Channel-forming toxins in Siphonops skin secretion Skin secretion of Siphonops paulensis ( Gymnophiona , Amphibia ) forms voltage-dependent ionic channels in lipid membranes

The effect of the skin secretion of the amphibian Siphonops paulensis was investigated by monitoring the changes in conductance of an artificial planar lipid bilayer. Skin secretion was obtained by exposure of the animals to ether-saturated air, and then rinsing the animals with distilled water. Artificial lipid bilayers were obtained by spreading a solution of azolectin over an aperture of a Delrin cup inserted into a cut-away polyvinyl chloride block. In 9 of 12 experiments, the addition of the skin secretion to lipid bilayers displayed voltage-dependent channels with average unitary conductance of 258 ± 41.67 pS, rather than nonspecific changes in bilayer conductance. These channels were not sensitive to 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid or tetraethylammonium ion, but the experimental protocol used does not permit us to specify their characteristics. Correspondence


• Siphonops
Amphibian skin secretion is considered to be a rich source of bioactive substances, which include biogenic amines, sterols, alkaloids, peptides, and proteins.These compounds have different biological activities, such as, myo-, cardio-, or neurotoxic, cholinomimetic, sympathomimetic, anesthetic, hemolytic, hallucinogenic, cytotoxic, and antibiotic activities (1).Most studies have focused on anuran species, whereas there are only a few studies on the toxicity of the skin of caecilian amphibians, although in 1940 Sawaya (2) demonstrated the cardiotoxicity of the skin secretion of Siphonops annulatus.A similar cardiotoxic action of S. paulensis skin secretion on toad heart bioassays was reported more recently (3).It was shown that the blockade of electrical activity of toad heart was due to the hemolytic activity on red blood cells, leading to an increase in K + concentration in the medium (4).It was also shown that the treatment of S. paulensis skin secretion with red blood cell ghosts inhibited hemolysis (3).On the basis of these results, we decided to investigate the effect of S. paulensis skin secretion on the conductance of planar lipid membranes.
Adult S. paulensis were collected in Formosa (GO, Brazil), and maintained alive in native soil in the Laboratory of Toxinology, University of Brasília.In order to obtain the skin secretion the animals were carefully washed with distilled water and exposed to diethyl ether-saturated air in a closed container.Then, the animals were rinsed with distilled water.The protein content of the S. paulensis skin secretion was measured (5) using bovine serum albumin as a standard and was used as reference for toxin content.Artificial lipid bilayers were obtained by spreading a solution of azolectin dissolved in n-decane (10 mg/ml) over an aperture (0.33 mm) of a Delrin cup, which was inserted into a cut-away polyvinyl chloride block, delimiting two compartments, i.e., cis-side and trans-side.These two chambers were filled with 3.5 ml each of buffered salt solutions, pH 7 (10 mM HEPES-Tris), of the following compositions: 150 mM KCl, 15 mM NaCl plus 100 µM CaCl 2 on the cisside, and 150 mM NaCl, 15 mM KCl plus 100 µM CaCl 2 on the trans-side.Membrane formation was followed by measuring membrane capacitance.After the addition of 10 µl of S. paulensis skin secretion solution to the cis-compartment, changes in bilayer conductance were monitored using an Axopatch 200A amplifier, visualized on a digital oscilloscope (Gould Instruments Systems, Valley View, OH, USA), and recorded in digital form on videotape (Sharp, Mahwah, NJ, USA) employing an analog-digital converter (PCMb2, Medical Systems Corp., Greenvale, NY, USA).For analysis, the analog signal derived from the digital record was passed through an 8-pole Bessel filter, with the corner frequency (-3 dB) set at 500 Hz, digitized using an analog interface (Digidata 1200, Union City, CA, USA) at a frequency of 3000 Hz and recorded directly on a personal computer.Data were analyzed using the pCLAMP-6 system (Axon Instruments, Union City, CA, USA).V c corresponds to the potential difference between the cis-and the trans-side (ground).By convention, a positive current reflects the flow of cations from the cis-to the trans-compartment or the flow of anions in the opposite direction.
In 9 of 12 experiments, addition of S. paulensis skin secretion at a final concentration of 6.4 µg/ml to the cis-compartment of the lipid bilayer led to changes in membrane conductance.However, instead of nonspecific changes, typical channel activities were observed up to 15 min after the addition of S. paulensis skin secretion (Figure 1).Moreover, the activity of the channel was voltage-dependent.Clearly, the open probability is reduced as the voltage increases over the range of -30 mV to 30 mV.Near zero mV and at positive voltages, transitions to the open state were greatly reduced (Figure 2A).When the voltage was clamped at zero there was no current, although the asymmetrical distribution for sodium and potassium provided chemical gradients for ion flows.Tetraethylammonium ion (TEA, 0.1 mM, added to the ciscompartment), a potassium channel blocker, had no effect on open probability.A commonly used chloride channel blocker, 4acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS, 1 mM, added to the ciscompartment) did not modify the channel activity.A typical current-voltage relation for a single channel is plotted in Figure 2B.Under the experimental conditions used, the I c vs V c relations were nearly linear.From the slope of the linear regression curve of the I c vs V c relations the single channel conductance averaged 258 ± 41.67 pS (N = 6).
In the present report we describe the occurrence of a channel-forming compound in an amphibian skin secretion.The skin secretion of the caecilian S. paulensis has been shown to contain a hemolytic substance (3).This skin secretion was able to form voltage-dependent channels in azolectin membranes.Although S. paulensis hemolysin has not yet been purified, we suggest that the mechanism of red blood cell lysis involves the insertion of the hemolysin in the membrane and channel formation.
As can be seen in Figure 1, the addition of S. paulensis skin secretion to the lipid bilayer resulted in the incorporation of voltage-dependent channels with unitary conductances of 258 ± 41.67 pS.It can also be observed that for null electrical gradients the current is zero.This probably means that i) the channel is specific for chloride, ii) it does not discriminate between sodium and potassium, having the same conductance for these two cations, or iii) the channel is closed at this voltage.The probability of the channels to be open was found to be voltage-dependent (Figure 2A).At close to zero and positive voltages the open probability is minimal.Additions of ion channel blockers such as TEA and SITS did not modify the open probability.The experimental protocol used does not permit to characterize the channel.It suggests, however, that this could be a chloride channel which would not be blocked by SITS, or a cation channel that does not discriminate between Na + and K + ions.
Further studies are necessary to purify and isolate the hemolytic compound and clarify the ion selectivity and mechanism of action of the channel-forming compound found in the skin secretion of S. paulensis.

Figure 1 .
Figure 1.Typical example of the channel activity formed by toxin(s) from skin secretion of Siphonops paulensis incorporated into the lipid bilayer.The channel clearly shows voltage dependence.In C, the channel is in the closed state, and in O the channel is open.C

Figure 2 .
Figure 2. A, An example of the effect of V c on the open probability (Po) in asymmetrical solutions for sodium and potassium.The channel is virtually closed at positive voltages.B, Typical single-channel current (I c ) vs clamped voltage (V c ) relation in asymmetrical solutions for Na + and K + .The average singlechannel conductance was 258 ± 41.67 pS.Buffered salt solutions: 150 mM KCl, 15 mM NaCl plus 100 µM CaCl 2 on the cisside, and 150 mM NaCl, 15 mM KCl plus 100 µM CaCl 2 on the trans-side.