Effects of veratridine on sodium currents and fluxes.

Veratridine causes Na+ channels to stay open during a sustained membrane depolarization by abolishing inactivation. The consequential Na+ influx, either by itself or by causing a maintained depolarization, leads to many secondary effects such as increasing pump activity, Ca2+ influx, and in turn exocytosis. If the membrane is voltage clamped in the presence of the alkaloid, a lasting depolarizing impulse induces, following the "normal" transient current, another much more slowly developing Na+ current that reaches a constant level after a few seconds. Repolarization then is followed by an inward tail current that slowly subsides. Development of these slow currents is enhanced by additional treatment with agents that inhibit inactivation. Most of these phenomena can be satisfactorily explained by assuming that Na+ channels must open before veratridine binds to them, and that the slow current changes reflect the kinetics of binding and unbinding. It is unclear, however, where the alkaloid stays when it is not bound. Although the effect sets in promptly, once this pool is filled, access to it from outside must be impeded since in most preparations veratridine can only partially be washed out. Cooling acts as if the available concentration is reduced, but this reversible "reduction" takes much longer to develop than the cold-induced changes in kinetics. Several authors assume that the binding site, site 2, is accessed from the lipid phase of the membrane. Considerations of this kind are often based on experiments with batrachotoxin, the widely used site-2 ligand which has a much higher affinity and acts as a full agonist in contrast to the partial agonist veratridine. Batrachotoxin thus lends itself to binding studies using radiolabeled derivatives. Such experiments may eventually lead to the characterization of neurotoxin site 2; the first promising steps have been taken. Modern techniques of molecular biology will almost certainly be successful, and one hopes for point-mutated channels with distinctly different reactions also to veratridine. A considerable amount of research is still required to clarify the structural basis for the numerous allosteric interactions with other sites, the mechanism of the very large potential shift of activation, the reduced single-channel conductance and selectivity, and the chemical nature of the different affinities of the site-2 toxins. Note Added in Proof. A report on point mutations with effects on neurotoxin site 2 (see Sect. 8) has just appeared: Wang S-Y, Wang GK (1988) Point mutations in segment I-S6 render voltage-gated Na+ channels resistant to batrachotoxin. Proc Natl Acad USA 95:2653-2658. In microliter muscle Na+ channels expressed in mammalian cells, mutation Asn434Lys leads to complete, Asn434Ala to partial insensitivity to 5 mM batrachotoxin. (Asn434 corresponds to Asn419 of Trainer et al. 1996). The mutant channel displays almost normal current kinetics and in the presence of veratridine little, if any, slow tail current. However, veratridine inhibits peak Na+ currents in the mutant which may point to a complex structure of site 2.

The inactivation of sodium channels in the node of Ranvier and its chemical modification.

The many experimental studies reported demonstrate the complexity of what is termed inactivation, the decrease of current flow through sodium channels at maintained depolarization. Even at the normal resting potential of, say, -70 mV for a frog node of Ranvier, ca. 20% of the channels are closed and inactivated, i.e., incapable of passing current on a sudden depolarization, in contrast to the remaining 80% of closed but resting channels. The term inactivation has thus evolved from bulk current ("macroscopic") phenomena and is applied to channels although its single-channel ("microscopic") basis is not entirely clear and may even vary among preparations. It is conceivable that the macroscopic phenomenon may have more than a single microscopic cause; this point will probably not be settled until a physical description of the conformational states of the channel macromolecule becomes available. At any rate, channel transition into an inactivated closed state can be easily affected by numerous reagents of highly diverse chemical nature and, most likely, different primary sites of action as already suggested by the sidedness of effective application, e.g., iodate and endopeptidases to the inside, polypeptide toxins to the outside. But also the search for a common denominator, a secondary target of all these treatments, has not been very successful as demonstrated by the experiments with group-specific reagents. Since modification of inactivation is often accompanied by shifts in the voltage dependence of gating parameters, a target could be the "voltage sensor" of the channel, charged and/or dipolar components of the channel macromolecule that, by being moved in the electric field, somehow induce gating and whose movement is measured as gating current (e.g, Hille, 1984). The fraction of open channels as a function of membrane potential, F(E), may serve as an indicator. It may be simply shifted (to more negative potentials) as by veratridine (Leibowitz et al., 1987) or flattened (reduction of gating charge?) and shifted (in the positive direction) as by Anemonia sulcata toxin II (Ulbricht and Schmidtmayer, 1981) or chloramine-T (Drews, 1987). On the other hand, the steady-state inactivation curve is shifted to more negative potentials by the toxin (Ulbricht and Schmidtmayer, 1981), but to more positive potentials by chloramine-T (Wang, 1984a; Schmidtmayer, 1985). Obviously, modifiers may affect activation and inactivation quite differently, a result that touches on the question as to what extent inactivation derives its potential dependence from activation.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

Interaction of lidocaine and benzocaine in blocking sodium channels.

