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Effects of strychnine on the potassium conductance of the frog node of Ranvier.

The nature of the block of potassium conductance by strychnine in frog node of Ranvier was investigated. The block is voltage-dependent and reaches a steady level with a relaxation time of 1 to several ms. Block is increased by depolarization or a reduction in [K+]O as well as by increasing strychnine concentration. A quaternary derivative of strychnine produces a similar block only when applied intracellularly. In general and in detail, strychnine block resembles that produced by intracellular application of the substituted tetraethylammonium compounds extensively studied by C.M. Armstrong (1969. J. Gen Physiol. 54:553-575. 1971. J. Gen. Physiol. 58:413-437). The kinetics, voltage dependence, and dependence on [K+]O of strychnine block are of the same form. It is concluded that tertiary strychnine must cross the axon membrane and block from the axoplasmic side in the same fashion as these quaternary amines.


INTRODUCTION
Strychnine recently has been shown to affect both sodium and potassium currents in the squid axon membranes (Shapiro et al., 1974). Earlier experiments had shown that strychnine has some effects on the action potentials in toad myelinated fibers (Maruhashi et al., 1956). This present paper is intended to extend the study to the effects of strychnine on a voltage-clamped frog node of Ranvier. It is further designed to answer several questions raised by the previous work on squid axons. First is whether strychnine produces its voltage-and timedependent block of potassium channels in a manner resembling that of certain quaternary ammonium ions (Armstrong, 1971). These ions act from the inside of the axon, moving into potassium channels after the channels are open for ion flow. The block is counteracted by the external potassium ions. These compounds act similarly on frog nodes of Ranvier (Armstrong and Hille, 1972). Armstrong (1971) has estimated rate constants for the blocking reaction. The present paper reports similar experiments and analyses to compare the details of strychnine blockage with that produced by the quaternary ammonium (QA) ions.

MATERIALS AND METHODS
The methods for voltage clamping the frog (Rana pipiens) node of Ranvier are those of Hille (1971). This is a modification of the voltage clamp apparatus of Dodge and  Frankenhaeuser (1958) and involves the use of four fluid pools separated by three Vaseline gaps. Two high-bandwidth, high-gain feedback amplifiers control the voltage in two of the pools. Current is measured by the change in voltage in one of the end pools. The internode in that pool serves as a resistive lead and the conversion of measured pool voltage into current depends on measurement of the resistance of that lead.
Since the axon is cut in the two end pools, the internal composition at the start of the experiment can be varied by altering the contents of these pools. As a rule, the axon is cut in isotonic (120 mM) KC1. In a few experiments N-methylstrychnine (NMS) was added to one end pool after initial voltage clamp data were obtained. In these cases the axon in the current-passing pool was recut in a new solution containing NMS. Since the resistance of the current-passing pathway was changed by this procedure, recalibration was necessary. Several minutes are required for the NMS to diffuse the approximately 500 ~m from the cut end to the region inside the node.
The standard Ringer's solution bathing the node contained 115 mM NaC1, 2 mM KCI, 2 mM CaCI2, and 2 mM Tris (hydroxymethyl) aminomethane buffer, pH 7.4. In high-KC1 solutions, some of the sodium was replaced by potassium. The external calcium and buffer concentrations were maintained throughout. Experiments on potassium currents were performed with 200 nM tetrodotoxin (TTX) (Sigma Chemical, Corp., St. Louis, Mo.). Contamination with the sodium channel current was minimal. Leakage current, along with some capacity current, was subtracted electronically in the manner of Armstrong and Hille (1972). The measured leakage value was used to obtain the resistance of the axon lead from the current pool. All measurements were at 10°C.
Strychnine base and strychnine sulfate were obtained from ICN K & K Laboratories Inc., Plainview, N.Y. Strychnine contains a tertiary nitrogen with a pKa of 7.45 in 120 mM KC1. We had previously postulated (Shapiro et al., 1974) that strychnine acts in the charged form on the inside of the membrane. Since strychnine is effective when applied externally we proposed that it crosses the membrane, probably largely or entirely in the uncharged form. In order to test this hypothesis I synthesized a simple quaternary derivative, N-methylstrychnine by dissolving the free base in a large excess of iodomethane (Eastman Kodak Corp., Organic Chemicals Div., Rochester, N.Y.). The reaction occurs at room temperature and yields a fine precipitate which was filtered and washed copiously with cold methanol. The precipitate was the~a dissolved in water and an aliquot was titrated. No group with a pKa between pH 5 and pH 9 was found. The precision of the titration was such that unreacted strychnine must have been <0.1% of the product.

