Single Na+ channels activated by veratridine and batrachotoxin

Voltage-sensitive Na+ channels from rat skeletal muscle plasma membrane vesicles were inserted into planar lipid bilayers in the presence of either of the alkaloid toxins veratridine (VT) or batrachotoxin (BTX). Both of these toxins are known to cause persistent activation of Na+ channels. With BTX as the channel activator, single channels remain open nearly all the time. Channels activated with VT open and close on a time scale of 1-10 s. Increasing the VT concentration enhances the probability of channel opening, primarily by increasing the rate constant of opening. The kinetics and voltage dependence of channel block by 21-sulfo-11-alpha-hydroxysaxitoxin are identical for VT and BTX, as is the ionic selectivity sequence determined by bi-ionic reversal potential (Na+ approximately Li+ greater than K+ greater than Rb+ greater than Cs+). However, there are striking quantitative differences in open channel conduction for channels in the presence of the two activators. Under symmetrical solution conditions, the single channel conductance for Na+ is about twice as high with BTX as with VT. Furthermore, the symmetrical solution single channel conductances show a different selectivity for BTX (Na+ greater than Li+ greater than K+) than for VT (Na+ greater than K+ greater than Li+). Open channel current-voltage curves in symmetrical Na+ and Li+ are roughly linear, while those in symmetrical K+ are inwardly rectifying. Na+ currents are blocked asymmetrically by K+ with both BTX and VT, but the voltage dependence of K+ block is stronger with BTX than with VT. The results show that the alkaloid neurotoxins not only alter the gating process of the Na+ channel, but also affect the structure of the open channel. We further conclude that the rate-determining step for conduction by Na+ does not occur at the channel's "selectivity filter," where poorly permeating ions like K+ are excluded.     

Purified and unpurified sodium channels from eel electroplax in planar lipid bilayers

Highly purified sodium channel protein from the electric eel, Electrophorus electricus, was reconstituted into liposomes and incorporated into planar bilayers made from neutral phospholipids dissolved in decane. The purest sodium channel preparations consisted of only the large, 260-kD tetrodotoxin (TTX)-binding polypeptide. For all preparations, batrachotoxin (BTX) induced long-lived single-channel currents (25 pS at 500 mM NaCl) that showed voltage-dependent activation and were blocked by TTX. This block was also voltage dependent, with negative potentials increasing block. The permeability ratios were 4.7 for Na+:K+ and 1.6 for Na+:Li+. The midpoint for steady state activation occurred around -70 mV and did not shift significantly when the NaCl concentration was increased from 50 to 1,000 mM. Veratridine-induced single-channel currents were about half the size of those activated by BTX. Unpurified, nonsolubilized sodium channels from E. electricus membrane fragments were also incorporated into planar bilayers. There were no detectable differences in the characteristics of unpurified and purified sodium channels, although membrane stability was considerably higher when purified material was used. Thus, in the eel, the large, 260-kD polypeptide alone is sufficient to demonstrate single-channel activity like that observed for mammalian sodium channel preparations in which smaller subunits have been found.     

Mouse brain synaptosomal sodium channels: activation by aconitine, batrachotoxin, and veratridine, and inhibition by tetrodotoxin

Batrachotoxin, veratridine and aconitine, activators of the voltage-dependent sodium channel in excitable cell membranes, increase the rate of 22Na+ uptake by mouse brain synaptosomes. Batrachotoxin was both the most potent (K0.5, 0.49 microM) and most effective activator of specific 22Na+ uptake. Veratridine (K0.5, 34.5 microM) and aconitine (K0.5, 19.6 microM) produced maximal stimulations of 22Na+ uptake that were 73% and 46%, respectively, of that produced by batrachotoxin. Activation of 22Na+ uptake by veratridine was completely inhibited by tetrodotoxin (I50, 6 nM ), a specific blocker of nerve membrane sodium channels. These results identify appropriate conditions for measuring sodium channel-dependent 22Na+ flux in mouse brain synaptosomes. The pharmacological properties of mouse brain synaptosomal sodium channels described here are distinct from those previously described for sodium channels in rat brain synaptosomes and mouse neuroblastoma cells.     

