Specificity of antibodies to sea anemone toxin III and immunogenicity of the pharmacological site of anemone and scorpion toxins

Toxin III (ATX III) of the sea anemone (Anemonia sulcata) is a polypeptide containing 27 amino acid residues. It has no sequence similarity with other toxins (ATX I and II) from the same species, or with scorpion toxins, although they apparently act in a similar manner by prolonging action potentials. The specificity of ATX III antibodies was characterized using ATX III, ATX I, native and chemically modified ATX II, and scorpion alpha-toxins. The results obtained suggest that a region of ATX III, partially or totally overlapping the pharmacological site shared with ATX I and ATX II, is immunogenic. It includes a guanidino and at least two carboxylate groups. The corresponding region is not immunogenic in ATX I and ATX II. Anti-(ATX III) antibodies recognize the similar regions of ATX I and ATX II and apparently do not recognize scorpion toxins.     

Action of different toxins from the scorpion Androctonus australis on a locust nerve-muscle preparation

The neuromuscular effects of four purified toxins and crude venom from the scorpion Androctonus australis were investigated in the extensor tibiae nerve-muscle preparation of the locust Locusta migratoria. Insect and crustacean toxin and the mammal toxins I and II which have previously been shown to act on fly larvae, isopods, and mice all paralyse locust larvae. The paralytic potencies decrease in the following order: insect toxin → mammal toxin I → crustacean toxin → mammal toxin II.The toxins and crude venom cause repetitive activity of the motor axons. This leads to long spontaneous trains of junction potentials in the case of crude venom and insect toxin. The other toxins chiefly cause short bursts of action and junction potentials following single stimuli.The ‘slow’ excitatory motor axon invariably is affected sooner than the inhibitory or the ‘fast’ excitatory one. The minimal doses of toxins required to affect the ‘slow’ motor axon decrease in an order somewhat different from that established for their paralytic potencies: insect toxin → crustacean toxin → mammal toxin I → mammal toxin II.Crude venom depolarises and destabilises the muscle membrane potential at low doses. At high doses it decreases the membrane resistance, whereas insect toxin leads to an increase.Crude venom and insect toxin enhance the frequency of mejps, whereas mammal toxin I leads to the occurrence of ‘giant’ mejps.The pattern of axonal activities indicates that the various peripheral branches of the motor nerve are the primary target of the toxins.The time course of nerve action potentials is affected by mammal toxin I and crustacean toxin which cause anomalous shapes and prolongations not caused by insect toxin.The results with other animals suggest that only the insect toxin is selective in its activity. The way it affects the axon might be quite different from that previously reported for scorpion venoms or toxins.     

Anemonia sulcata toxin (ATX II) enhances spontaneous electrical activity and tension in chronically denervated rat diaphragm

In isolated strips of rat diaphragm denervated 9-21 days prior to experimentation, spontaneous action potentials were recorded extracellularly and twitch and resting tension were measured. The sea anemone toxin ATX II enhances the occurrence of spontaneous action potentials, increases resting tension and depresses twitch tension. These effects are essentially irreversible. In low sodium solution substituted with sucrose the effects of ATX II are attenuated, however, they fully develop upon return to normal sodium solution with a marked transient increase in the incidence of spontaneous action potentials and in resting tension. ATX II remains uneffective after pretreatment with tetrodotoxin. Reelevation of the extracellular sodium concentration after exposure to low sodium solution per se causes a marked increase in occurrence of fibrillation potentials, however the transient increase in resting tension was much smaller than in the presence of ATX II. Substitution of chloride with the impermeable anion methylsulphate enhances spontaneous activity and resting tension without an effect on twitch tension. Addition of ATX II elevates resting tension although the concomitant further increase in incidence of spontaneous action potentials is small. It is concluded that the increase in resting tension reflects a summation of the fibrillatory activity, but fibrillations become more effective when the preparations are exposed to ATX II. This finding points at the possible r?le of sodium ions in excitation contraction coupling of denervated skeletal muscle.     