Strychnine Blocks Potassium Channels
The block of potassium channels by strychnine closely resembles that seen in squid axons. A typical family of currents following steps from -80 mV to more positive potentials is seen in Fig. 1. For depolarizations to positive membrane potentials, the currents have a characteristic shape, first rising to a peak with kinetics close to that of control currents and then declining with an exponential time course to a low plateau level. The heights of the peaks and plateaus as well as the exponential decay rates are all functions of step voltage.
In an earlier paper on squid giant axons (Shapiro et al., 1974) the parameter extracted from such records oflK was the ratio IK peak/IK control (steady state) as a function of E m. However, if the dip in IK is due to some first-order or pseudofirst-order block of the channels by strychnine, then the plateau level of IK is a measure of the equilibrium value of the block, and it is the ratio of these steady currents rather than the ratio o f peak currents which is of theoretical interest. T h e peak IK value is a function of the equilibrium level of the block as well as of the relative time constants of this block and of the potassium activation.
A plot o f the steady-state currents before and after application of 1 mM strychnine is shown in Fig. 2 and a plot o f the calculated potassium conductances for the same e x p e r i m e n t is shown in Fig. 3. T h e block is voltage d e p e n d e n t . At this strychnine concentration there is little block of IK at negative inside potentials. In contrast, with the highest depolarizations there is a 90% block OflK. T h e shape of the IK-Em curve after strychnine is variable. T h e magnitude of a negative slope region (as in Fig. 2) depends on the axon. However, nearly all axons exhibit a negative slope conductance above 100 mV, and often above 40 mV (Fig. 2). T h e shape of the GK-Em curve after strychnine is more consistent. T h e r e is always a segment over which GK decreases approximately exponentially with increasing depolarization. We define Y= as the fraction of unblocked channels in the steady state. T h e n the steady-state block with strychnine is 1 -Y0~. T h e voltage d e p e n d e n c e of block is graphed in Fig. 4.
In this and all other cases the observations are fit reasonably well by equations of the form: where Era, EA, and EB are in millivolts; EA and EB are constants which vary slightly from node to node; EA is the value o f the midpoint of the sigmoid curve; and EB is inversely related to the steepness of the sigmoid. T h e continuous line in Fig. 4 is the solution of Eq. (1) with EA = 46 mV and EB = 38 inV.
T h e constants were calculated for seven axons bathed in 1 mM strychnine and the results are tabulated in Table I  tion of molecules at a blocking site in the channel, 1 -Y=, is related to potential by the following equation: where W is the work required to move the strychnine from the axoplasm to the   blocking site when Era = 0; z is the valence of strychnine; e is the electron charge; is the fraction of the membrane field through which strychnine must be moved to block the channel; k is Boltzmann's constant; and T is the absolute temperature. At 10°C, kT/ze = 25 InV.
Increasing the strychnine concentration decreases EA. There is no obvious systematic change in EB. For lower strychnine concentrations Eq. (1) may cease to be valid since the block remains incomplete even at very positive voltages, i.e. the high voltage asymptote for 1 -Y~0 is less than 1.