1. Single myelinated nerve fibres of the frog, Rana esculenta, were investigated in voltage and current clamp experiments at pH 7.2 2. Measured with infrequent test pulses, 0.123 mM lidocaine reduced INa to 54%, 0.25 mM benzocaine to 40% and the mixture 0.125 mM lidocaine +/- 0.25 mM benzocaine to 31% of the control. When hyperpolarizing prepulses (V = -40 mV for 15 ms) preceded the test pulses the respective reductions were to 58%, 74% and 55% i.e. adding benzocaine to lidocaine had little additional effect. 3. Increasing the rate of the prepulse-test pulse pairs from 1 to 20 Hz did not change INa in benzocaine but gradually relieved block by lidocaine; in the mixture this change was much reduced or absent. 4. Switching off prepulses (at 20 Hz) led to a gradual decrease of INa in lidocaine but to a prompt fall in benzocaine and in the mixture. 5. 0.25 mM lidocaine and 0.5 mM benzocaine were approximately equieffective in reducing INa (no prepulse) to 29% and 24%; a one-to-one mixture of the two solutions (0.125 mM lidocaine + 0.25 mM benzocaine) reduced to 27%. 6. In current clamp experiments 0.25 mM lidocaine and 0.36 mM benzocaine reduced the maximum rate of rise of the action potential to 32% and 30%, the mixture of solutions (0.125 mM lidocaine + 0.18 mM benzocaine) to 29%. 7. These results are fully compatible with the idea of a single common binding site for which lidocaine and benzocaine compete.

Action of benzocaine on sodium channels of frog nodes of Ranvier treated with chloramine-T.

Single myelinated nerve fibres of the frog Rana esculenta were voltage clamped in solutions containing 10 mM TEA to block potassium channels. Reduction of peak INa was measured after equilibrating the membrane in solution containing benzocaine between 0.025 and 2.0 mM. A Hill plot of this effect revealed a slope nH approximately 1 between 0.25 and 1.0 mM but showed deviations for the lowest concentrations (nH less than 1) and the highest concentration (nH greater than 1). Treatment with 0.6 mM chloramine-T irreversibly partially inhibited inactivation of sodium channels leading to a large INa component persisting during depolarization. After treatment the benzocaine effect on peak INa (tested with 0.25-1.0 mM) was unchanged but the persistent component was much more depressed. Benzocaine shifted the steady-state inactivation curve to more negative potentials. This was also observed after chloramine-T treatment which itself produced a curve of decreased slope, shifted to more positive potentials. Recovery from inactivation was studied at different levels of hyperpolarization; it was diphasic in anaesthetic-free solutions before and after chloramine-T treatment although slowed in the latter case. In benzocaine recovery started with a delay (less than 0.6 ms at 16-18 degrees C) and proceeded with a single time constant that decreased with increasing hyperpolarization, was independent of benzocaine concentration and not affected by chloramine-T. The results are compatible with the idea that the affinity of the binding site for benzocaine increases when the channel state changes from resting to open to inactivated with equilibrium dissociation constants of the reaction with resting channels, KR = 0.7 mM and with inactivated channels, KI = 0.04 mM.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of Na+ and Li+ ions on the kinetics of sodium channel block by tetrodotoxin and saxitoxin.

Voltage clamp experiments were done on single myelinated frog nerve fibres. The rate of block of Na+ channels by tetrodotoxin (TTX) was obtained from changes in peak Na+ current during 1-Hz trains of depolarizing impulses. In hypertonic Na(+)-rich solution (216 mM) the stationary block was reduced compared with Na(+)-poor solutions (54 mM or less; tetramethylammonium ions substituting for Na+). Washout in 216 mM Na+ was faster than in 54 mM Na+. Concentration of Na+ [( Na+]) little affected onset of block. After equilibration in Na(+)-poor TTX solution, a sudden application of Na(+)-rich toxin solution led to a partial relief from block that proceeded faster than the onset in the latter solution. Comparable results were obtained with saxitoxin (STX) and in analogous Li+ solutions. Most of the observed phenomena could be quantitatively fitted by a cyclic model in which cations favour the transition of channels (unblocked and blocked) from a high- to a low-affinity state from which toxin dissociates faster.

[Migraine accompagnée]. / Migraine accompagnée.

