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.     

Evidence for a direct interaction between internal tetra-alkylammonium cations and the inactivation gate of cardiac sodium channels

The effects of internal tetrabutylammonium (TBA) and tetrapentylammonium (TPeA) were studied on human cardiac sodium channels (hH1) expressed in a mammalian tsA201 cell line. Outward currents were measured at positive voltages using a reversed Na gradient. TBA and TPeA cause a concentration-dependent increase in the apparent rate of macroscopic Na current inactivation in response to step depolarizations. At TPeA concentrations < 50 microM the current decay is well fit by a single exponential over a wide voltage range. At higher concentrations a second exponential component is observed, with the fast component being dominant. The blocking and unblocking rate constants of TPeA were estimated from these data, using a three-state kinetic model, and were found to be voltage dependent. The apparent inhibition constant at 0 mV is 9.8 microM, and the blocking site is located 41 +/- 3% of the way into the membrane field from the cytoplasmic side of the channel. Raising the external Na concentration from 10 to 100 mM reduces the TPeA-modified inactivation rates, consistent with a mechanism in which external Na ions displace TPeA from its binding site within the pore. TBA (500 microM) and TPeA (20 microM) induce a use-dependent block of Na channels characterized by a progressive, reversible, decrease in current amplitude in response to trains of depolarizing pulses delivered at 1-s intervals. Tetrapropylammonium (TPrA), a related symmetrical tetra-alkylammonium (TAA), blocks Na currents but does not alter inactivation (O'Leary, M. E., and R. Horn. 1994. Journal of General Physiology. 104:507-522.) or show use dependence. Internal TPrA antagonizes both the TPeA-induced increase in the apparent inactivation rate and the use dependence, suggesting that all TAA compounds share a common binding site in the pore. A channel blocked by TBA or TPeA inactivates at nearly the normal rate, but recovers slowly from inactivation, suggesting that TBA or TPeA in the blocking site can interact directly with a cytoplasmic inactivation gate.     

Frequency domain analysis of asymmetry current in squid axon membrane.

The change in capacity of squid axon membrane during hyper- and depolarizations was investigated in the absence of ionic currents after the membrane was treated with pronase. In the presence of the inactivation process (h parameter), failure to observe the gating current in the frequency domain was attributed to the rapid attenuation of the possible capacity change during depolarizations, which is likely to be due to the sodium activation process. Elimination of the h process would therefore enable us to observe the gating current in the frequency domain as the change in the capacitance component of membrane admittance. However, even after the inactivation process was abolished by pronase, the capacity of the axon membrane remained constant when ionic currents were blocked by external tetrodotoxin and internal Cs+ ion. Actually capacity was observed to decrease slightly with depolarization, contrary to the prediction based on the magnitude of gating currents.     

Binding of scorpion toxin to receptor sites associated with sodium channels in frog muscle. Correlation of voltage-dependent binding with activation.

Purified scorpion toxin (Leiurus quinquestriatus) slows inactivation of sodium channels in frog muscle at concentrations in the range of 17-170 nM. Mono[125I]iodo scorpion toxin binds to a single class of sites in frog sartorius muscle with a dissociation constant of 14 nM and a binding capacity of 13 fmol/mg wet weight. Specific binding is inhibited more than 90% by 3 microM sea anemone toxin II and by depolarization with 165 mM K+. Half-maximal inhibition of binding is observed on depolarization to -41 mV. The voltage dependence of scorpion toxin binding is correlated with the voltage dependence of activation of sodium channels. Removal of calcium from the bathing medium shifts both activation and inhibition of scorpion toxin binding to more negative membrane potentials. The results are considered in terms of the hypothesis that activation of sodium channels causes a conformational change in the scorpion toxin receptor site resulting in reduced affinity for scorpion toxin.     

Modification of sodium inactivation in myelinated nerve by Anemonia toxin II and iodate. Analysis of current fluctuations and current relaxations

