Saffron and retinal neurodegenerative diseases: Relevance of chemical composition.

Saffron is an ancient spice largely used in traditional medicine. It has been found to be effective in treatment of retinal neurodegenerative diseases like age-related macular degeneration and Stargardt. In the present manuscript, it is shown that saffron's neuroprotective power is strongly related to the bioactivity of all its chemical components. Nuclear magnetic resonance spectroscopy and "in vitro" experiments confirm the relevance of crocins for saffron efficacy. These results underline the importance of strictly defining the chemical composition of the natural compounds in saffron to optimize their effectiveness in the treatment of diseases.

Saffron reduces ATP-induced retinal cytotoxicity by targeting P2X7 receptors.

P2X7-type purinergic receptors are distributed throughout the nervous system where they contribute to physiological and pathological functions. In the retina, this receptor is found in both inner and outer cells including microglia modulating signaling and health of retinal cells. It is involved in retinal neurodegenerative disorders such as retinitis pigmentosa and age-related macular degeneration (AMD). Experimental studies demonstrated that saffron protects photoreceptors from light-induced damage preserving both retinal morphology and visual function and improves retinal flicker sensitivity in AMD patients. To evaluate a possible interaction between saffron and P2X7 receptors (P2X7Rs), different cellular models and experimental approaches were used. We found that saffron positively influences the viability of mouse primary retinal cells and photoreceptor-derived 661W cells exposed to ATP, and reduced the ATP-induced intracellular calcium increase in 661W cells. Similar results were obtained on HEK cells transfected with recombinant rat P2X7R but not on cells transfected with rat P2X2R. Finally, patch-clamp experiments showed that saffron inhibited cationic currents in HEK-P2X7R cells. These results point out a novel mechanism through which saffron may exert its protective role in neurodegeneration and support the idea that P2X7-mediated calcium signaling may be a crucial therapeutic target in the treatment of neurodegenerative diseases.

Interaction of the scorpion toxin discrepin with Kv4.3 channels and A-type K(+) channels in cerebellum granular cells.

BACKGROUND: The peptide discrepin from the α-KTx15 subfamily of scorpion toxins preferentially affects transient A-type potassium currents, which regulate many aspects of neuronal function in the central nervous system. However, the specific Kv channel targeted by discrepin and the molecular mechanism of interaction are still unknown. METHODS: Different variant peptides of discrepin were chemically synthesized and their effects were studied using patch clamp technique on rat cerebellum granular cells (CGC) and HEK cells transiently expressing Kv4.3 channels. RESULTS: Functional analysis indicated that nanomolar concentrations of native discrepin blocked Kv4.3 expressed channels, as previously observed in CGC. Similarly, the apparent affinities of all mutated peptides for Kv4.3 expressed channels were analogous to those found in CGC. In particular, in the double variant [V6K, D20K] the apparent affinity increased about 10-fold, whereas in variants carrying a deletion (ΔK13) or substitution (K13A) at position K13, the blockage was removed and the apparent affinity decreased more than 20-fold. CONCLUSION: These results indicate that Kv4.3 is likely the target of discrepin and highlight the importance of the basic residue K13, located in the α-helix of the toxin, for current blockage. GENERAL SIGNIFICANCE: We report the first example of a Kv4 subfamily potassium channel blocked by discrepin and identify the amino acid residues responsible for the blockage. The availability of discrepin variant peptides stimulates further research on the functions and pharmacology of neuronal Kv4 channels and on their possible roles in neurodegenerative disorders.

Solution structure of native and recombinant expressed toxin CssII from the venom of the scorpion Centruroides suffusus suffusus, and their effects on Nav1.5 sodium channels.

The three-dimensional structures of the long-chain mammalian scorpion ß-toxin CssII from Centruroides suffusus suffusus and of its recombinant form, HisrCssII, were determined by NMR. The neurotoxin CssII (nCssII) is a 66 amino acid long peptide with four disulfide bridges; it is the most abundant and deadly toxin from the venom of this scorpion. Both native and recombinant CssII structures were determined by nuclear magnetic resonance using a total of 828 sequential distance constraints derived from the volume integration of the cross peaks observed in 2D NOESY spectra. Both nCssII and HisrCssII structures display a mixed α/ß fold stabilized by four disulfide bridges formed between pairs of cysteines: C1-C8, C2-C5, C3-C6, and C4-C7 (the numbers indicate the relative positions of the cysteine residues in the primary structure), with a distortion induced by two cis-prolines in its C-terminal part. The native CssII electrostatic surface was compared to both the recombinant one and to the Cn2 toxin, from the scorpion Centruroides noxius, which is also toxic to mammals. Structural features such N- and C-terminal differences could influence toxin specificity and affinity towards isoforms of different sub-types of Na(v) channels.

Scorpion toxins that block transient currents (I(A)) of rat cerebellum granular cells.