Calitoxin, a neurotoxic peptide from the sea anemone Calliactis parasitica: amino acid sequence and electrophysiological properties

We have isolated a new toxin, calitoxin (CLX), from the sea anemone Calliactis parasitica whose amino acid sequence differs greatly from that of other sea anemone toxins. The polypeptide chain contains 46 amino acid residues, with a molecular mass of 4886 Da and an isoelectric point at pH 5.4. The amino acid sequence determined by Edman degradation of the reduced, S-carboxymethylated polypeptide chain and tryptic and chymotryptic peptides is Ile-Glu-Cys-Lys-Cys-Glu-Gly-Asp-Ala-Pro-Asp-Leu-Ser-His-Met-Thr-Gly-Thr- Val-Tyr - Phe-Ser-Cys-Lys-Gly-Gly-Asp-Gly-Ser-Trp-Ser-Lys-Cys-Asn-Thr-Tyr-Thr-Ala- Val-Ala - Asp-Cys-Cys-His-Glu-Ala. No cysteine residues were present in the peptide. Similarly to other sea anemone toxins, calitoxin interacts, in crustacean nerve muscle preparations, with axonal and not with muscle membranes, inducing a massive release of neurotransmitter that causes a strong muscle contraction. The low homology of CLX with RP II and ATX II toxins has implications regarding the role played by particular amino acid residues.     

Effect of ciguatoxins on the cardiocirculatory system

The aim of the present review was to collect the main observations reported until now concerning the cardio-circulatory effects of polyether toxins, called ciguatoxins, which are involved in an endemic intoxication named ciguatera found in tropical and subtropical countries. Ciguatera is caused by the ingestion of fishes contaminated with the dinoflagellate Gamberdiscus toxicus. Due to both tropical fish exportation destined for food and tourism, the disease has now spread out to temperate areas. Several toxins have been isolated and purified from different fish species living in different geographical areas. They are classified into three main groups by the nature of certain cycles of their carbon skeleton. Clinical reports show evidence that ciguatera intoxication affect both electrocardiograms and blood pressure. In most cases, ciguateric intoxication mainly evoked bradycardia, hypotension, and the alteration of S-T segment in the electrocardiogram. Isolated and purified ciguatoxins strongly altered the morphology of cardiac tissue inducing swelling of the cells and alterations of cellular organelles. These toxins impair the conduction of cardiac nerves and increase the opening probability of Na+ channels in intracardiac ganglions. Depending on the concentration applied, the substances exerted either a fast positive inotropic effect or a negative inotropic effect on the contraction of mammalian atrial and ventricular cardiac muscle. These effects were attributed to a release of noradrenaline and acetylcholine from neural terminals of the autonomic nervous system present in cardiac tissue. They also exert a slow delayed inotropic effect on the contraction which has been attributed to a direct effect of the toxins on tetrodotoxin-sensitive voltage-dependent Na+ channels of cardiac membranes. Ciguatoxins depolarized the membrane of mammalian atrial and ventricular preparations and shifted the threshold of sodium current activation to more negative membrane potentials. In conclusion, the inotropic effects of ciguatoxins on cardiac tissues mainly depend on the toxin concentration sensitivity of autonomic nerve terminals, which released noradrenaline and/or acetylcholine, while the ciguatoxin-induced increase of the sodium influx could be involved in the cardiac cell swelling which coincides with reports in which ciguatoxins induced a mannitol-inhibited swelling of the Node of Ranvier.     

Permeation and metabolism of Alternaria mycotoxins with perylene quinone structure in cultured Caco-2 cells

The absorption of four Alternaria toxins with perylene quinone structures, i.e. altertoxin (ATX) I and II, alteichin (ALTCH) and stemphyltoxin (STTX) III, has been determined in the Caco-2 cell Transwell system, which represents a widely accepted in vitro model for human intestinal absorption and metabolism. The cells were incubated with the four mycotoxins on the apical side, and the concentration of the toxins in the incubation media of both chambers and in the cell lysate were determined by liquid chromatography coupled with diode array detection and mass spectrometry (LC-DAD-MS) analysis. ATX I and ALTCH were not metabolised in Caco-2 cells, but ATX II and STTX III were partly biotransformed by reductive de-epoxidation to the metabolites ATX I and ALTCH, respectively. Based on the apparent permeability coefficients (P_app), the following ranking order for the permeation into the basolateral compartment was obtained: ATX I > ALTCH >> ATX II > STTX III. Total recovery of the four toxins decreased in the same order. It is assumed that the losses of STTX III, ATX II and ALTCH in Caco-2 cells are caused by covalent binding to cell components due to the epoxide group and/or the α,β-unsaturated carbonyl group present in these toxins. We conclude from this study that ATX I and ALTCH are well absorbed from the intestinal lumen into the portal blood in vivo. For ATX II and STTX III, intestinal absorption of the parent toxins is very low, but these toxins are partly metabolised to ATX I and ALTCH, respectively, in the intestinal epithelium and absorbed as such.     

