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 In this revision some unpublished original data are also included to foster future work on the field, as well as a short discussion on some relevant aspects still pending and possible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.

Intracellular proton regulation of ClC-0.

Some CLC proteins function as passive Cl(-) ion channels whereas others are secondary active chloride/proton antiporters. Voltage-dependent gating of the model Torpedo channel ClC-0 is modulated by intracellular and extracellular pH, possibly reflecting a mechanistic relationship with the chloride/proton coupling of CLC antiporters. We used inside-out patch clamp measurements and mutagenesis to explore the dependence of the fast gating mechanism of ClC-0 on intracellular pH and to identify the putative intracellular proton acceptor(s). Among the tested residues (S123, K129, R133, K149, E166, F214L, S224, E226, V227, C229, R305, R312, C415, H472, F418, V419, P420, and Y512) only mutants of E166, F214, and F418 qualitatively changed the pH(int) dependence. No tested amino acid emerged as a valid candidate for being a pH sensor. A detailed kinetic analysis of the dependence of fast gate relaxations on pH(int) and [Cl(-)](int) provided quantitative constraints on possible mechanistic models of gating. In one particular model, a proton is generated by the dissociation of a water molecule in an intrapore chloride ion binding site. The proton is delivered to the side chain of E166 leading to the opening of the channel, while the hydroxyl ion is stabilized in the internal/central anion binding site. Deuterium isotope effects confirm that proton transfer is rate limiting for fast gate opening and that channel closure depends mostly on the concentration of OH(-) ions. The gating model is in natural agreement with the finding that only the closing rate constant, but not the opening rate constant, depends on pH(int) and [Cl(-)](int).

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.

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.