure–activity relationships (SAR), and the discovery of potent exemplars (up to pEC₅₀ = 9). Details of the SAR and optimization of this series are presented herein.     

Evaluation of Antiinflammatory Activity of 4'-О-Methylhonokiol Derivatives in a Neuroinflammation Model

Russian Journal of Bioorganic Chemistry - The synthesis of two derivatives of lignan 4'-O-methylhonokiol (MH), i.e., 4'-methoxy-5-propyl-1,1'-biphenyl-2-ol (III) and...     

Carnosine is a quencher of 4-hydroxy-nonenal: through what mechanism of reaction?

The aim of this study was to understand the mechanism of action through which carnosine (beta-alanyl-L-histidine) acts as a quencher of cytotoxic alpha,beta-unsaturated aldehydes, using 4-hydroxy-trans-2,3-nonenal (HNE) as a model aldehyde. In phosphate buffer solution (pH 7.4), carnosine was 10 times more active as an HNE quencher than L-histidine and N-acetyl-carnosine while beta-alanine was totally inactive; this indicates that the two constitutive amino acids act synergistically when incorporated as a dipeptide and that the beta-alanyl residue catalyzes the addition reaction of the histidine moiety to HNE. Two reaction products of carnosine were identified, in a pH-dependent equilibrium: (a) the Michael adduct, stabilized as a 5-member cyclic hemi-acetal and (b) an imine macrocyclic derivative. The adduction chemistry of carnosine to HNE thus appears to start with the formation of a reversible alpha,beta-unsaturated imine, followed by ring closure through an intra-molecular Michael addition. The biological role of carnosine as a quencher of alpha,beta-unsaturated aldehydes was verified by detecting carnosine-HNE reaction adducts in oxidized rat skeletal muscle homogenate.     

Contribution of Position α4S336 on Functional Expression and Up-regulation of α4β2 Neuronal Nicotinic Receptors

Phosphorylation of the nicotinic acetylcholine receptor (nAChR) is believed to play a critical role in its nicotine-induced desensitization and up-regulation. We examined the contribution of a consensus PKC site in the α4 M3/M4 intracellular loop (α4S336) on the desensitization and up-regulation of α4β2 nAChRs expressed in oocytes. Position α4S336 was replaced with either alanine to abolish potential phosphorylation at this site or with aspartic acid to mimic phosphorylation at this same site. Mutations α4S336A and α4S336D displayed a threefold increase in the ACh-induced response and an increase in ACh EC₅₀. Epibatidine binding revealed a three and sevenfold increase in surface expression for the α4S336A and α4S336D mutations, respectively, relative to wild-type, therefore, both mutations enhanced expression of the α4β2 nAChR. Interestingly, the EC₅₀’s and peak currents for nicotine activation remained unaffected in both mutants. Both mutations abolished the nicotine-induced up-regulation that is normally observed in the wild-type. The present data suggest that adding or removing a negative charge at this phosphorylation site cannot be explained by a simple straightforward on-and-off mechanism; rather a more complex mechanism(s) may govern the functional expression of the α4β2 nAChR. Along the same line, our data support the idea that phosphorylation at multiple consensus sites in the α4 subunit could play a remarkable role on the regulation of the functional expression of the α4β2 nAChR.