Developmental changes in inotropic actions of neurotoxins observed in ventricular muscle of rat heart.

Developmental changes in functions of myocardial sodium channels were examined from inotropic effects of several neurotoxins in ventricular muscle preparations obtained from prenatal (20-22 day gestation) or adult (3-4 months old) rat hearts. Tetrodotoxin caused a negative inotropic effect in low concentrations and a loss of muscle responsiveness to electrical stimulation in high concentrations in preparations obtained from either prenatal or adult rat heart. The tetrodotoxin concentration that caused a 50% decrease in developed tension was higher in prenatal rats. Anemonia sulcata toxin, Androctonus australis toxin, veratridine, and Centruroides sculpturatus toxin all produced positive inotropic effects in adult rat heart. The effects were largest with A. sulcata and A. australis toxins, intermediate with veratridine, and smallest with C. sculpturatus toxin. Prenatal heart required higher concentrations of either veratridine, or A. sulcata or A. australis toxins to produce comparable positive inotropic effects. With C. sculpturatus toxin, no significant positive inotropic effect was observed in prenatal heart muscle preparations. These results indicate that cardiac sodium channels undergo significant functional changes during development and that negative and positive inotropic effects of neurotoxins resulting from inhibition and enhancement of fast Na+ channels reflect developmental changes in the cardiac sodium channels.     

Effects of pH and temperature on cardioactive polypeptides from sea anemones: a 1H-NMR study

The effects of pH and temperature on the 300-MHz ¹H-nmr spectra of three cardioactive polypeptides from sea anemones, anthopleurin-A from Anthopleura xanthogrammica (AP-A) and Anemonia sulcata toxins I and II (ATX I and II), are described. AP-A and ATX II exhibit major spectral heterogeneity. Evidence from the pH and temperature studies and from high performance liquid chromatography indicates that this heterogeneity is conformational rather than chemical in origin. By contrast, purified isotoxins of ATX I show no evidence of conformational heterogeneity. The pKa values of most of the ionizable groups in these polypeptides are not strongly perturbed by interactions in the tertiary structure, with the exception of one of the Asp carboxylates, which has a pKa of ? 2 in AP-A and ATX II and 3.0 in ATX I. Protonation of this carboxylate, suggested to be Asp-9, leads to a conformational change in all three molecules. All three polypeptides are thermally stable, showing some conformational changes but not major unfolding at elevated temperatures.     

Interaction of scorpion alpha-toxins with cardiac sodium channels: binding properties and enhancement of slow inactivation

The effects of the scorpion alpha-toxins Lqh II, Lqh III, and LqhalphaIT on human cardiac sodium channels (hH1), which were expressed in human embryonic kidney (HEK) 293 cells, were investigated. The toxins removed fast inactivation with EC(50) values of <2.5 nM (Lqh III), 12 nM (Lqh II), and 33 nM (LqhalphaIT). Association and dissociation rates of Lqh III were much slower than those of Lqh II and LqhalphaIT, such that Lqh III would not dissociate from the channel during a cardiac activation potential. The voltage dependence of toxin dissociation from hH1 channels was nearly the same for all toxins tested, but it was different from that found for skeletal muscle sodium channels (muI; Chen et al. 2000). These results indicate that the voltage dependence of toxin binding is a property of the channel protein. Toxin dissociation remained voltage dependent even at high voltages where activation and fast inactivation is saturated, indicating that the voltage dependence originates from other sources. Slow inactivation of hH1 and muI channels was significantly enhanced by Lqh II and Lqh III. The half-maximal voltage of steady-state slow inactivation was shifted to negative values, the voltage dependence was increased, and, in particular for hH1, slow inactivation at high voltages became more complete. This effect exceeded an expected augmentation of slow inactivation owing to the loss of fast inactivation and, therefore, shows that slow sodium channel inactivation may be directly modulated by scorpion alpha-toxins.     