The exponential decay rates of the currents are themselves voltage dependent. Inspection of the current time courses in Fig. 1 shows that for small  I  I  I  I  I  I  I  I  I  iii  I  I  I   0  2  3  4  5  6  7  8  9  I0  12 13 14 Prepulse Durofion (ms) FIGURE 5. Potassium channel block as a function of pulse duration./tat is value of peak test current. Itest,= is It~t for very long conditioning pulses. Pulses sequence as in inset. 1 m in inset is not data but for illustration only. Circles, 5 mV (Era) prepulse; triangles, 21 mV (Era) prepulse. Arrows indicate time of prepulse peaks.
depolarizations, to negative Era, there is no hump in the current trace. The final current values are approached monotonically although they are significantly reduced from the controls. For example, equilibrium currents at -8 mV are reduced by 15%. Although the decay time constants vary with the axon, typical values from the currents plotted in Fig. 5 are: 48 mV, 2.7 ms; 72 mV, 1.5 ms; 88 mV, 1.1 ms; 200 mV, 1.1 ms. A saturation of the blocking rate at high voltages was observed in all cases. The limiting value in nine cases of nodes bathed in 1 mM strychnine ranged between 0.8 and 1.2 ms. The block is faster at higher strychnine concentrations, but no quantitative estimation of this effect was made.
The block does not begin at the onset of depolarization. In order to study the development of block more clearly, strychnine-treated nodes were depolarized in two steps. The first, smaller, conditioning step was of variable duration. The second, test step was to a potential at which the potassium activation was several times faster than the strychnine block, usually +80-90 mV. The equilibrium potassium current was unaffected by the conditioning pulse. In contrast, the peak test current varied with the duration of the conditioning pulse. After long conditioning pulses the peak test current approaches a minimum value (/test, 0~). After very short conditioning pulses there was little reduction in peak test cfirrent. The value Itest-Itest. ~, is plotted against prepulse duration for two prepulse potentials in Fig. 5. On a semilogarithmic plot the two functions closely approximate a straight line for prepulse durations over 3 ms, indicating that block develops exponentially. Conditioning pulses shorter than 1 ms do not reduce the height of the test pulse. The delay observed in the case of strychnine block corresponds roughly to the time to peak current in the conditioning pulse. The delay is less after larger depolarizing conditioning pulses.
An experiment similar to that in Fig. 5 was performed except that the amplitude rather than the duration of the conditioning pulse was varied. All conditioning pulses were 25 ms long, sufficient to attain a near-equilibrium block level. The ratio of peak test pulse to that seen with no conditioning pulse was calculated and subtracted from unity. The resulting value, representing the fraction of channels blocked during the conditioning pulse, is plotted against conditioning pulse voltage in Fig. 6. The function is sigmoid and strongly resembles that of Eq. (1). It is consistent with a model in which the prepulse determines the number of blocked channels. The same experiment also provides information on the equilibrium potassium conductances as a function of conditioning pulse voltage. Instead of measuring the peak test current, I took the current flowing immediately after the step to the test voltage. This function should resemble the conductance-potential curve calculated from the plateau levels of the partially blocked potassium currents. Such measurements describe a curve (Fig. 7) which is almost superimposable (except for a voltage shift) on the steady-state conductance-voltage curve in Fig. 3, obtained from a different node.
The strychnine block appears to be a genuine conductance decrease. The block developing at one voltage reduces the current seen in steps to other voltages, although any change in voltage will eventually alter the degree of block and, of course, the number of activated channels. The block develops witla firstorder kinetics with one major qualification. There is a 1 to several ms lag before the blocking reaction commences.

The Block Depends on [K]o
So far, a dependence on voltage, time, and strychnine concentration has been described. Block also depends on the external concentration of potassium ion. Other ions such as sodium, lithium, and tetramethylammonium seem to be  contrast, block is decreased by elevating [K]o. Frankenhaeuser (1962) showed in normal fibers that IK varies with [K]o in a manner satisfactorily described by the "independence principle." I have confirmed this. Changes of [K]o from 10 to 60 to 120 mM in normal fibers produce no systematic change in current after correction according to the equation. However, with nodes bathed in strychnine at different external potassium concentrations, the corrected currents were larger for greater [K]o. One such experiment is shown in Fig. 8. The lower the external potassium the stronger the block of potassium channels. Qualitatively the effect is best shown at very positive membrane potentials. Under such conditions an increase in external potassium ions results in an increase in outward potassium current. This is of course a change in the direction exactly opposite to that predicted from the independence relation.
Potassium clarrents in high potassium and strychnine display another interesting feature which can be seen in Fig. 9. In normal fibers the tail currents after repolarization to -80 mV decline monotonically. But after strychnine they increase to a peak and then smoothly decline. The decline is also slower than that of the control.