(1) Na+ currents and Na+ current fluctuations were measured in single myelinated nerve fibres of Rana esculenta under voltage-clamp conditions. The process of Na+ inactivation was modified by external treatment with 7 microM Anemonia Toxin II or by internal application of 20 or 40 mM IO3(-). (2) At depolarization of 24 and 32 mV the spectral density of Na+ current fluctuations could be described as the sum of two contributions, Sh(f) and Sm(f), representing the spectrum from fluctuations of the inactivation (h) and activation (m) gates, respectively. At higher depolarizations of 40 and 48 mV the low frequency (h) fluctuations could be better fitted by the sum, Sh1(f)+Sh2(f), of two separate Lorentzian functions. (3) The Na+ current and the variance of Na+ current fluctuations between 150 and 450 ms after depolarization are increased by one order of magnitude after application of Anemonia Toxin II or IO3(-). (4) The kinetics of Na+ current inactivation were described as A1 x exp(-t/tau h1) + A2 x exp(-t/tau h2) + B. The constant, tau h1, of fast Na+ inactivation was the same in normal and modified nerve fibres. The slow inactivation time constant, tau h2, increased with increasing depolarizations in modified fibres but decreased under control conditions. In all cases tau h2 showed a similar voltage dependence as the time constant found by fitting the low frequency fluctuations of Na+ current with one Lorentzian function, Sh(f). (5) It is concluded that Anemonia Toxin II and IO3(-) modify a fraction of Na+ channels in an all-or-none manner. A lower limit of the number of modified Na+ channels is estimated from the Na+ current and the variance Na+ current fluctuations. 7 microM external Anemonia Toxin II modifies more than 17% and 20 or 40 mM internal IO3(-) more than 8% of all Na+ channels. The inactivation gates in modified channels experience an electric field different from that in normal fibres.     

Modification of sodium channel currents by lanthanum and lanthanide ions in human heart cells

I examined the effects of 100 microM extracellular lanthanum and lanthanide ions on the fast transmembrane sodium channel currents of human heart cell segments. The experiments were conducted under control of the transmembrane electrical and chemical gradients. Lanthanum and lanthanide ion exposure decreased the amplitude and increased the inactivation time constant of the sodium current. Only a transient increase occurred for the activation time constant of the sodium current. The dependence of peak sodium current on excitatory and holding potentials (steady-state activation and inactivation curves, respectively) was transiently shifted to less negative potentials during the first 3 min of exposure, as if these cations were momentarily neutralizing the effective negative charges at the extracellular side of the membrane. The curves then returned to their original position and only the inactivation curves continued shifting progressively towards a limit at more negative membrane potentials. Membrane capacitance was always reduced and this may explain these late effects in terms of changes in membrane dielectric properties and free and bound charges, in addition to traditional screening and binding concepts. These effects were related to the electronic structure of these ions.     

Ionic currents in dispersed chemoreceptor cells of the mammalian carotid body

Ionic currents of enzymatically dispersed type I and type II cells of the carotid body have been studied using the whole cell variant of the patch-clamp technique. Type II cells only have a tiny, slowly activating outward potassium current. By contrast, in every type I chemoreceptor cell studied we found (a) sodium, (b) calcium, and (c) potassium currents. (a) The sodium current has a fast activation time course and an activation threshold at approximately -40 mV. At all voltages inactivation follows a single exponential time course. The time constant of inactivation is 0.67 ms at 0 mV. Half steady state inactivation occurs at a membrane potential of approximately -50 mV. (b) The calcium current is almost totally abolished when most of the external calcium is replaced by magnesium. The activation threshold of this current is at approximately -40 mV and at 0 mV it reaches a peak amplitude in 6-8 ms. The calcium current inactivates very slowly and only decreases to 27% of the maximal value at the end of 300-ms pulses to 40 mV. The calcium current was about two times larger when barium ions were used as charge carriers instead of calcium ions. Barium ions also shifted 15-20 mV toward negative voltages the conductance vs. voltage curve. Deactivation kinetics of the calcium current follows a biphasic time course well fitted by the sum of two exponentials. At -80 mV the slow component has a time constant of 1.3 +/- 0.4 ms whereas the fast component, with an amplitude about 20 times larger than the slow component, has a time constant of 0.16 +/- 0.03 ms. These results suggest that type I cells have predominantly fast deactivating calcium channels. The slow component of the tails may represent the activity of a small population of slowly deactivating calcium channels, although other possibilities are considered. (c) Potassium current seems to be mainly due to the activity of voltage-dependent potassium channels, but a small percentage of calcium-activated channels may also exist. This current activates slowly, reaches a peak amplitude in 5-10 ms, and thereafter slowly inactivates. Inactivation is almost complete in 250-300 ms. The potassium current is reversibly blocked by tetraethylammonium. Under current-clamp conditions type I cells can spontaneously fire large action potentials. These results indicate that type I cells are excitable and have a variety of ionic conductances. We suggest a possible participation of these conductances in chemoreception.     