Voltage-dependent membrane ionic currents in lamprey striated muscle

Membrane ionic currents in striated muscle bundles of lamprey suction apparatus were recorded using a double sucrose gap technique. Transmembrane currents in a single muscle fiber and a fiber bundle in the frog were compared so as to check the validity of current measurement in multicell preparations. It was found that fast inward sodium currents arise in the lamprey muscle membrane in response to depolarization together with a delayed outward potassium current, with steady-state characteristics resembling those of membrane currents in frog muscle. The only difference consisted of a flatter curve for steady-state inactivation of potassium current, probably indicative of greater density of potassium channels. Both the changes in reversal potential and the speed of potassium current deactivation occurring during protracted stimuli point to the presence of two fractions in this current. No functioning voltage-dependent calcium channels are found in the lamprey muscle membrane.I. M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 18, No. 5, pp. 629–636, September–October, 1986.     

Modification of sodium channels in myelinated nerve by Anemonia sulcata toxin II

1. Single myelinated nerve fibres of the frog, Rana esculenta, were investigated predominantly in voltage clamp experiments. 2. Sodium current (INa) inactivation was measured in the presence of 10 mM TEA to suppress IK. Inactivation was diphasic but complete in toxin-free solution; it was delayed and became incomplete in Anemonia sulcata toxin II (ATX II) leading to persistent INa flow even during long depolarizations. The effects were reversible. Activation was not affected. 3. The persistent INa component increased with increasing toxin concentration and saturated at ca. 15 microM. The lowest concentration yielding unequivocal effects in the voltage clamp was 0.5 microM. 4. The curve relating the steady-state inactivation parameter, h infinity to the conditioning potential V became non-monotonic in ATX II i.e. dh infinity/dV greater than 0 for V greater than 30 mV. 5. Inactivation could be formally described by a three-state model with two conducting (h2 and h2) and one closed state (x) in the sequence h1 in equilibrium x in equilibrium h2. 6. Ca2+ modifies h2(V) more than h1(V) whose reaction to Ca2+ is similar to h(V) in toxin-free solution. The Ca2+ effect is very rapid and reversible.     

Birefringence signals in mammalian and frog myocardium. E-C coupling implications

Birefringence signals from mammalian and frog hearts were studied. The period between excitation and the onset of contraction in which optical signals were free of movement artifact was determined by changes in scattered incandescent light and changes in laser diffraction patterns. The birefringence signal preceding contraction was found to behave as a change in retardation and was not contaminated measurably by linear dichroic or isotropic absorption changes. There were two components of the birefringence signal in mammalian heart muscles but only one component in the frog heart. The first component of the birefringence signals in both mammalian and frog hearts had a time course coincident with the action potential upstroke. The second component in mammalian preparations was sensitive to inotropic interventions, such as variation of extracellular Ca2+, stimulation frequency, temperature, and epinephrine, in a manner that correlated with the maximum rate of rise of tension. Caffeine (2-10 mM) not only failed to generate a second component in the frog heart, but also suppressed the second component in the mammalian heart while potentiating twitch tension. The results suggest that the second component of the birefringence signal in the mammalian myocardium is related to Ca2+ release from the sarcoplasmic reticulum.     

Effects of a neurotoxin isolated from the sea anemone, Anemonia sulcata, at frog neuromuscular junction (author's transl)]

ATX II is a toxin extracted from tentacles of Anemonia sulcata. It was known that this protein displays neurotoxic effects on frog isolated neuromuscular preparation (Fig. 1, 2) and that muscular contractures observed with ATX II are blocked by d-tubocurarine (Fig. 3) or on a 40-days-denervated gastrocnemius (Fig. 4). Part of these experiments has already appeared. 1. These effects of ATX II depend on calcium concentration in the bathing medium, as is the case for transmitter release. The same results were observed when we substituted strontium to calcium. 2. On an intact sciatic sartorius preparation, ATX II does not act on the amplitude of the miniature endplate potentials (mepps, Fig. 6). The muscular action potential is not modified by this toxin. 3. ATX II increases the frequency of the mepps (Fig. 5). The evoked transmitter release (quantal content) after ATX II is also largely increased (Fig. 7). 4. In conclusion, it is suggested that ATX II acts indirectly on the muscle through an increase in acetylcholine release from the motor nerve terminals.     

