Ectatomin (m = 7928 Da) is a toxic component from the Ectatomma tuberculatum ant venom containing two homologous polypeptide chains (37 and 34 residues) linked to each other by a disulfide bond. In aqueous solution it forms a four a-helix bundle. At concentrations of 0.05±0.1 mm, ectatomin forms channels in cellular and artificial bilayer membranes. Immunochemical analysis of the intracellular distribution of ectatomin showed that the toxin gets efficiently inserted into the plasma membrane at a concentration of 5 £ 10 27 m and does not penetrate inside the cell. The effect of ectatomin on cardiac L-type calcium current was studied. Calcium currents (I Ca ) in isolated rat cardiac ventricular myocytes were measured using the whole-cell perforated patch-clamp technique. It was shown that ectatomin at concentrations of 0.01±10 nm inhibited I Ca after a latency of few seconds. I Ca was decreased twofold by 10 nm ectatomin. However, the most prominent effect of ectatomin was observed after stimulation of I Ca by isoproterenol, an agonist of b-adrenoreceptors, or forskolin, a stimulator of adenylate cyclase. At a concentration of 1 nm, ectatomin abolished the isoproterenol-and forskolin-sensitive components of I Ca . The inhibitory effect of ectatomin was partially reversed by subsequent application of 2 mm of forskolin, whereas subsequent isoproterenol application did not produce the same effect.Keywords: ectatomin; cytotoxicity; calcium channels; b-adrenergic receptor.Ectatomin is a highly basic protein toxin (m = 7928 Da, pI 9.95) isolated from the venom of Ectatomma tuberculatum ants. The amino acid sequence and spatial structure were determined by protein chemistry methods and by twodimensional NMR spectroscopy techniques [1,2]. The protein consists of two highly homologous amphiphilic polypeptide chains (37 and 34 amino acid residues) connected by a disulfide bond (A) Cys22±Cys20 (B). Each chain comprises two anti-parallel a-helices linked by a hinge region of four amino acid residues and a disulfide bridge (A) Cys12±Cys34 (B) Cys10±Cys32.GVIPKKIWETVCPTVEPWAKKCSGDIATYIKRECGKL A-chain WSTIVKLTICPTLKSMAKKCEGSIATMIKKKCDK B-chain Ectatomin is responsible for the major toxic effect of the venom in both mammals and insects. Treatment of Xenopus laevis oocytes or rat myocardial cells with ectatomin (0.5±1 mm) induced a gradual irreversible increase in the ion leakage across the membrane, a decrease in membrane resistance and cell death [3]. Patch-clamp experiments have revealed that ectatomin has channel-forming activity. At a concentration of 0.05±0.1 mm, it forms nonselective cationic channels in X. laevis oocyte membranes and in model membrane systems. The channel formation depends on the membrane potential and occurs only at positive cis-potential (i.e. in physiological conditions). Analysis of the correlation between the membrane permeability and the ectatomin concentration revealed that each pore is formed by two ectatomin molecules. Channel-forming properties of ectatomin may account at least partially for its ...

An undefined property of L-type Ca2+ channels is believed to underlie the unique phenotype of hibernating hearts. Therefore, L-type Ca2+ channels in single cardiomyocytes isolated from hibernating versus awake ground-squirrels (Citellus undulatus) were compared using the perforated mode of the patch-clamp technique, and interpreted by way of a kinetic model of Ca2+ channel behavior based upon the concept of independence of the activation and inactivation processes. We find that, in hibernating ground-squirrels, the cardiac L-type Ca2+ current is lower in magnitude when compared to awake animals. Both in the awake or hibernating states, kinetics of L-type Ca2+ channels could be described by a d2f1(2)f2 model with an activation and two inactivation processes. The activation (or d) process relates to the movement of the gating charge. The slow (or f1) inactivation is associated with movement of gating charge and is current-dependent. The rapid (or f2) inactivation is a complex process which cannot be represented as a single-step conformational transition induced by the gating charge movement, and is regulated by beta-adrenoceptor stimulation. When compared to awake animals, the kinetic properties of Ca2+ channels from hibernating ground-squirrels differed in the following parameters: (1) pronounced shift (15-20 mV) toward depolarization in the normalized conductance of both inactivation components, and moderate shift in the activation component; (2) 1.5-2-fold greater time constants; and (3) two-fold greater activation gating charge. Thus, L-type Ca2+ channels apparently switch their phenotype during the hibernating transition. Stimulation of beta-adrenoceptors by isoproterenol, reversed the hibernating kinetic- (but not amplitude-) phenotype toward the awake type. Therefore, an aberrance in the beta-adrenergic system can not fully explain the observed changes in the L-type Ca2+ current. This suggests that during hibernation additional mechanisms may reduce the single Ca2+ channel-conductance and/or keep a fraction of the cardiac L-type Ca2+ channel population in a non-active state.

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