The symptomatology of migraine accompagnée is described. The order of the symptoms, the suspected localisation and the possibility of permanent defects, heredity and treatment are discussed. The results obtained by traditional methods of examination such as blood flow measurements, arteriography and EEG are presented, as are those of more modern techniques such as skull rheography and computed tomography.

[Adipocyte volume and count in obesity during weight reduction]. / Untersuchungen von Adipositasvolumen und -zahl bei Adipositas während Gewichtsreduktion.

In the subcutaneous fatty tissue of 27 adipose patients the number and volume of adipocytes were determined before and after a stationary weight reduction. 47 normal test persons served as comparison. Under stationary weight reduction the volume of adipocytes significantly decreased, whereas the number of adipocytes remained constant. The subdivision of the obesity according to the cellularity of the fatty tissue into a hypertrophic and a hypertrophic hypercellular form is proposed. A possible indication for the determination of the volume of fat cells in adipose patients is given.

[Behavior of the volume and number of fat cells in obese patients in middle age in inpatient weight reduction]. / Verhalten des Volumens und der Zahl der Fettzellen bei adipösen Patienten im mittleren Lebensalter unter stationärer Gewichtsreduktion.

Fat cell volume and fat cell quantity were determined in subcutaneous fatty tissue of 27 obese patients (22 female and 5 male aged 42 +/- 8) before and after in-patient weight reduction. 47 normal probands of the same age served as a control group. With in-patient weight reduction, fat cell volume decreased significantly, while fat cell quantity remained constant. Division of obesity into hypertrophical and hypertrophical-hypercellular types based on fatty tissue cellularity is suggested. Possible indication for the determination of fat cell volume in obese patients is shown.

The reaction between tetrodotoxin and membrane sites at the node of Ranvier: its kinetics and dependence on pH.

Voltage clamp experiments were done on single nodes of Ranvier to study the inhibition of the sodium permeability by tetrodotoxin (TTX). Equilibrium results could be excellently fitted on the assumption that a sodium channel is blocked when one toxin molecule binds to it, the equilibrium dissociation constant, KT of this reaction being 3.6 nm at 20 degrees C. Onset and offset of block could be quantitatively interpreted to be determined by the rates of the TTX-channel reaction whose average constants, at room temperature, were 3 times 10-6M-minus 1s-minus 1 for the association (k1) and 1.4 times 10-minus 2 s-minus 1 for the dissociation (k2). The dependence of the constants on temperature could be described by Arrhenius plots yielding activation energies of 29.3, 85.5 and 41.0 (57.3) kJ/mol for KT, k2 and k1 (K1 derived from onset alone), respectively. At low pH the relative TTX effect was clearly less than at neutral pH. These results could be explained by a model involving the competition of TTX and protons for the same receptor to which protons bind as a function of membrane potential.

The influence of pH on equilibrium effects of tetrodotoxin on myelinated nerve fibres of Rana esculenta.

1. The experiments were done on single nodes of Ranvier of Rana esculenta. The effects of tetrodotoxin and H ions were determined either by the reduction of the maximum rate of rise, VA, of action potentials evoked with threshold stimuli or in the voltage clamp by the decrease of the peak Na permeability, PNa. 2. With the tetrodotoxin sample used throughout the investigation the equilibrium dissociation constant, KT, of the toxin-receptor reaction at neutral pH was determined to be 2-8 nM. Between 1-55 and 15-5 nM tetrodotoxin the normalized value, A, of VA, was found to be related to the normalized toxin concentration cT = [TTX]/2-8 nM by the empirical equation log [(1-A)/A] = 1-22 log cT-0-573. 3. On increasing the pH (up to 8-8) the effect of tetrodotoxin diminished as revealed by an increase in A. The apparent reduction of cT (as calculated from A) suggests that the toxin is active only in its cationic forms. 4. Weakly acid tetrodotoxin solutions (7-3 less than pH less than or equal to 5-5) reduced A to a lesser degree than did neutral toxin solutions in spite of the inherent depressing effect of acid pH on A (A = 0-5 at about pH 5-5). In more acid toxin solutions A decreased again and at pH 4-6 it was about equal to the value in toxin-free solution. 5. When, after equilibrium in an acid toxin solution, the perfusate was suddenly changed to neutral Ringer solution A jumped to a higher value A' as measured 1 sec after the switch. Since the blocking effect of hydrogen ions subsided within a fraction of a second while the time constant of the toxin washout is of the order of 1 min, A' reflects the number of Na channels blocked by tetrodotoxin at acid pH. 6. In acid toxin-free solution the peak PNa as obtained in voltage clamp experiments was reduced by a voltage-dependent factor (cH + 1)-1 with CH = [H+]/KH(E) and KH(E) = 2-04 muM exp (0-34 EF/RT). Adding tetrodotoxin resulted in another reduction by a constant factor p'T. 7. Experiments employing various combinations of toxin concentration (3-1-93 nM) and pH values (7-3-5-2) confirm the decreased toxin effect at low pH. Moreover, p'T was smaller (the additional toxin effect larger) when the membrane had been kept depolarized and thus cH reduced during equilibration. This suggests that tetrodotoxin cations and H ions compete for the same blocking site. A quantitative fit, however, requires additional assumptions.

