Calcicludine, a venom peptide of the Kunitz-type protease inhibitor family, is a potent blocker of high-threshold Ca2+ channels with a high affinity for L-type channels in cerebellar granule neurons.

Abstract

(3,12). This paper reports the isolation of a toxin, calcicludine (CaC), from the venom of the green mamba Dendroaspis angusticeps. This toxin is a potent blocker of all types of HVA Ca2+ channels (L-, N-, and P-type). However, L-type Ca2+ channels from cerebellar granule cells appear to be the preferential target of CaC. MATERIALS AND METHODSPurification of CaC. The crude venom (500 mg) of D. angusticeps (Latoxan, Rosans, France) was dissolved in 1% acetic acid and chromatographed onto a Sephadex G50 column. The peptidic fraction was directly loaded onto a TSK (Toyosoda, Japan) SP 5PW (21.5 x 150 mm) column equilibrated with 1% acetic acid. Peptide fractions were then eluted (Fig. 1 Top), with a linear gradient from 1% acetic acid to 1 M ammonium acetate at a flow rate of 8 ml/min. The fractions obtained (horizontal bars) were designated A-R. Fraction Q was lyophilized, redissolved in 1 ml of 0.5% trifluoroacetic acid plus 0.9%6 triethylamine in water, and loaded on a Lichrosorb RP18 7-ikm (250 x 10 mm) column (Merck, Darmstadt, Germany) and eluted ( Fig. 1 Middle) at aflow rate of3 ml/min with a lineargradient from 10%6 to 40% of0.5% trifluoroacetic acid plus 0.9% triethylamine in acetonitrile. Fraction Q1 is CaC.The primary structure of CaC (Fig.

Proteomics and Deep Sequencing Comparison of Seasonally Active Venom Glands in the Platypus Reveals Novel Venom Peptides and Distinct Expression Profiles

“…Serine protease inhibitors are found in the venoms of snakes, where they play key roles in blood coagulation (39,40). They have also been identified in sea anemone (41) and wasp (42) venoms. The platypus serpin does not belong to the Kunitz family of serine protease inhibitors found in snake and anemone venoms, but instead is a member of MEROPS inhibitor family 14, clan ID.…”

“…Other Kunitz-type protease inhibitors were previously identified in the platypus venom gland (5). The ancestral function of these proteins is the inhibition of serine proteases, such as those involved in hemostasis, but some venom Kunitz-domain proteins have evolved ion-channel blocking activity (41,47,48). These toxin proteins are well documented in snakes but are also found in the venoms of spiders, cone snails, and sea anemones (41,47,48).…”

Delineation of the Functional Site of α-Dendrotoxin

“…Clearly, therefore, the surface of the BPTI/DTX fold can accommodate distinct functional sites in unrelated regions, a scenario that agrees with previous proposals made for other toxin folds (68 -70). It will now be interesting to examine whether additional regions of the DTX/BPTI fold are associated with other functions, such as the calcium channel-blocking activity of calcicludine (34). In any case, the available data suggest that the DTX/BPTI fold undergoes a natural "engineering" resulting in divergent functional topographies.…”

Calcicludine Binding to the Outer Pore of L-type Calcium Channels Is Allosterically Coupled to Dihydropyridine Binding

“…Because of its folding pattern, calcicludine is classified as a bovine pancreatic trypsin inhibitor (BPTI)/Kunitz-like toxin (14)(15)(16)(17) and is structurally homologous to the K + channel selective dendrotoxins (18). Schweitz et al reported that N-and P/Q-type currents are effectively blocked by calcicludine (16). In contrast, Stotz et al found that currents through Ca V 2.1, Ca V 2.2 and Ca V 2.3 Ca 2+ channels are only modestly inhibited (<10%) by 100 nM calcicludine (19).…”