Spontaneous opening at zero membrane potential of sodium channels from eel electroplax reconstituted into lipid vesicles

Summary The voltage-dependent sodium channel from the eel electroplax was purified and reconstituted into vesicles of varying lipid composition. Isotopic sodium uptake experiments were conducted with vesicles at zero membrane potential, using veratridine to activate channels and tetrodotoxin to block them. Under these conditions, channel-dependent uptake of isotopic sodium by the vesicles was observed, demonstrating that a certain fraction of the reconstituted protein was capable of mediating ion fluxes. In addition, vesicles untreated with veratridine showed significant background uptake of sodium; a considerable proportion of this flux was blocked by tetrodotoxin. Thus these measurements showed that a significant subpopulation of channels was present that could mediate ionic fluxes in the absence of activating toxins. The proportion of channels exhibiting this behavior was dependent on the lipid composition of the vesicles and the temperature at which the uptake was measured; furthermore, the effect of temperature was reversible. However, the phenomenon was not affected by the degree of purification of the protein used for reconstitution, and channels in resealed electroplax membrane fragments or reconstituted, solely into native eel lipids did not show this behavior. The kinetics of vesicular uptake through these spontaneously-opening channels was slow, and we attribute this behavior to a modification of sodium channel inactivation.     

Reserpine: interactions with batrachotoxin and brevetoxin sites on voltage-dependent sodium channels

Reserpine inhibited batrachotoxin-elicited sodium influx in guinea pig brain synaptoneurosomes with an IC₅₀ of about 1 M. In the presence of brevetoxin the IC₅₀ increased to about 80 M. Reserpine inhibited binding of batrachotoxinin-A [³H]benzoate ([³H]BTX-B) binding in a complex manner causing a partial inhibition from 0.001 to 0.08 M, then a rebound stimulation from 0.1 to 0.8 M, followed by complete inhibition by 80 M. The stimulation was prevented by the presence of brevetoxin; reserpine then smoothly inhibited binding with an IC₅₀ of about 1 M. Reserpine at 1 M slightly reduced the off-rate of [³H]BTX-B binding measured in the presence of veratridine, while at a concentration of 50 M it enhanced the off-rate, presumably by an allosteric mechanism. Reserpine at 0.3–10 M elicited a partial inhibition of the binding of [³H]brevetoxin-3. The local anesthetic dibucaine had effects similar to reserpine: It partially inhibited binding of [³H]brevetoxin. The presence of brevetoxin reduced the potency of dibucaine as an inhibitor of batrachotoxin-elicited sodium influx from an IC₅₀ of about 2 M to an IC₅₀ of about 50 M. The results suggest that reserpine binds at both a local anesthetic site to cause allosteric inhibition of batrachotoxin-binding and action, but that it also binds to another site causing, like brevetoxin, an enhancement of batrachotoxin-binding and action. Local anesthetics also may bind to the brevetoxin site.     

A study of properties of batrachotoxin modified sodium channels

A further analysis of the effects of the steroidal alkaloid batrachotoxin (BTX) on sodium channels in frog node of Ranvier has been carried out under voltage-clamp conditions. The main properties of modified channels as compared with those of normal ones are as follows: The rate of channel closing is drastically decreased, whereas that of opening is changed slightly if at all; The steady-state voltage dependence of channel activation is shifted towards more negative potentials by 60-70 mV; Currents through modified channels do not show a decay during maintained depolarization as it is typical for normal channels. However modified channels retain the ability to partial inactivation as shown by experiments with depolarizing prepulses; Sodium against potassium selectivity beyond--20 mV suggesting either nonhomogeneity of the modified channels as for their kinetic and selectivity properties or potential-dependence of ionic selectivity for each channel; The selectivity sequence determined from peak current reversal potential measurements is as follows: H: Na :NH4:K = 528:1:0.47: :0.19; The effective pK value of proton block is decreased by about 0.4; 7) The sensitivity of the channels to tetrodotoxin (TTX) block is practically unchanged.     