Effects of several sea anemone and scorpion toxins on excitability and ionic currents in the giant axon of the cockroach

The membrane effects of 4 sea anemone and 6 scorpion toxins have been studied under current clamp and voltage clamp conditions. Micromolar concentrations of the purified toxins were applied externally on single giant axons of the american cockroach. Periplaneta americana in a double oil-gap arrangement and the effects on the resting potential, action potential and underlying currents analysed. The 4 sea anemone toxins (Condylactis toxin, Anemonia toxin 2, Anthopleurin toxin A and Parasicyonis toxin) were found to considerably prolong the action potential. This effect is frequency dependent and long plateau spikes (100-500 ms in duration) are consistently seen for frequencies lower than 0.2 Hz. This effect is due to a considerable delay in the turning-off of the sodium current during square membrane depolarizations associated, for large concentrations, with a decrease in the potassium conductance. Toxin effects on the sodium current are not prevented by pretreatment with STX. From the 4 purified toxins extracted from the venom of the scorpion, Androctonus australis Hector, 3 (Mammal toxins 1 and 2 and crustacean toxin) were found to have sea anemone toxin like effects and to induce long duration plateau action potentials. As for sea anemone toxins, this effect is due to a lengthening of the falling phase of the sodium current associated with a small decrease in the potassium conductance. The 4th toxin (insect toxin or ITAaH) depolarizes the membrane and induces repetitive firing of short action potentials.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sea Anemone Toxin (ATX II) Modulation of Heart and Skeletal Muscle Sodium Channel α-Subunits Expressed in tsA201 Cells

We have expressed recombinant α-subunits of hH1 (human heart subtype 1), rSkM1 (rat skeletal muscle subtype 1) and hSkM1 (human skeletal muscle) sodium channels in human embryonic kidney cell line, namely the tsA201 cells and compared the effects of ATX II on these sodium channel subtypes. ATX II slows the inactivation phase of hH1 with little or no effect on activation. At intermediate concentrations of ATX II the time course of inactivation is biexponential due to the mixture of free (fast component, τ^fast _h ) and toxin-bound (slow component, τ^slow _h ) channels. The relative amplitude of τ^slow _h allows an estimate of the IC₅₀ values ∼11 nm. The slowing of inactivation in the presence of ATX II is consistent with destabilization of the inactivated state by toxin binding. Further evidence for this conclusion is: (i) The voltage-dependence of the current decay time constants (τ _h ) is lost or possibly reversed (time constants plateau or increase at more positive voltages in contrast to these of untreated channels). (ii) The single channel mean open times are increased by a factor of two in the presence of ATX II. (iii) The recovery from inactivation is faster in the presence of ATX II. Similar effects of ATX II on rSkM1 channel behavior occur, but only at higher concentrations of toxin (IC₅₀= 51 nm). The slowing of inactivation on hSkM1 is comparable to the one seen with rSkM1. A residual or window current appears in the presence of ATX II that is similar to that observed in channels containing mutations associated with some of the familial periodic paralyses. Received: 5 December 1995/Revised: 1 March 1996     

Phylogenetic changes in sensitivity to Anemonia sulcata toxin (ATX II), and impact of first interaction with the toxin (imprinting) on later response to it

The Anemonia sulcata toxin ATX II is cardiotoxic and neurotoxic, and--at a high dose level--even lethal for the mouse, neurotoxic, but non-lethal for the frog, and has no adverse influence whatever on the Planaria and Tetrahymena; it even stimulates the growth of the Tetrahymena at a low dose level. It also induces imprinting in the Tetrahymena, as judged from the altered response of the latter to ATX II on re-exposure. No similar imprinting effect was demonstrable in mice.     

Modulation of human Nav1.7 channel gating by synthetic α-scorpion toxin OD1 and its analogs

Nine different voltage-gated sodium channel isoforms are responsible for inducing and propagating action potentials in the mammalian nervous system. The Na_v1.7 channel isoform plays an important role in conducting nociceptive signals. Specific mutations of this isoform may impair gating behavior of the channel resulting in several pain syndromes. In addition to channel mutations, similar or opposite changes in gating may be produced by spider and scorpion toxins binding to different parts of the voltage-gated sodium channel. In the present study, we analyzed the effects of the α-scorpion toxin OD1 and 2 synthetic toxin analogs on the gating properties of the Na_v1.7 sodium channel. All toxins potently inhibited channel inactivation, however, both toxin analogs showed substantially increased potency by more than one order of magnitude when compared with that of wild-type OD1. The decay phase of the whole-cell Na⁺ current was substantially slower in the presence of toxins than in their absence. Single-channel recordings in the presence of the toxins revealed that Na⁺ current inactivation slowed due to prolonged flickering of the channel between open and closed states. Our findings support the voltage-sensor trapping model of α-scorpion toxin action, in which the toxin prevents a conformational change in the domain IV voltage sensor that normally leads to fast channel inactivation.     