Effect of pH on fast sodium channels in neurons of the rat dorsal root ganglion

Ionic currents through fast sodium channels in the neuronal somatic membrane were measured under voltage clamp conditions using external solutions of normal and low pH. Voltage-dependent inhibition of ionic currents through open channels was observed in acidic solutions. The voltage-dependent block of sodium channels may be explained by the presence of two acid groups at the channel. The parameters of the inner and outer acid groups calculated according to this model are similar to those reported for the nodal membrane.     

Properties of the sodium channels of the membrane of neuroblastoma clone N18 A-1 cultured cells

Ionic currents through sodium channels of dialyzed mouse neuroblastoma N18 A-1 cells were measured under voltage clamp conditions. The PNa/PK ratio evaluated by reversal potential shifts was 10.4 +/- 0.7. Parameters of steady-state fast inactivation curves (h--V) and peak sodium conductance curves (gNa--V) were determined. The inactivation kinetics had usually a two-exponential time course. The internal perfusion of cells by trypsin and pronase caused a slowing-down of the sodium current falling phase, pronase being more specific in this respect. An external application of the purified scorpion toxin in concentration of 1.42 X 10(-7) M leads to a fast and sharp slowing-down of sodium inactivation. The same toxin in concentration of 5 X 10(-6) M, applied internally was quite unaffective. Experimental results demonstrate similarities in the main features between the sodium channels of neuroblastoma cells and those of other excitable cell membranes.     

Effects of toxin II from the scorpion Androctonus australis Hector on sodium current in neuroblastoma cells and their modulation by oleic acid

The effects of toxin II (AaH II) isolated from the scorpion Androtonus australis Hector on sodium current in neuroblastoma X glioma NG 108-15 hybrid cells were analysed under patch clamp conditions in the whole cell configuration. AaH II (70 nM)_induced a maintained sodium current, as well as increasing both fast and slow inactivation time constants and the amplitude of the peak current. This latter effect occurred via a shift of the activation-voltage curve towards negative voltage values by about 9 mV. Oleic acid (5 M), which had no effect on I_Na under control conditions, decreased the AaH II-induced maintained current. It also reversed, or prevented the increase of the peak current induced by AaH II. However, it neither prevented nor modified the AaH II-induced increase in inactivation time constants. The binding of the toxin to its specific site and the number of binding sites for AaH II were not significantly modified by oleic acid. The oleic acid-induced effects could not be related to the activation of protein kinase C since PMA, a potent activator of this enzyme, did not produce oleic acid-like effects. From these results, it is concluded that AaH II has several independent effects on sodium channels, some of which could be modulated by the lipid environment of sodium channels in the membrane.     

Potential-dependent interaction of toxin from venom of the scorpion Buthus eupeus with sodium channels in myelinated fibre: voltage clamp experiments

1. The steady-state characteristics of the sodium channel gating in the nodal membrane were determined under voltage clamp conditions before and after treatment with toxins from the venom of scorpion, Buthus eupeus. 2. The apparent binding constant (KA) of the toxin was determined for different levels of the membrane potential. At potentials more negative than -120 mV, KA tends to a constant level. KA is maximum at about -80 mV, and it decreases as the potential is teduced to 0 mV. 3. A model assuming that the voltage dependency of KA is mainly due to the difference in electrical energy between inactivated states of normal and poisoned channels is proposed. An additional decrease in overall binding of toxin results from the transition of a fraction of the sodium channels into the state of slow inactivation.     

Hydrogen ion block of single sodium channels in neuroblastoma cells

The effects of pH of the external medium on amplitude of currents through single sodium channels at the membrane of cultured neuroblastoma cells were investigated in mice belonging to strain C 1300, clone N18A-1. Currents through single sodium channels in isolated membrane segments (outside-out configuration) were registered with normal (7.2) and reduced (5.4) pH levels in the external medium. Reducing the pH to 5.4 was found to decrease current amplitude reversibly by about twofold (–10 to –30 mV for test potentials). Findings would confirm that the depression of macroscopic sodium currents produced by reducing the pH of the extracellular solution is due to a decline in ionic flow through single open sodium channels.Institute of Cytology, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 21, No. 1, pp. 101–105, January–February, 1989.     

Intracellular pH and ionic channels in the Loligo vulgaris giant axon.