Reconstituted voltage-sensitive sodium channels from eel electroplax: Activation of permeability by quaternary lidocaine,N-bromoacetamide,and N-bromosuccinimide

Summary We have investigated the ion permeability properties of sodium channels purified from eel electroplax and reconstituted into liposomes. Under the influence of a depolarizing diffusion potential, these channels appear capable of occasional spontaneous openings. Fluxes which result from these openings are sodium selective and blocked (from opposite sides of the membrane) by tetrodotoxin (TTX) and moderate concentrations of the lidocaine analogue QX-314. Low concentrations of QX-314 paradoxically enhance this channel-mediated flux. N-bromoacetamide (NBA) and N-bromosuccinimide (NBS), reagents which remove inactivation gating in physiological preparations, transiently stimulate the sodium permeability of inside-out facing channels to high levels. The rise and subsequent fall of permeability appear to result from consecutive covalent modifications of the protein. Titration of the protein with the more reactive NBS can be used to produce stable, chronically active forms of the protein. Low concentrations of QX-314 produce a net facilitation of channel activation by NBA, while higher concentrations produce block of conductance. This suggests that rates of modifications by NBA which lead to the activation of permeability are influenced by conformational changes induced by QX-314 binding.     

Veratridine modification of the purified sodium channel alpha- polypeptide from eel electroplax

In the interest of continuing structure-function studies, highly purified sodium channel preparations from the eel electroplax were incorporated into planar lipid bilayers in the presence of veratridine. This lipoglycoprotein originates from muscle-derived tissue and consists of a single polypeptide. In this study it is shown to have properties analogous to sodium channels from another muscle tissue (Garber, S. S., and C. Miller. 1987. Journal of General Physiology. 89:459-480), which have an additional protein subunit. However, significant qualitative and quantitative differences were noted. Comparison of veratridine-modified with batrachotoxin-modified eel sodium channels revealed common properties. Tetrodotoxin blocked the channels in a voltage-dependent manner indistinguishable from that found for batrachotoxin-modified channels. Veratridine-modified channels exhibited a range of single-channel conductance and subconductance states. The selectivity of the veratridine-modified sodium channels for sodium vs. potassium ranged from 6-8 in reversal potential measurements, while conductance ratios ranged from 12-15. This is similar to BTX-modified eel channels, though the latter show a predominant single-channel conductance twice as large. In contrast to batrachotoxin-modified channels, the fractional open times of these channels had a shallow voltage dependence which, however, was similar to that of the slow interaction between veratridine and sodium channels in voltage-clamped biological membranes. Implications for sodium channel structure are discussed.     

Biosynthesis of electroplax sodium channels in Electrophorus electrocytes and Xenopus oocytes

We have synthesized the eel electroplax sodium channel core polypeptide in both a cell-free and a frog oocyte system and report it does not possess the unusual electrophoretic properties of the mature, native sodium channel polypeptide isolated from electroplax membranes. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the mature channel polypeptide exhibits both a diffuse banding pattern (microheterogeneity) and an extremely high electrophoretic free mobility. In contrast, the core polypeptide synthesized in vitro or in vivo migrates as a sharp band with a near-normal electrophoretic free mobility (Mr 230,000). The microheterogeneity of the mature peptide has been inferred to result from varying degrees of glycosylation of the channel polypeptide [Miller, J.A., Agnew, W.S., & Levinson, S.R. (1983) Biochemistry 22, 462-470]. We present evidence here that the anomalously high electrophoretic free mobility is due to the binding of large amounts of sodium dodecyl sulfate to posttranslationally modified domains on the protein. In addition, we have followed the posttranslational processing of eel sodium channels in both the eel electrocyte and the frog oocyte. Using lectin binding and Ferguson analysis, we found that the channel was processed relatively rapidly to an intermediate form in the Golgi apparatus that apparently contained fewer carbohydrate and hydrophobic domains than the mature channel. The further addition of carbohydrate and hydrophobic domains, which are required before the channel acquires its characteristic physicochemical properties, proceeded relatively slowly in the electrocyte and appeared not to have occurred to the majority of intermediately processed channels in the frog oocyte.     

