No abstract

We describe the first potent and selective blocker of the class E Ca2+channel. SNX-482, a novel 41 amino acid peptide present in the venom of the African tarantula, Hysterocrates gigas, was identified through its ability to inhibit human class E Ca2+ channels stably expressed in a mammalian cell line. An IC50 of 15-30 nM was obtained for block of the class E Ca2+ channel, using either patch clamp electrophysiology or K+-evoked Ca2+ flux. At low nanomolar concentrations, SNX-482 also blocked a native resistant or R-type Ca2+ current in rat neurohypophyseal nerve terminals, but concentrations of 200-500 nM had no effect on R-type Ca2+ currents in several types of rat central neurons. The peptide has the sequence GVDKAGCRYMFGGCSVNDDCCPRLGCHSLFSYCAWDLTFSD-OH and is homologous to the spider peptides grammatoxin S1A and hanatoxin, both peptides with very different ion channel blocking selectivities. No effect of SNX-482 was observed on the following ion channel activities: Na+ or K+ currents in several cultured cell types (up to 500 nM); K+ current through cloned potassium channels Kv1.1 and Kv1. 4 expressed in Xenopus oocytes (up to 140 nM); Ca2+ flux through L- and T-type Ca2+ channels in an anterior pituitary cell line (GH3, up to 500 nM); and Ba2+ current through class A Ca2+ channels expressed in Xenopus oocytes (up to 280 nM). A weak effect was noted on Ca2+ current through cloned and stably expressed class B Ca2+ channels (IC50 > 500 nM). The unique selectivity of SNX-482 suggests its usefulness in studying the diversity, function, and pharmacology of class E and/or R-type Ca2+ channels.

A 30-residue amphipathic peptide was designed to interact with uncharged bilayers in a pH-dependent fashion. This was achieved by a pH-induced random coil-alpha-helical transition, exposing a hydrophobic face in the peptide. The repeat unit of the peptide, glutamic acid-alanine-leucine-alanine (GALA), positioned glutamic acid residues on the same face of the helix, and at pH 7.5, charge repulsion between aligned Glu destabilized the helix. A tryptophan was included at the N-terminal as a fluorescence probe. The rate and extent of peptide-induced leakage of contents from large, unilamellar vesicles composed of egg phosphatidylcholine were dependent on pH. At pH 5.0 with a lipid/peptide mole ratio of 500/1, 100% leakage of vesicle contents occurred within 1 min. However, no leakage of vesicle contents occurred at pH 7.5. Circular dichroism measurements indicated that the molar ellipticity at 222 nm changed from about -4000 deg cm2 dmol-1 at pH 7.6 to -11,500 deg cm2 dmol-1 at pH 5.1, indicating a substantial increase in helical content as the pH was reduced. Changes in molar ellipticity were most significant over the same pH range where a maximum change in the extent and rate of leakage occurred. The tryptophan fluorescence emission spectra and the circular dichroism spectra of the peptide, in the presence of lipid, suggest that GALA did not associate with the bilayer at neutral pH. A change in the circular dichroism spectrum and a blue shift of the maximum of the tryptophan fluorescence emission spectra at pH 5.0, in the presence of lipid, indicated an association of GALA with the bilayer.(ABSTRACT TRUNCATED AT 250 WORDS)

The nerve endings of rat neurohypophyses were acutely dissociated and a combination of pharmacological, biophysical and biochemical techniques was used to determine which classes of Ca2+ channels on these central nervous system (CNS) terminals contribute functionally to arginine vasopressin (AVP) and oxytocin (OT) secretion. Purified neurohypophysial plasma membranes not only had a single high‐affinity binding site for the N‐channel‐specific ω‐conopeptide MVIIA, but also a distinct high‐affinity site for another ω‐conopeptide (MVIIC), which affects both N‐ and P/Q‐channels. Neurohypophysial terminals exhibited, besides L‐ and N‐type currents, another component of the Ca2+ current that was only blocked by low concentrations of MVIIC or by high concentrations of ω‐AgaIVA, a P/Q‐channel‐selective spider toxin. This Ca2+ current component had pharmacological and biophysical properties similar to those described for the fast‐inactivating form of the P/Q‐channel class, suggesting that in the neurohypophysial terminals this current is mediated by a ‘Q’‐type channel. Pharmacological additivity studies showed that this Q‐component contributed to rises in intraterminal Ca2+ concentration ([Ca2+]i) in only half of the terminals tested. Furthermore, the non‐L‐ and non‐N‐component of Ca2+‐dependent AVP release, but not OT release, was effectively abolished by the same blockers of Q‐type current. Thus Q‐channels are present on a subset of the neurohypophysial terminals where, in combination with N‐ and L‐channels, they control AVP but not OT peptide neurosecretion.

We report here the solution structure of omega-conotoxin GVIA, a peptide antagonist of the N-type neuronal voltage-sensitive calcium channel. The structure was determined using two-dimensional NMR in combination with distance geometry and restrained molecular dynamics. The full relaxation matrix analysis program MARDIGRAS was used to generate maximum and minimum distance restraints from the crosspeak intensities in NOESY spectra. The 187 restraints obtained were used in conjunction with 23 angle restraints from vicinal coupling constants as input for the structure calculations. The backbones of the best 21 structures match with an average pairwise RMSD of 0.58 A. The structures contain a short segment of triple-stranded beta-sheet involving residues 6-8, 18-21, and 24-27, making this the smallest published peptide structure to contain a triple-stranded beta-sheet. Conotoxins have been shown to be effective neuroprotective agents in animal models of brain ischemia. Our results should aid in the design of novel nonpeptide compounds with potential therapeutic utility.