AbeTx1 is a novel sea anemone toxin with a dual mechanism of action on Shaker-type K+ channels activation

Voltage-gated potassium (KV) channels regulate diverse physiological processes and are an important target for developing novel therapeutic approaches. Sea anemone (Cnidaria, Anthozoa) venoms comprise a highly complex mixture of peptide toxins with diverse and selective pharmacology on KV channels. From the nematocysts of the sea anemone Actinia bermudensis, a peptide that we named AbeTx1 was purified and functionally characterized on 12 different subtypes of KV channels (KV1.1–KV1.6; KV2.1; KV3.1; KV4.2; KV4.3; KV11.1; and, Shaker IR), and three voltage-gated sodium channel isoforms (NaV1.2, NaV1.4, and BgNaV). AbeTx1 was selective for Shaker-related K+ channels and is capable of inhibiting K+ currents, not only by blocking the K+ current of KV1.2 subtype, but by altering the energetics of activation of KV1.1 and KV1.6. Moreover, experiments using six synthetic alanine point-mutated analogs further showed that a ring of basic amino acids acts as a multipoint interaction for the binding of the toxin to the channel. The AbeTx1 primary sequence is composed of 17 amino acids with a high proportion of lysines and arginines, including two disulfide bridges (Cys1–Cys4 and Cys2–Cys3), and it is devoid of aromatic or aliphatic amino acids. Secondary structure analysis reveals that AbeTx1 has a highly flexible, random-coil-like conformation, but with a tendency of structuring in the beta sheet. Its overall structure is similar to open-ended cyclic peptides found on the scorpion κ-KTx toxins family, cone snail venoms, and antimicrobial peptides

Mechanisms of noncovalent β subunit regulation of NaV channel gating

Voltage-gated Na(+) (NaV) channels comprise a macromolecular complex whose components tailor channel function. Key components are the non-covalently bound β1 and β3 subunits that regulate channel gating, expression, and pharmacology. Here, we probe the molecular basis of this regulation by applying voltage clamp fluorometry to measure how the β subunits affect the conformational dynamics of the cardiac NaV channel (NaV1.5) voltage-sensing domains (VSDs). The pore-forming NaV1.5 α subunit contains four domains (DI-DIV), each with a VSD. Our results show that β1 regulates NaV1.5 by modulating the DIV-VSD, whereas β3 alters channel kinetics mainly through DIII-VSD interaction. Introduction of a quenching tryptophan into the extracellular region of the β3 transmembrane segment inverted the DIII-VSD fluorescence. Additionally, a fluorophore tethered to β3 at the same position produced voltage-dependent fluorescence dynamics strongly resembling those of the DIII-VSD. Together, these results provide compelling evidence that β3 binds proximally to the DIII-VSD. Molecular-level differences in β1 and β3 interaction with the α subunit lead to distinct activation and inactivation recovery kinetics, significantly affecting NaV channel regulation of cell excitability

Studying the role of negatively charged membranes on the mode of action of Esc 1b (1-18)

FAPESPCNPqCAPESUniv Fed Sao Paulo, Dept Biofis, Sao Paulo, BrazilUniv Fed Sao Paulo, Dept Biofis, Sao Paulo, BrazilFAPESPCNPqCAPESWeb of Scienc

Luminescent Ru(II) Phenanthroline Complexes as a Probe for Real-Time Imaging of A beta Self-Aggregation and Therapeutic Applications in Alzheimer's Disease

The complexes, cis-[Ru(phen)(2)(Apy)(2)](2+), Apy = 4-aminopyridine and 3,4-aminopyridine, are stable in aqueous solution with strong visible absorption. They present emission in the visible region with long lifetime that accumulates in the cytoplasm of Neuro2A cell line without appreciable cytotoxicity. The complexes also serve as mixed-type reversible inhibitors of human AChE and BuChE with high active Site contact. cis-[Ru(phen)(2)(3,4Apy)(2)](2+) competes efficiently with DMPO by the OH center dot radical. Luminescence using fluorescence lifetime imaging (FLIM) enables real-time imaging of the onformational Changes of the self-aggregation, of A beta with incubation of complexes (0-24 h) in phosphate buffer at micromolar concentrations. By this technique, we identified protofibrillsin the self-assembly of A beta(1-40) and globular structures in the short fragment A beta(15-21) in aqueous solution.FAPESP [2014/12538-8, 2014/07935-8, 2012/02065-0, 2014/24643-0, 2014/26895-7]CNPq [304981/2012-5]CAPESUniv Fed Sao Carlos, Dept Quim, BR-13565905 Sao Carlos, SP, BrazilUniv Sao Paulo, Dept Fis, Fac Filosofia Ciencias & Letras Ribeirao Preto, BR-14040901 Ribeirao Preto, SP, BrazilUniv Fed Sao Paulo, Dept Biofis, Escola Paulista Med, BR-04023062 Sao Paulo, SP, BrazilUniv Fed Ceara, Dept Quim Organ & Inorgan, BR-60451970 Fortaleza, Ceara, BrazilDepartamento de Biofísica, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, São Paulo 04023-062, BrazilFAPESP: 2014/12538-8FAPESP: 2014/07935-8FAPESP: 2012/02065-0FAPESP: 2014/24643-0FAPESP: 2014/26895-7CNPq: 304981/2012-5Web of Scienc

Formation of inverse topology lyotropic phases in dioleoylphosphatidylcholine/oleic acid and dioleoylphosphatidylethanolamine/oleic acid binary mixtures

The addition of saturated fatty acids (FA) to phosphatidylcholine lipids (PC) that have saturated acyl chains has been shown to promote the formation of lyotropic liquid-crystalline phases with negative mean curvature. PC/FA mixtures may exhibit inverse bicontinuous cubic phases (Im3m, Pn3m) or inverse topology hexagonal phases (H-II), depending on the length of the acyl chains/fatty acid. Here we report a detailed study of the phase behavior of binary mixtures of dioleoylphosphatidylcholine (DOPC)/oleic acid (OA) and dioleoylphosphatidylethanolamine (DOPE)/oleic acid at limiting hydration, constructed using small-angle X-ray diffraction (SAXD) data. The phase diagrams of both systems show a succession of phases with increasing negative mean curvature with increasing OA content. At high OA concentrations, we have observed the occurrence of an inverse micellar Fd3m phase in both systems. Hitherto, this phase had not been reported for phosphatidylethanolamine/fatty acid mixtures, and as such it highlights an additional route through which fatty acids may increase the propensity of bilayer lipid membranes to curve. We also propose a method that uses the temperature dependence of the lattice parameters of the H-II phases to estimate the spontaneous radii of curvature (R-0) of the binary mixtures and of the component lipids. Using this method, we calculated the R-0 values of the complexes comprising one phospholipid molecule and two fatty acid molecules, which have been postulated to drive the formation of inverse phases in PL/FA mixtures. These are -1.8 nm (+/-0.4 nm) for DOPC(OA)(2) and -1.1 nm (+/-0.1 nm) for DOPE(OA)(2). R-0 values estimated in this way allow the quantification of the contribution that different lipid species make to membrane curvature elastic properties and hence of their effect on the function of membrane-bound proteins