Sodium channels in planar lipid bilayers. Channel gating kinetics of purified sodium channels modified by batrachotoxin

Single channel currents of sodium channels purified from rat brain and reconstituted into planar lipid bilayers were recorded. The kinetics of channel gating were investigated in the presence of batrachotoxin to eliminate inactivation and an analysis was conducted on membranes with a single active channel at any given time. Channel opening is favored by depolarization and is strongly voltage dependent. Probability density analysis of dwell times in the closed and open states of the channel indicates the occurrence of one open state and several distinct closed states in the voltage (V) range-120 mV less than or equal to V less than or equal to +120 mV. For V less than or equal to 0, the transition rates between stages are exponentially dependent on the applied voltage, as described in mouse neuroblastoma cells (Huang, L. M., N. Moran, and G. Ehrenstein. 1984. Biophysical Journal. 45:313-322). In contrast, for V greater than or equal to 0, the transition rates are virtually voltage independent. Autocorrelation analysis (Labarca, P., J. Rice, D. Fredkin, and M. Montal. 1985. Biophysical Journal. 47:469-478) shows that there is no correlation in the durations of successive open or closing events. Several kinetic schemes that are consistent with the experimental data are considered. This approach may provide information about the mechanism underlying the voltage dependence of channel activation.     

An asymmetrical kinetic model for veratridine interactions with sodium channels in molluscan neurons

Veratridine alkaloid induces bi-stability or saw-tooth-shaped long potential waves in molluscan neurons. Voltage clamp experiments reveal the production of a slow sodium current whose changes are described by an asymmetric kinetic diagram relating the states of the sodium channels. Methods of the qualitative theory of differential equations were used to determine the condition necessary for such a model to have either an oscillatory solution or a bi-stable behavior. The kinetic diagram was modified to account for the frequency dependence of the slow sodium current production upon repeated short depolarizations. The modified kinetic diagram suggests that open and inactivated sodium channels are turned into channels with slow kinetic parameters; the transition from open channels would be fast, irreversible and restricted to part of the open channels, whereas that from inactivated channels would be slow and fully reversible upon repolarization.     

A comparison of eel electroplax and snake venom acetylcholinesterase

The effects of some cholinergic ligands, harmala alkaloids and local anesthetics on the activity of eel electroplax and Naja naja siamensis venom acetylcholinesterase have been studied. In most cases, eel electroplax was found to be more susceptible towards inhibition than the venom acetylcholinesterase. No major difference was observed with respect to the type of inhibition in both enzymes. The activation of the two enzyme preparations by inorganic cations (Ca2+, Mg2+ and Na+) showed a similar pattern. In both preparations, the onset of activation was detectable at much lower concentration with the divalent metal ions than with the monovalent Na+. Antagonism between Ca2+ and decamethonium, tubocurarine and tetracaine in both enzymes approached competitive kinetics. The onset of substrate inhibition is delayed by Ca2+ (30 mM) in both enzymes. It is suggested that the Ca2+ binding site overlaps with the substrate inhibitory site. It is concluded that cobra venom acetylcholinesterase has similar allosteric binding sites to those of eel electroplax.     

Neuraminidase treatment modifies the function of electroplax sodium channels in planar lipid bilayers

Sodium channels from several sources are covalently modified by unusually large numbers of negatively charged sialic acid residues. In the present studies, purified electroplax sodium channels were treated with neuraminidase to remove sialic acid residues and then examined for functional changes in planar lipid bilayers. Neuraminidase treatment resulted in a large depolarizing shift in the average potential required for channel activation. Additionally, desialidated channels showed a striking increase in the frequency of reversible transitions to subconductance states. Thus it appears that sialic acid residues play a significant role in the function of sodium channels, possibly through their influence on the local electric field and/or conformational stability of the channel molecule.     