Effects of isoprenaline on tonic tension and Na-Ca exchange in frog atrial fibres

The action of isoprenaline, a purely beta-agonist, was investigated on frog atrial fibres under voltage clamp conditions; tonic tension was induced by long depolarizing pulses and the outward delayed current simultaneously developed. The cumulative dose-response curves showed that isoprenaline increased the peak of tonic tension in the concentration range 10(-8) to 3. 10(-6) mol.l-1, with a maximum effect for 10(-6) mol.l-1. The positive inotropic action of isoprenaline was associated with an increase in the rates of tension rise and of relaxation. Isoprenaline also increased the amplitude of the outward delayed current in a dose-dependent manner. The effects of isoprenaline (10(-6) mol.l-1) on tonic tension and outward delayed current were not observed in the presence of propranolol (10(-7) mol.l-1). Experiments carried out in low-sodium solution demonstrated that the action of isoprenaline on tonic tension can be explained by activation of Na-Ca exchange; the enhanced relaxation might result from the same process. These results suggested that the positive inotropic action of isoprenaline is mediated not only by the well-known increase in the slow inward current but also by activation of the Na-Ca exchange mechanism.     

Conotoxin modulation of voltage-gated sodium channels

The rising phase of the action potential in excitable cells is mediated by voltage-gated sodium channels (VGSCs), of which there are nine mammalian subtypes with distinct tissue distribution and biophysical properties. The involvement of certain VGSC subtypes in disease states such as pain and epilepsy highlights the need for agents that modulate VGSCs in a subtype-specific manner. Conotoxins from marine snails of the Conus genus constitute a promising source of such modulators, since these peptide toxins have evolved to become selective for various membrane receptors, ion channels and transporters in excitable cells. This review covers the structure and function of three classes of conopeptides that modulate VGSCs: the pore-blocking mu-conotoxins, the delta-conotoxins which delay or inhibit VGSC inactivation, and the muO-conotoxins which inhibit VGSC Na(+) conductance independent of the tetrodotoxin binding site. Some of these toxins have potential therapeutic and research applications, in particular the muO-conotoxins, which may develop into potential drug leads for the treatment of pain states.     

Anemone toxin II receptor site of the lobster nerve sodium channel. Studies in membrane vesicles and in proteoliposomes

The receptor-site for the sea anemone toxin II from Anemonia sulcata (ATX) and its functional relationship with the Na+ channel were studied in plasma membrane preparations from lobster walking leg nerves. The modification of the 22Na influx by ATX was determined in membrane vesicles and in proteoliposomes prepared by reconstitution of detergent-extracted, unfractionated membrane particles into soybean liposomes. The effects of two other toxins, veratridine (VER) and tetrodotoxin (TTX), which bind to Na+ channel receptor-sites other than that for polypeptide toxins, were also studied, ATX and VER stimulated 22Na flux into membrane vesicles with K0.5 values in the order of 10(-7) and 10(-5) M, respectively. Positive cooperativity among these toxins was also seen; ATX displaces the K0.5 for VER towards lower VER concentrations. TTX abolishes the 22Na influx increment caused by ATX and/or VER with a K0.5 in the order of 10(-8) M. In proteoliposomes, in contrast, ATX modified the 22Na influx only at high concentrations (greater than 1 microM) and in the presence of VER. VER stimulation and TTX inhibition of the VER and the VER plus ATX modified fluxes, had the same characteristics as in the vesicle preparations. Measurable ATX and VER toxin effects were only seen in the presence of an outwardly directed K+ gradient for both vesicles and proteoliposomes. Detergent treatment and the reconstitution procedure seem to affect the functional properties of the ATX receptor site whereas the VER and the TTX sites remain unaltered.