Squid giant axons were used to investigate the reversible effects of intracellular pH(pHi) on the kinetic properties of ionic channels. The pharmacologically separated K+ and Na+ currents were measured under: (a) internal perfusion, (b) enzymatic Pronase treatment, and (c) continuous estimate of periaxonal ion accumulation. Variation of internal pH from 4.8 to 11 resulted in: (a) a decrease of steady-state sodium inactivation at positive potentials similar to the effect of the proteolytic enzyme Pronase, (b) a shift of the h infinity (E) curve toward depolarizing voltages, and (c) a decrease of the time constant of inactivation for potentials below -20 mV (an increase above). A plot of the steady-state sodium conductance at E = +40 mV as a function of pHi suggests that two groups with pKa 10.4 and 5.6 affect respectively the inactivation gate and the rate constants for the transition from the inactivated to the second open state (h2) (Chandler and Meves, 1970b). The voltage shifts of the kinetic parameters predicted by the Gouy-Chapman-Stern theory are well satisfied at high pHi and less at low. Once corrected for voltage shifts, the forward rate constants for channel opening were found to be slowed with the acidity of the internal or external solution.     

Open- and closed-state fast inactivation in sodium channels: differential effects of a site-3 anemone toxin

The role of sodium channel closed-state fast inactivation in membrane excitability is not well understood. We compared open- and closed-state fast inactivation, and the gating charge immobilized during these transitions, in skeletal muscle channel hNa(V)1.4. A significant fraction of total charge movement and its immobilization occurred in the absence of channel opening. Simulated action potentials in skeletal muscle fibers were attenuated when pre-conditioned by sub-threshold depolarization. Anthopleurin A, a site-3 toxin that inhibits gating charge associated with the movement of DIVS4, was used to assess the role of this voltage sensor in closed-state fast inactivation. Anthopleurin elicited opposing effects on the gating mode, kinetics and charge immobilized during open- versus closed-state fast inactivation. This same toxin produced identical effects on recovery of channel availability and remobilization of gating charge, irrespective of route of entry into fast inactivation. Our findings suggest that depolarization promoting entry into fast inactivation from open versus closed states provides access to the IFMT receptor via different rate-limiting conformational translocations of DIVS4.     

Sea anemone toxin and scorpion toxin share a common receptor site associated with the action potential sodium ionophore.

Toxin II isolated from the sea anemone Anemonia sulcata enhances activation of the action potential sodium ionophore of electrically excitable neuroblastoma cells by veratridine and batrachotoxin. This heterotropic cooperative effect is identical to that observed previously with scorpion toxin but occurs at a 110-fold higher concentration. Depolarization of the neuroblastoma cells inhibits the effect of sea anemone toxin as observed previously for scorpion toxin. Specific scorpion toxin binding is inhibited by sea anemone toxin with KD approximately equal to 90 nM. These results show that the polypeptides scorpion toxin and sea anemone toxin II share a common receptors site associated with action potential sodium ionophores.     

Multiple-state model of ionic channel in reproducing the time course of electrophysiological events.

BACKGROUND: The predictions of the Hodgkin-Huxley model do not accurately fit all the measurements of voltage-clamp currents, gating charge and single-channel currents. There are many quantitative differences between the predicted and measured characteristics of the sodium and potassium channels. For example, the two-state gate model has exponential onset kinetics, whereas the sodium and potassium conductances show S-shaped activation and the sodium conductance shows an exponential inactivation. In this paper we shall examine a more general channel model that can more faithfully represent the measured properties of ionic channels in the membrane of the excitable cell. METHODS: The model is based on the generalisation of the notion of a channel with a discrete set of states. Each state has state attributes such as the state conductance, state ionic current and state gating charge. These variables can have quite different waveforms in time, in contrast with a two-state gate channel model, in which all have the same waveforms. RESULTS: The kinetics of all variables are equivalent: gating and ionic currents give equivalent information about channel kinetics; both the equilibrium values of the current and the time constants are functions of membrane potential. The results are in almost perfect concordance with the experimental data regarding the characteristics of nerve impulse. CONCLUSIONS: The expected values of the gating charge and the ionic conductance are weighted sums of the state occupancy probabilities, but the weights differ: for the expected value of the gating charge the weights are the state gating charges and for the expected value of the ionic conductance the weights are the state conductances. Since these weights are different, the expected values of the gating charge and the ionic conductance will differ.     

Voltage-dependent removal of sodium inactivation by N-bromoacetamide and pronase.