Activation of the action potential Na+ ionophore of cultured neuroblastoma cells by veratridine and batrachotoxin.

The activation of the action potential Na+ ionophore by veratridine and batrachotoxin is time- and concentration-dependent and completely reversible. Batrachotoxin acts more slowly than veratridine. The concentration dependence of activation at equilibrium suggests reversible interaction of each toxin with a single class of independent sites having dissociation constants at physiologic ion concentrations of 80 plus or minus 13 muM for veratridine and 0.4 plus or minus muM for batrachotoxin. The maximum velocity of Na+ uptake at 50 mM Na+ is 128 plus or minus 12 nmol/min/mg in the presence of batrachotoxin compared to 48 plus or minus 4 nmol/min/mg in the presence of veratridine. Treatment of cells with excess veratridine in addition to batrachotoxin inhibits batrachotoxin-dependent 22-Na+ uptake. The concentration dependence of this inhibition suggests that it reflects competitive displacement of batrachotoxin from its binding site by veratridine. The activation by veratridine and batrachotoxin is inhibited in a competitive manner by divalent cations. The inhibition by divalent cations exhibits significant ion specificity with Mn-2+ greater than Co-2+ greater than Ni-2+ greater than Ca-2+ greater than Mg-2+ greater than Sr-2+. The inhibition constants (KI) for Ca-2+ are 0.84 mM for veratridine-dependent 22-Na+ uptake and 1.2 mM for batrachotoxin-dependent 22-Na+ uptake. The activation by veratridine and batrachotoxin is inhibited in a noncompetitive manner by tetrodotoxin. The apparent KD for tetrodotoxin as 11 plus or minus 1 nM in the presence of 150 mM Na+ and approximately 8.5 nM in 50 mM Na+. Divalent cations do not affect the apparent KD for tetrodotoxin. A hypothesis is presented which suggests that batrachotoxin, veratridine, and divalent cations interact with an activation site associated with the action potential Na+ ionophore, whereas tetrodotoxin interacts with a physically and functionally independent site involved in the transport of monovalent cations by the ionophore.     

Pharmacological modifications of the sodium channels of frog nerve

Voltage clamp measurements on myelinated nerve fibers show that tetrodotoxin, saxitoxin, and DDT specifically affect the sodium channels of the membrane. Tetrodotoxin and saxitoxin render the sodium channels impermeable to Na ions and to Li ions and probably prevent the opening of individual sodium channels when one toxin molecule binds to a channel. The apparent dissociation constant of the inhibitory complex is about 1 nM for the cationic forms of both toxins. The zwitter ionic forms are much less potent. On the other hand, DDT causes a fraction of the sodium channels that open during a depolarization to remain open for a longer time than is normal. The effect cannot be described as a specific change in sodium inactivation or as a specific change in sodium activation, for both processes continue to govern the opening of the sodium channels and neither process is able to close the channels. The effects of DDT are very similar to those of veratrine.     

Voltage activation of purified eel sodium channels reconstituted into artificial liposomes

We report here a characterization of the voltage-activated behavior of sodium channels purified from the electroplax of Electrophorus electricus. Single-channel activity in response to depolarizing pulses was recorded from patches excised from liposomes containing the reconstituted channel. Strong hyperpolarizations were required to elicit channel activity. Channels exhibited two typical gating patterns. They either would open in brief bursts upon depolarization and then inactivate (fast) or would stay opened for prolonged periods that frequently lasted several consecutive depolarizations and showed intense flickering (slow). The single-channel conductance estimated from the slope of the I-V curves ranged between 15 and 30 pS under several experimental conditions. Channels gating in either mode, fast or slow, were indistinguishable in terms of their sizes. No clear difference in their mean open times was observed. In addition to the two gating patterns, we also found a very clear tendency of the channels to stay quiet for long periods.