The transient receptor potential ankyrin-repeat 1 (TRPA1) is an important player in pain and inflammatory pathways. It is a promising target for novel drug development for the treatment of a number of pathological states. A novel peptide producing a significant potentiating effect on allyl isothiocyanate- and diclofenac-induced currents of TRPA1 was isolated from the venom of sea anemone It is a 35-amino acid peptide cross-linked by two disulfide bridges named τ-AnmTX Ms 9a-1 (short name Ms 9a-1) according to a structure similar to other sea anemone peptides belonging to structural group 9a. The structures of the two genes encoding the different precursor proteins of Ms 9a-1 were determined. Peptide Ms 9a-1 acted as a positive modulator of TRPA1 but did not cause pain or thermal hyperalgesia when injected into the hind paw of mice. Intravenous injection of Ms 9a-1 (0.3 mg/kg) produced a significant decrease in the nociceptive and inflammatory response to allyl isothiocyanate (the agonist of TRPA1) and reversed CFA (Complete Freund's Adjuvant)-induced inflammation and thermal hyperalgesia. Taken together these data support the hypothesis that Ms 9a-1 potentiates the response of TRPA1 to endogenous agonists followed by persistent functional loss of TRPA1-expressing neurons. We can conclude that TRPA1 potentiating may be useful as a therapeutic approach as Ms 9a-1 produces significant analgesic and anti-inflammatory effects in mice models of pain.
A 600 MHz 1H NMR study of toxin OSK1, blocker of small-conductance Ca2+-activated K+ channels, is presented. The unambiguous sequential assignment of all the protons of the toxin was obtained using TOCSY, DQF-COSY, and NOESY experiments at pH 3.0 (10, 30, and 45 degrees C) in aqueous solution. 3J(N alpha), 3J(alphabeta) vicinal spin coupling constants were determined in high-resolution spectra. The cross-peak volumes in NOESY spectra and the coupling constants were used to define the local structure of the protein by the program HABAS and to generate torsion angle and interproton distance constraints for the program DIANA. Hydrogen-deuterium exchange rates of amide protons showed possible locations of hydrogen bonds. The hydrogen bond acceptors and disulfide bridges between residues 8-28, 14-33, and 18-35 were determined when analyzing distance distribution in preliminary DIANA structures. All constraints were used to obtain a set of 30 structures by DIANA. The resulting rms deviations over 30 structures are 1.30 A for the heavy atoms and 0.42 A for the backbone heavy atoms. The structures were refined by constrained energy minimization using the SYBYL program. Their analysis indicated the existence of the alpha-helix (residues 10-21) slightly distorted at the Cys14 residue, two main strands of the antiparallel beta-sheet (24-29, 32-38), and the extended fragment (2-6). The motif is stabilized by the disulfide bridges in the way, common to all known scorpion toxins. Using the fine spatial toxin structure, alignment of the homologues, mutagenesis analysis, and comparison of scorpion toxin family functions, we delineate some differences significant for the toxin specificity.
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