This communication is a revision of the state-of-the-art knowledge of the field of scorpion toxins specific for the K(+)-channels, responsible for the I(A) currents of granular cells of rat cerebellum, maintained in vitro culture. There are 6 members of the sub-family alpha-KTx15 known to affect the I(A) currents. They are: toxins Aa1 from Androctonus australis Garzoni, BmTx3 from Buthusmartensi Karch, AmmTx3 from Androctonus mauretanicus mauretanicus, AaTx1 and AaTx2 from A. australis Garzoni and Discrepin from Tityus discrepans. They share high sequence similarity, apart from Discrepin, which causes an irreversible effect on the I(A) currents and is the most thoroughly studied toxin of the sub-family alpha-KTx15. The three-dimensional structure of Discrepin was determined and a series of mutants were synthesized and assayed in the system with the aim of identifying possible amino acids or sequence segments responsible for the irreversible effect found. In this revision some unpublished original data are also included to foster future work on the field, as well as a short discussion on some relevant aspects still pending and possible limitations associated with the strategy proposed.

A positive charge at the N-terminal segment of Discrepin increases the blocking effect of K+ channels responsible for the IA currents in cerebellum granular cells.

Discrepin is a scorpion peptide that blocks preferentially the IA currents of the voltage-dependent K+ channel of rat cerebellum granular cells. It was isolated from the venom of the buthid scorpion Tityus discrepans and contains 38 amino acid residues with a pyroglutamic acid at the N-terminal site. Discrepin has the lowest sequence identity (approx. 50%) among the six members of the alpha-KTx15 sub-family of scorpion toxins. In order to find out which residues are important for the blocking effects of Discrepin, six mutants were chemically synthesized (V6K, I19R, D20K, T35V, I19R-D20K, I19R-D20K-R21V), correctly folded and their physiological properties were examined. Substitution of residues V6 and D20 for basically charged amino acids increases the blocking activity of Discrepin, specially the mutation V6K at the N-terminal segment of the toxin. Analysis of 3D-structure models of the mutants V6K and D20K supports the idea that basic residues improve their blocking activities, similarly to what happens with BmTx3, a toxic peptide obtained from Buthus martensi scorpion, which has the highest known blocking effects of IA currents in K+ channels of rat cerebellum granular cells.

Solution structure of discrepin, a new K+-channel blocking peptide from the alpha-KTx15 subfamily.

Discrepin, isolated from the venom of the Venezuelan scorpion Tityus discrepans, blocks preferentially the I(A) currents of the voltage-dependent K+ channel of rat cerebellum granular cells in an irreversible way. It contains 38 amino acid residues with a pyroglutamic acid as the N-terminal residue [D'Suze, G., Batista, C. V., Frau, A., Murgia, A. R., Zamudio, F. Z., Sevcik, C., Possani, L. D., and Prestipino, G. (2004) Arch. Biochem. Biophys. 430, 256-63]. It is the most distinctive member of the alpha-KTx15 subfamily of scorpion toxins. Six members of the alpha-KTx15 subfamily have been reported so far to be specific for this subtype of the K+ channel; however, none of them have had their three-dimensional structure determined, and no information for the residues possibly involved in channel recognition and binding is available. Natural discrepin (n-discrepin) was prepared from scorpion venom, and its synthetic analogue (s-discrepin) was obtained by solid-phase synthesis. Analysis of two-dimensional 1H NMR spectra of n- and s-discrepin indicates that both peptides have the same structure. Here we report the solution structure of s-discrepin determined by NMR using 565 meaningful distance constraints derived from the volume integration of the two-dimensional NOESY spectrum, 22 dihedrals, and three hydrogen bonds. Discrepin displays the alpha/beta scaffold, characteristic of scorpion toxins. Some features of the proposed interacting surface between the toxin and channel as well as the opposite "alpha-helix surface" are discussed in comparison with those of other alpha-KTx15 members. Both n- and s-discrepin exhibit similar physiological actions as verified by patch-clamp and binding and displacement experiments.

Discrepin, a new peptide of the sub-family alpha-ktx15, isolated from the scorpion Tityus discrepans irreversibly blocks K+ -channels (IA currents) of cerebellum granular cells.

A new peptide was purified from the venom of the Venezuelan scorpion Tityus discrepans, by high-performance liquid chromatography and its amino acid sequence was completed by Edman degradation and mass spectrometry analysis. It contains 38 amino acid residues with a molecular weight of 4177.7 atomic mass units, tightly folded by three disulfide bridges, and has a pyroglutamic acid at the N-terminal region. This peptide, named Discrepin, was shown to block preferentially the IA currents of the voltage-dependent K+ -channel of rat cerebellum granular cells in culture. The K+ -currents are inhibited in an apparently irreversible manner, whose 50% inhibitory effect is reached with a 190 nM toxin concentration. The systematic nomenclature proposed for this toxin is alpha-KTx15.6.

