Interactions of monovalent cations with sodium channels in squid axon. II. Modification of pharmacological inactivation gating

The time-, frequency-, and voltage-dependent blocking actions of several cationic drug molecules on open Na channels were investigated in voltage-clamped, internally perfused squid giant axons. The relative potencies and time courses of block by the agents (pancuronium [PC], octylguanidinium [C8G], QX-314, and 9-aminoacridine [9-AA]) were compared in different intracellular ionic solutions; specifically, the influences of internal Cs, tetramethylammonium (TMA), and Na ions on block were examined. TMA+ was found to inhibit the steady state block of open Na channels by all of the compounds. The time-dependent, inactivation-like decay of Na currents in pronase-treated axons perfused with either PC, 9-AA, or C8G was retarded by internal TMA+. The apparent dissociation constants (at zero voltage) for interaction between PC and 9-AA with their binding sites were increased when TMA+ was substituted for Cs+ in the internal solution. The steepness of the voltage dependence of 9-AA or PC block found with internal Cs+ solutions was greatly reduced by TMA+, resulting in estimates for the fractional electrical distance of the 9-AA binding site of 0.56 and 0.22 in Cs+ and TMA+, respectively. This change may reflect a shift from predominantly 9-AA block in the presence of Cs+ to predominantly TMA+ block. The depth, but not the rate, of frequency-dependent block by QX-314 and 9-AA is reduced by internal TMA+. In addition, recovery from frequency-dependent block is not altered. Elevation of internal Na produces effects on 9-AA block qualitatively similar to those seen with TMA+. The results are consistent with a scheme in which the open channel blocking drugs, TMA (and Na) ions, and the inactivation gate all compete for a site or for access to a site in the channel from the intracellular surface. In addition, TMA ions decrease the apparent blocking rates of other drugs in a manner analogous to their inhibition of the inactivation process. Multiple occupancy of Na channels and mutual exclusion of drug molecules may play a role in the complex gating behaviors seen under these conditions.     

Activation, inactivation and recovery in the sodium channels of the squid giant axon dialysed with different solutions.

Comparisons were made between families of ion currents recorded in voltage-clamped squid axons dialysed with 20 mM NaF and 330 mM CsF or TMAF, and bathed in a solution in which four fifths of the Na was replaced by Tris. The permeability coefficient PNa,fast for the fast-inactivating current in the initial open state was calculated as a function of test potential from the size of the initial peak of INa. The permeability coefficient PNa,non for the non-inactivating open state was calculated from the steady-state INa that persisted until the end of the test pulse. Dialysis with TMA had no direct effect on the QV curve for gating charge. The reversal potential for INa,non was always lower than that for INa,fast, the mean difference being about -9 mV when dialysing with Cs, but only about -1 mV with TMA. Except close to threshold, PNa,fast was roughly halved by dialysis with TMA as compared with Cs, but PNa,non was substantially increased. The time constant tau h inactivation of the sodium system was slightly increased during dialysis with TMA in place of Cs, and there were small shifts in the steady-state inactivation curve, but the rate of recovery from inactivation was not measurably altered. The flattening off of the tau h curve at increasingly positive test potentials corresponded to a steady reduction of the apparent inactivation charge until a value of about 0.2e was reached for pulses to 100 mV. The instantaneous I-V relationship in the steady state was also investigated. The results have a useful bearing on the effects of dialysis with TMA, on the differences between the initial and steady open states of the sodium channel, and on the relative voltage-dependences of the transitions in each direction between the resting and inactivated states.     

Saturation transfer electron paramagnetic resonance detection of sickle hemoglobin aggregation during deoxygenation.

In Myxicola axons, substitution of tetramethylammonium (TMA+) for Cs+ alters intramembrane charge movements (gating currents). Although the total charge moved during and following a depolarizing step remains constant, with TMA+ the ON response has additional slower component(s), and the OFF response is retarded. Concommitantly, TMA+ produces the same voltage-dependent block of Na+ inactivation in Myxicola as has been observed in other preparations. At large positive potentials as many as 70% of the Na+ channels fail to inactivate in the steady state. In addition, TMA+ slows Na+ activation, retards the inactivation of those Na+ channels that remain able to inactivate, and decreases the maximum Na+ conductance. The steady-state Na+ conductance induced by internal TMA+ or Na+ is consistent with a scheme in which these internal cations simply modify Na+ channels in an all-or-none fashion so that a fraction become incapable of inactivating.     

Internal cations, membrane current, and sodium inactivation gate closure in Myxicola giant axons.