The influence of pH on the rate of tetrodotoxin action on myelinated nerve fibres.

1. The experiments were done on single myelinated nerve fibres of Rana esculenta. The rates of toxin effect were studied either by measuring the maximum rate of rise, VA, of repetitively evoked action potentials or by measuring Na currents during periodic impulses in the voltage clamp. 2. VA measurements showed that in alkaline solutions (pH up to 8-8) the offset rate was unchanged while the onset was slowed in quantitative agreement with an assumed decrease in the active cationic form of tetrodotoxin. 3. Both VA measurements and those in the voltage clamp revealed a decrease in T'off, the offset time constant and in increase in the onset time constant, T'on, as the pH was lowered. 4. For tetrodotoxin concentrations, [TTX], up to 400 nM and pH values down to 5-3 the simple relation T'on/T'off = p'R held, where p'T is the constant factor by which the Na permeability was reduced at equilibrium with a given [TTX]. 5. The agreement between kinetic and equilibrium results was also valid when, at constant [TTX] and pH. p'T was modified by the holding potential during equilibration. 6. No unequivocal explanation of the results can be given but some of their features resemble acid catalysis.

The rates of saxitoxin action and of saxitoxin-tetrodotoxin interaction at the node of Ranvier.

Voltage clamp experiments were done on single nodes of Ranvier of Rana esculenta. Equilibrium effects were obtained from INa-V curves, the rates of action from changes in INa on changing solutions during repetitive depolarizing pulses. 2. Saxitoxin (STX) exclusively and reversibly blocked Na channels, the effect being fully described by a one-to-one reaction between STX and a receptor at the channel with an equilibrium dissociation constant, Ks, of 1.4 nM; the mean offset rate constant k2s, was 1.76 X 10(-2) sec-1 (16 +/- 1 degree C;pH 7.2), 1.7 times the value for tetrodotoxin (TTX). 3. At pH 5.6, K2S WAs increased by a factor of 1.33 while the equilibrium STX effects was decreased in a way suggesting competition between STX and protons. 4. After pretreatment of nodes with 3.1 nM TTX the extra block on adding 9.0 nM STX as well as its relief on taking out STX of the TTX-STX mixture revealed transients in the time course of receptor occupation. 5. These non-monotonic time courses are incompatible with the idea of two independent blocking sites (for STX and TTX) per channel but could be quantitatively fitted by analog-computed curves assuming competition between STX and TTX for the same site.

Block of potassium channels of the nodal membrane by 4-aminopyridine and its partial removal on depolarization.

1. Voltage clamp experiments were done on single myelinated nerve fibres of the frog, Rana esculenta. 2. 53 muM 4-aminopyridine (4-AP) reduced IK to about one-fifth if tested with infrequent (1/min) and short (10 ms) depolarizing pulses; the onset time constant under these circumstances was ca. 160 s (14-15 degrees C). After prolonged treatment the effect was virtually irreversible. 3. At equilibrium with 4-AP, increasing the frequency of short pulses removed part of the block, the block removal accelerating with increasing pulse duration and frequency. 4. In 53 muM 4-AP unblocking of K channels during long (0.8 s) depolarizing pulses proceeded with a time constant, taur, of ca. 0.2 s. Restoration of block at the resting potential proceeded with a much larger time constant, tau'r, of ca. 1 min. 5. The stationary fraction, rinfinity, of K channels conducting in 53 muM 4-AP was 0.66, 0.41, and 0.24 at V = 120, 50, and 0 mV, respectively. 6. In a series of experiments with [4-AP] varying between 13.3 and 848 muM, taur decreased from 0.25 to 0.10 s (V = 130 mV, ca. 17 degrees C) while rinfinity followed the empirical relation 1/rinfinity = 1 + ct + cv exp(-0.77 EF/RT) with E = V - 70 mV. ct and cv are dimensionless quantities that increase with [4-AP] and reflect the voltage-independent and voltage-dependent component, respectively, of block. 7. Block of K channels and partial removal are also observed with inward IK at raised [K+]O. Removal proceeds on depolarization even if IK is additionally but temporarily suppressed by tetraethylammonium. Hence neither direction nor amplitude of IK but only the pulse potential seems to determine the extent of block for a given [4-AP].