Definition of the alpha-KTx15 subfamily.

Three novel scorpion toxins, Aa1 from Androctonus australis, BmTX3 from Buthus martensi and AmmTX3 from Androctonus mauretanicus were shown able to selectively block A-type K+ currents in cerebellum granular cells or cultured striatum neurons from rat brain. In electrophysiology experiments, the transient A-current completely disappeared when 1 microM of the toxins was applied to the external solution whereas the sustained K+ current was unaffected. The three toxins shared high sequence homologies (more than 94%) and constituted a new 'short-chain' scorpion toxin subfamily: alpha-KTx15. Monoiododerivative of 125I-sBmTX3 specifically bound to rat brain synaptosomes. Under equilibrium binding conditions, maximum binding was 14 fmol/mg of protein and the dissociation constant (Kd) was 0.21 nM. This Kd value was confirmed by kinetic experiments (kon = 6.0 x 10(6) M(-1) s(-1) and koff = 6.0 x 10(-4) s(-1)). Competitions with AmmTX3 and Aa1 with 125I-sBmTX3 bound to its receptor on rat brain synaptosomes showed that they fully inhibited the 125I-sBmTX3 binding (Ki values of 20 and 44 pM, respectively), demonstrating unambiguously that the three molecules shared the same target in rat brain. A panel of toxins described as specific ligands for different K+, Na+ and Ca2+ channels were not able to displace 125I-sBmTX3 from its binding site. Thus, 125I-sBmTX3 is a new ligand for a still unidentified target in rat brain. In autoradiography, the distribution of 125I-sBmTX3 binding sites in the adult rat brain indicated a high density of 125I-sBmTX3 receptors in the striatum, hippocampus, superior colliculus, and cerebellum.

Amino acid sequence and function of a new alpha-toxin from the Amazonian scorpion Tityus cambridgei.

A toxic peptide earlier denominated Tc48b [Toxicon 40 (2002) 557] was purified to homogeneity and its amino acid sequence determined. It has 64 amino acid residues stabilized by four disulfide bridges with a molecular weight of 7,385.2 atomic mass units (a.m.u.). It affects Na(+)-permeability in pituitary GH3 cells in culture, in a similar fashion as those reported for alpha-scorpion toxins, contrary to most of the New World scorpion toxins that are beta-toxins.

Anticonvulsant phenytoin affects voltage-gated potassium currents in cerebellar granule cells.

The anticonvulsant drug phenytoin (diphenylhydantoin, DPH) was examined for its action on potassium currents in cerebellar granule cells using the whole-cell patch-clamp technique. Granular cells expressed two main types of voltage-dependent potassium currents: the first, sensitive to Tetraethylammonium ion (TEA), resembles a delayed rectifier K(+) channel (I(d)); the second shows biophysical and pharmacological properties similar to an I(A)-type potassium current. Phenytoin blocks the I(A) current in a dose-dependent manner, with an apparent dissociation constant K(d) of (73+/-7) microM. The drug shifts the steady-state inactivation curves towards a more negative potential, stabilizing the inactivated state, while the activation kinetics remain unaffected. The estimated K(d) when the cell is held to -100 mV (closed state of the channel) is 145+/-8 microM which decreases to 35+/-10 microM at -80 mV holding potential (partial inactivation of the channel). Phenytoin shows a discriminant behaviour between the two different types of potassium channels because at high concentration the effect of the drug on the delayed rectifier K(+) channel is negligible.

Scorpion toxins from Tityus cambridgei that affect Na(+)-channels.

By means of high performance liquid chromatography (HPLC) the soluble venom of the Amazonian scorpion Tityus cambridgei was fractionated into over 50 different components. Four toxic and/or lethal peptides to mice were obtained in pure form and sequenced. Mass spectrometry analysis showed molecular weights of 7310, 7151, 7259 and 7405, respectively, for toxins Tc48a, Tc49a, Tc54 and Tc49b. The N-terminal amino acid sequence was obtained for the three first toxins mentioned, whereas the full primary structure was determined for Tc49b. It contains 64 amino acid residues, closely packed by four disulfide bridges. Sequence comparison analysis showed similarities around 50% with other toxins from scorpions of the genus Tityus of Brazil. It is lethal to mice at doses of 20 microg per 20 g mouse. The toxin was shown to affect the Na(+)-currents permeability of rat cerebellum granular cells in culture. Almost a complete elimination of current was observed with 100 nM toxin concentration. This effect was partially reversible. Furthermore, this toxin does not modify the function of the Shaker B K(+)-channels expressed on Sf9 cells, nor does it modify the Na(+)-channel function in a similar manner as those reported for the alpha-scorpion toxins purified from other scorpions.