N-Methylstrychnine Produces a Voltage-Dependent Block when Applied Internally
The charged quaternary derivative of strychnine should cross membranes less readily than the parent compound. If its action resembles that of strychnine it should provide an opportunity to determine whether strychnine acts from the inside or outside of the membrane. Current-voltage measurements were made on a normal node and then NMS was introduced into one end pool and the axon was recut. Fig. I0 shows a tracing of the potassium current 20 min after exposure to NMS. The current shows the peak and subsequent exponential decay to a plateau characteristic of external strychnine application. Since only 50/zM NMS was applied to the cut end of the fiber, the actual level at the inner surface of the nodal membrane was probably smaller. The voltage-dependence of the conductance block can be seen in Fig. 11. In this experiment the axon was actually cut twice in two different NMS concentrations. The equilibrium current-voltage curve shows a clear negative slope region at high potential after introduction of NMS at concentrations below 1 mM. In contrast, NMS applied externally never produces a voltage-dependent block or a hump or peak of the potassium currents. If the external concentration is greater than 500 /~M some relatively Potassium current 20 min after recutting axon in 50/zM NMS.
voltage-independent block is produced (Fig. 12). As in untreated nodes the potassium conductance in such NMS-treated nodes approaches a maximum at large membrane potentials.

Strychnine Can Washout Rapidly
Although there is evidence that strychnine may block from the inside of the axon, in most cases it can be removed rapidly, upon washing. Such a washout is shown in Fig. 13. The node had been exposed to 5 x 10 -4 M strychnine for 17 I00-  rain. Then it was washed and the recovery monitored by measuring the equilibrium potassium current to a pulse to 48 mV every 10 s. The smooth curve is for a simple exponential removal with a time constant of 125 s. This is close to that for removal of TTX but several orders of magnitude slower than for the removal of tetraethylammonium ion (TEA). suggests a first-order or pseudofirst-order process. The lag time is consistent with a requirement that the channel be open before it can be blocked. The simplest form of Armstrong's (1969) model for the block of potassium channels by internal QA ions is:

DISCUSSION
Armstrong concluded that only open channels could be blocked. Conversely, blocked channels had to be unblocked before they could close. In a later paper (Armstrong, 1971) Armstrong has proposed an additional reaction: k+2 open blocked channel , ~ closed blocked channel.
(4) k-2 However, at -60 mV and standard [K]0, k+2 is 0.01 ms -1 and this reaction is less significant than the blocking reaction. To a first approximation Eq. (3) gives a reasonable fit of the data. The blocking reaction is assumed to occur with the QA in great excess and thus is pseudofirst order, much like an enzyme-substrate reaction with an excess of substrate. The model predicts that as the concentration of QA is increased the rate of blocking should be increased and the peak and equilibrium currents decreased. The initial rate of rise of potassium currents should be unaffected.
The kinetic scheme in Eq.
(3) also implies that if a sufficient number of channels is blocked, then upon repolarization there may be a transient increase in the tail current rather than a monotonic ¢lecrease. This would occur if the rate of channel unblocking exceeded the rate of closing and were predicated on the assumption that blocked channels must pass through an open state before closing. Another prediction is that the rate of decline in tail currents should be different in the presence of QA since the transition blocked-to-open may be rate limiting.
Nodes treated with strychnine resemble those treated with QA (Armstrong and Hille, 1972) in several basic features. In both cases two pulse experiments ( Fig. 5; Armstrong, 1969) show that block begins after a delay and is part of the basis for the kinetic model which states that only open channels can be blocked. Also, in both cases potassium currents may reach a peak and then decline exponentially to a plateau level. For small depolarizing pulses or for low QA or strychnine concentrations the potassium current approaches its equilibrium state monotonically. Armstrong (1969) and Armstrong and Hille (1972) have modeled this behavior and demonstrated that the presence of a peak in the potassium current depends on the relative values of the normal H-H potassium rate constants, C~n and/3, and the QA blocking rate constants, k+l and k-1. Armstrong (1971) found that an increase in QA concentration does increase the blocking rate and decrease the peak and equilibrium potassium currents. Strychnine has the same effect. Armstrong observed characteristic humps in the current tails after repolarization. These tail currents declined more slowly than those seen before QA perfusion. A similar change in the current tails is seen after exposure to strychnine.