When perfused internally through crayfish giant axons, pronase removed sodium inactivation more than three times as fast at -100 mV as compared with -30 mV. N-bromoacetamide, applied internally, removed sodium inactivation twice as fast at -100 mV as at -30 mV, and the relative rate of removal declined with membrane depolarization in proportion to steady-state sodium inactivation. We conclude that in the closed conformation the sodium inactivation gate is partially protected from destruction by N-bromoacetamide and pronase.     

Blockade of sodium channels by Bistramide A in voltage-clamped frog skeletal muscle fibres.

The effect of Bistramide A, a toxin isolated from Bistratum lissoclinum Sluiter (Urochordata), on the peak sodium current (INa) of frog skeletal muscle fibres was studied with the double sucrose gap voltage clamp technique. External or internal application of Bistramide A inhibited INa without alteration of the kinetic parameters of the current nor of the apparent reversal potential for Na. The steady-state activation curve of INa was unchanged while the steady-state inactivation curve of INa was shifted towards more negative membrane potentials. Dose-response curves indicated an apparent dissociation constant for Bistramide A of 3.3 microM and a Hill coefficient of 1.2 which suggested a one to one relation between the toxin and Na channel. The inhibition of INa occurred at rest, and was more important at more positive holding potentials. Bistramide A exhibited only a weak frequency-dependent effect. The toxin did not interact with the use-dependent effect of lidocaine. It mainly blocked Na channels at more depolarized holding potentials. The toxin blocked Na channels when it was internally applyed and when the inactivation gating system has been previously destroyed by internal diffusion of iodate. The data suggest that Bistramide A inhibited the Na channel both at rest and in the inactivated state and occupied a site which was not located on the inactivation gate.     

Ionic currents of the nodal membrane underlying the fastest saltatory conduction in myelinated giant nerve fibers of the shrimp Penaeus japonicus.

The myelinated giant nerve fiber of the shrimp, Penaeus japonicus, is known to have the fastest velocity of saltatory impulse conduction among all nerve fibers so far studied, owing to its long distances between nodal regions and large diameter. For a better understanding of the basis of this fast conduction, a medial giant fiber of the ventral nerve cord of the shrimp was isolated, and ionic currents of its presynaptic membrane (a functional node) were examined using the sucrose-gap voltage-clamp method. Inward currents induced by depolarizing voltage pulses had a maximum value of 0.5 microA and a reversal potential of 120 mV. These currents were completely suppressed by tetrodotoxin and greatly prolonged by scorpion toxin, suggesting that they are the Na current. Both activation and inactivation kinetics of the Na current were unusually rapid in comparison with those of vertebrate nodes. According to a rough estimation of the excitable area, the density of Na current reached 500 mA/cm2. In many cases, the late outward currents were induced only by depolarizing pulses larger than 50 mV in amplitude. The slope conductance measured from late currents were mostly smaller than that measured from the Na current, suggesting a low density of K channels in the synaptic membrane. These characteristics are in good harmony with the fact that the presynaptic membrane plays a role as functional node in the fastest impulse conduction of this nerve fiber.     

Depolarization Differentially Affects Allosteric Modulation by Neurotoxins of Scorpion α-Toxin Binding on Voltage-Gated Sodium Channels

Abstract: Voltage-gated sodium channels serve as a target for many neurotoxins that bind to several distinct, allosterically interacting receptor sites. We examined the effect of membrane potentials (incited by increasing external K⁺ concentrations) on the binding modulation by veratridine, brevetoxin, and tetrodotoxin of the scorpion α-toxin AaH II to receptor site 3 on sodium channels of rat brain synaptosomes. Depolarization is shown to differentially modulate neurotoxin effects on AaH II binding: Veratridine increase is potentiated, brevetoxin's inhibitory effect is reduced, and tetrodotoxin enhancement is evident mainly at resting membrane potential (5 m M K⁺). Both tetrodotoxin and veratridine apparently reverse the inhibition of AaH II binding by brevetoxin at resting membrane potential, but only veratridine is able to partially restore AaH II binding at 0 mV (135 m M K⁺). Thus, the allosteric interactions are grouped into two categories, depending on the membrane potential. Under depolarized conditions, the cooperative effects among veratridine and brevetoxin on AaH II binding fit the previously described two-state conformational model. At resting membrane potential, additional interactions are revealed, which may be explained by assuming that toxin binding induces conformational changes on the channel structure, in addition to being state-dependent. Our results provide a new insight into neurotoxin action and the complex dynamic changes underlying allosteric coupling of neurotoxin receptor sites, which may be related to channel gating.