Saxitoxin and procaine act independently on separate sites of the sodium channel.

1. Voltage clamp experiments were done on single myelinated nerve fibres of the frog, Rana esculenta. 2. The time course of procaine action (1.0 mM at pH 7.2) was obtained from changes in INa on changing solutions during repetitive (1 HZ) depolarizing pulses of constant amplitude following hyperpolarizing prepulses. The mean half times of onset and offset of procaine block were 3.7 and 28 s, respectively. In the presence of 1.4 nM saxitoxin (STX) the corresponding times were virtually the same, 3.1 and 27 s. 3. Similarly, the time course of partial relief from procaine block that is obtained by increasing the frequency of the prepulse-test pulse pairs from 1-10 HZ was unaffected in the presence of STX. 4. Comparison of the equilibrium effects of procaine concentrations ranging from 0.03-1.0 mM suggest a one-to-one drug-receptor reaction. The fraction of Na channels blocked at equilibrium with 1.0 mM procaine, 1.4 nM STX, and 1.0 mM procaine + 1.4 nM STX was 0.81, 0.49, and 0.90, respectively. This result and the kinetic behaviour fully agree with the idea of two separate and independent receptors for procaine and STX.

Chloramine-T effect on sodium conductance of neuroblastoma cells as studied by whole-cell clamp and single-channel analysis.

Patch-clamp experiments were done on sodium channels of neuroblastoma cells (N1E-115) in the presence of tetraethylammonium ions to block potassium channels. In Ringer solution whole-cell records revealed a diphasic INa inactivation with the fast (tau 0) component. being clearly larger than the slow (tau 1 approximately 3 tau 0) component. In single-channel studies on inside-out patches the mean open time, to, turned out to be only a fraction of tau 0 and almost independent of membrane potential. After external application of chloramine-T INa inactivation of whole cells was delayed with both tau 0 and tau 1 increased, and incomplete, i.e. a persistent current component emerged. The latter was maximal at a more positive membrane potential than the peak current. Also, after chloramine-T treatment the peak INa increased, particularly at weak depolarizations. In inside-out patches the equally effective internal application of chloramine-T led to bursting channel openings with mean burst times (tb) approximately 6 ms, and gap times (tg) approximately 20 ms, where gap is defined as a closure of greater than or equal to 1.5 ms. Within the bursts to was approximately 2 ms, again clearly shorter than tau 0; the mean close time, tc was approximately 0.5 ms. The single-channel conductance was approximately 13 pS and unaffected by chloramine-T. Diphasic INa inactivation and the fact that to less than tau 0 led to an extension of the model of Aldrich and Stevens [J Neurosci 7:418-431 (1987)], in which overall kinetics is determined by the openings rather than closures of the sodium channels.(ABSTRACT TRUNCATED AT 250 WORDS)

Distinctly different rates of benzocaine action on sodium channels of Ranvier nodes kept open by chloramine-T and veratridine.

Single myelinated nerve fibres of the frog, Rana esculenta, were voltage clamped in a fast-exchange chamber in the presence of 10 mM TEA to block potassium channels. After treatment with 0.6 mM chloramine-T for 1-4 min a sizeable INa component persisted even during a 14-s depolarizing impulse. Changing the perfusate to Ringer solution + 1 mM benzocaine resulted in a fast reduction (half time ca. 0.06 s) of the persistent INa, comparable to the rate of block of peak INa during a series of short impulses before chloramine-T. In the presence of 60 microM veratridine the peak INa was followed by a slow exponential (tau s) reincrease of inward current, Is, that did not appreciably inactivate. Application of 0.25 mM benzocaine during a 14-s depolarizing impulse caused Is to decrease exponentially with a large time constant, tau on of 4.3 s. Recovery on washout proceeded with tau off = 3.4s. Tau on was little dependent on benzocaine concentration and was 4.5 s on the average in 1 mM. Tau on in 25 microM was insignificantly (15%) larger than in 1 mM if tested on the same fibre. After equilibration in 25 microM, 0.25 mM and 1 microM, Is(t = 14s) was reduced to 0.69, 0.30, and 0.10, respectively, of the value without anaesthetic. Cooling by only 4-5 degrees C reduced Is and much increased tau s. tau on (1 mM benzocaine) increased almost in proportion to tau s. Tail currents during a series of pulses (1.1 s every 2.5 s) were reduced by 0.25 mM benzocaine clearly faster (tau on = 1.3 s) than Is during a long pulse of the same amplitude.(ABSTRACT TRUNCATED AT 250 WORDS)