The Block of Potassium Channels by QA or Strychnine is Voltage Dependent
The voltage dependence of the block is plotted in Fig. 4. The fit to the theory, Eq. (1), is reasonably good. The mean value of EB from Table I is 40.1 mV. Since this is approximately 25 mV/~, ~, the fraction of the membrane field through which strychnine must be moved to block a channel, is about 0.62. This lies between the values of~ ~zalculated by Hille (1975 a, b), 0.20 for TEA, and 0.70 for methylammonium ion, for a simple single-site block by these compounds. Other models could account for the voltage dependence of the strychnine-induced block. In one model a charged particle in the membrane might have to move through the field to expose or position a strychnine binding site. In a third model a voltage-dependent reaction could compete or otherwise interfere with strychnine block. For example, if external potassium ions moved toward the axoplasmic face of the membrane (presumably through ionic channels) to interfere with strychnine block we might observe a similar voltage dependence. Armstrong (1971) found that recovery from block was accelerated by increasing the external potassium concentration. The kinetic model in Eq. (3) and (4) fits both the high and low [K]o cases. Increasing potassium increases k-x but does not affect the other rate constants. Armstrong (1975) notes that this result indicates that potassium ions are displacing QA from the blocking site, not simply competing with them, but by a reaction of the form K~ut + QA -site ~.~ K + site + QA.
However, the evidence which Armstrong (1971) presents demonstrates the increase in k-a due to an increase in [K]o only for internally negative potentials, -60 mV and -100 mV, and his rate constants are therefore from recovery kinetics. The only other point tabulated is at +90 mV. In this case increasing [K]o had no effect on any calculated rate constants.
The experiments on the effect of [K]0 on strychnine block did not include observations on the rate of recovery. Rather, the amount of block was compared at three different external potassium concentrations (Fig. 8). At potentials above -20 mV increasing [K]o increased Y~. In the Armstrong kinetic equations this can result from a decrease in k+a and/or an increase in k-i. In order to calculate the rate constants, the rate of block as well as the extent of block must be measured. Because of experimental difficulties this was possible at only one potential in the experiment illustrated in Fig. 8. The time constant for the exponential approach to equilibrium block during a pulse to + 112 mV was decreased from 1.1 to 0.9 ms when [K]o was increased from 10 mM to 110 mM. It is immediately clear, since this can be produced only by an increase in either or both unidirectional rate constants, that k-a increases. Calculations of the rate constants, with no better than 10% precision gives the following values: 10 mM: k+l = 0.78 ms-l; k-1 = 0.09 ms -a, 110 mM: k+l = 0.79 ms-a; k-1 = 0.28 ms -1. This is consistent with the model which proposes that extracellular potassium ions displace strychnine from the blocking site, perhaps by electrostatic repulsion (Armstrong, 1975;Hille, 1975a). Strychnine may well differ from Armstrong's C9 (nonyltriethylammonium ion) in that even the small inward potassium flux at +112 mV can interfere with its binding. Of course, at +112 mV in essentially isotonic [K]o the net potassium flux through open channels is outward. The displacement of strychnine from blocking sites by external potassium ions at that potential may indicate that the block is primarily affected by the unidirectional flux and is relatively insensitive to outward flux, Of the four cations tested, only potassium interfered with block. This implies that either the inward flowing ion must pass the selectivity filter before getting sufficiently near the blocking site to displace strychnine, or the displacement reaction itself has a similar selectivity sequence.
The variable EA in Eq. (1) should be a function of the dissociation constant of the blocking site-strychnine complex and the concentration of strychnine in the axoplasm. An increase in strychnine concentration should move the curve to the left. This has been observed. An increase in the dissociation constant should move it to the right. If the concentration in the axoplasm is assumed to be the same as in the bath, then it is apparent that I~l~ is a little above 1 mM because a positive Era is required to produce 50% block. The Kaiss of the blocking site can be calculated to be about 2.5 mM.
Since we know the equilibrium levels of block and the time constants of association at each of several voltages we can calculate the unidirectional rate constants for the proposed pseudofirst-order blocking reaction. Typical values for one node are shown in Table II.
We can utilize this information to make a rough and speculative estimate of the energy barriers presented to a strychnine molecule approaching or leaving a blocking site. I assume a single energy barrier for the blocking reaction and apply absolute rate theory as used for a single barrier by Woodhull (1973), and for a multibarrier reaction by Hille (1975b). The assumptions other than those in the rate theory itself are that k+l and k_l measured are those of the actual binding, the "transmission coefficient" of the reaction is 1.0, and the concentration of the strychnine in the axoplasm is the same as in the bath. We can then calculate two numbers at each voltage. U+I is the voltage-independent barrier which axoplasmic strychnine must cross to reach the blocking site; U-1 is the voltageindependent, barrier which strychnine at the blocking site must cross to reach the axoplasm, Since the rate constants are determined for Em d= 0, we must subtract the energy involved in movement of strychnine through the electric field. I assumed that the energy barrier is half-way down the field between the axoplasm and binding site. The energies U+I and U-1 should be independent of the voltage if the correct 6 is chosen. A value of about 0.5 gave a good fit, slightly better than the 0.6 calculated from the fit to the Boltzmann equation alone. The calculated values for U+, and U_~ are 9.6 and -12.9 kcal/mol, respectively. The sum implies that the free energy of the strychnine-site complex is 3.3 kcal/mol less than that of unreacted strychnine in the cytoplasm (when Era = 0)-a number consistent with a dissociation constant of about 2 mM. The quaternary strychnine derivative NMS produces a voltage-dependent block only from the axoplasmic side of the node. Its action is so like that of strychnine that it is most probable that strychnine crosses the membrane in its uncharged form and then acts at the axoplasmic surface probably in the cationic form. Fig. 14 compares the molecular structure of the most potent of the QA ions tested by Armstrong, phenylpropyl triethylammonium ion (d)C3) with NMS. The 4~C3 molecule is almost superimposable on NMS except for the presence of a methyl group on NMS and an ethyl on ~C3. d~C3 would be superimposable on Nethylstrychnine.
NMS applied to the cut end of the axon has effects equivalent to strychnine applied at about 10 times the concentration in the bath. Either the concentration of intracellular strychnine is much lower than in the bath, or NMS is more potent or somehow concentrated as it travels down the core of the axon. If one ignores the last possibility.this means that NMS and possibly strychnine, too, have significantly different kaiss, k+~, and U+~ than those calculated above, kalss and U+~ would be smaller, k+l larger.
We have seen that strychnine block of potassium channels resembles that by QA ions and that, indeed, the molecular structure of the most potent QA, d)C3, is nearly superimposable on that of strychnine. Armstrong has estimated the (Arrnstrong,1971) N-Methylstrychnine value of the rate constants for several QA's. Typical values are, at 90 mV, k+l = 0.63 ms -1, k-1 = 0.03 ms -1 for nonyltriethylammonium ion (C9), and k-I = 0.60 ms -1 for pentyltriethylammonium (C5) ion. The general rule is that as the chain length of the R group of R-triethylammonium increases, k_l decreases but k+l is changed little if at all. Dissociation constants (measured at +50 mV) change with chain length and range: for straight chains, from 2 mM for C1 to 5 ~M for C9. 4~C3 binds even more tightly than C9. By assuming that potassium and QA ions enter potassium channels with equal ease Armstrong has calculated the single channel conductance for potassium ions. The figure, about 3 pmho, is in reasonable agreement with other more direct measurements. The estimates of the rate of strychnine block are subject to errors, the greatest of which is the estimation of axoplasmic strychnine concentration. Yet the agreement between the strychnine and QA constants is striking. The forward reaction rate is evidently similar for all the QA ions. The rate of removal of strychnine is between that of C~ and C9. As for C9, external potassium ions only affect k-1.
The parallels are so extensive that I conclude that strychnine must be blocking the channels by the same mechanism. Although a tertiary amine, strychnine must be crossing the membrane and, in its charged form, mimicking QA ions.