Steady state to peak Na current ratio (INa,/INapeak) in Myxicola is greater, under some conditions, in internal Cs than in K, indicating less steady state inactivation in Csi. Csi effects are selective for steady state inactivation, with negligible effects on single-pulse inactivation time constants (Th). Mean Th ratios (Csi to Ki) were 1.04 and 1.02 at 0 and 10 mV. Two pulse inactivation time constants were also little affected. Inactivation is blocked in an all or none manner. Ki has little effect on steady state inactivation in the presence of inward INa, with INa/INapeak often declining to zero at positive potentials and independent of external Na concentration from 1/4 to 2/3 artificial sea water (ASW). Cs also has little effect at more negative potentials, but more with either more positive potentials or Na reduction, both reducing inward INa. K effects are evident when Na channel current is outward. A site in the current pathway when occupied selectively blocks inactivation gate closure. As occupancy does not depend significantly on potential, the site must not be very deep into the membrane field. Inactivation gates may associate with these sites on closure. The inactivated state may consist of a positively-charged structure occluding the inner channel mouth.     

The dual effect of internal tetramethylammonium ions on the open states of the sodium channel in the squid giant axon.

Voltage-clamp recordings of INa in squid axons dialysed with Cs or TMA, and bathed in low Na choline seawater, showed that, except close to threshold, the initial peak of fast-inactivating current was invariably decreased by TMA, whereas the non-inactivating current in the steady state was simultaneously increased. The results suggest that although TMA does not act directly on the movements of the voltage sensors that activate the sodium system, it blocks single-channel conductance in a voltage-dependent fashion in both the open states of the Na channel, while it has an entirely different type of action by increasing the probability of late openings in the steady state. Another difference between the two open states was that the sodium permeability coefficient had a Q10 of 1.8 in the initial open state, whereas in the steady state the effect of temperature was much smaller or even negative.     

Internal cesium and the sodium inactivation gate in Myxicola giant axons.

Internal cesium (CSi), relative to internal potassium (Ki), alters Na current (INa) time course in internally perfused Myxicola giant axons. CSi slows the time to peak INa, slows its decline from peak and increases the steady state to peak current ratio, INainfinity/INapeak. Neither activation nor deactivation kinetics are appreciably affected by CSi. Na current rising phases, times to half maximum and tail current time courses are similar in CSi and Ki. Inactivation time constants determined by both one (tau h) and two (tau c) pulses are also little changed by CSi. The CSi effects are due largely or entirely to an increased INainfinity/INapeak. CSi decreases the steady level of inactivation reached during a step in potential, preventing some fraction of inactivation gates from closing at all, the rest apparently closing normally. Inactivation block in CSi decreases with increasing inward current magnitude and in Ki inactivation block is appreciable only for outward Na channel current, suggesting the site of action is located somewhere in the current pathway. If this site mediates the normal operation of the inactivation gate, then a possible mechanism for gate closure could involve a positively charged structure moving to associate with a negative site near or into the inner channel mouth.     

Internal cesium alters sodium inactivation in Myxicola.

When Myxicola giant axons are internally dialyzed with Cs+ as the sole cation, the time-course of prepulse inactivation is selectively accelerated compared to its rate with K+ dialysis in the same axons. This decrease in tauph occurs without any change in the magnitude or time-course of INa during step depolarizations and results in tauph/taush ratios near unity over most of the potential range in Cs+ dialyzed axons.     

Inorganic, monovalent cations compete with agonists for the transmitter binding site of nicotinic acetylcholine receptors.

The properties of adult mouse recombinant nicotinic acetylcholine receptors activated by acetylcholine (ACh+) or tetramethylammonium (TMA+) were examined at the single-channel level. The midpoint of the dose-response curve depended on the type of monovalent cation present in the extracellular solution. The shifts in the midpoint were apparent with both inward and outward currents, suggesting that the salient interaction is with the extracellular domain of the receptor. Kinetic modeling was used to estimate the rate constants for agonist binding and channel gating in both wild-type and mutant receptors exposed to Na+, K+, or Cs+. The results indicate that in adult receptors, the two binding sites have the same equilibrium dissociation constant for agonists. The agonist association rate constant was influenced by the ionic composition of the extracellular solution whereas the rate constants for agonist dissociation, channel opening, and channel closing were not. In low-ionic-strength solutions the apparent association rate constant increased in a manner that suggests that inorganic cations are competitive inhibitors of ACh+ binding. There was no evidence of an electrostatic potential at the transmitter binding site. The equilibrium dissociation constants for inorganic ions (Na+, 151 mM; K+, 92 mM; Cs+, 38 mM) and agonists (TMA+, 0.5 mM) indicate that the transmitter binding site is hydrophobic. Under physiological conditions, about half of the binding sites in resting receptors are occupied by Na+.     

Modification of inactivation in cardiac sodium channels: ionic current studies with Anthopleurin-A toxin

The site 3 toxin, Anthopleurin-A (Ap-A), was used to modify inactivation of sodium channels in voltage-clamped single canine cardiac Purkinje cells at approximately 12 degrees C. Although Ap-A toxin markedly prolonged decay of sodium current (INa) in response to step depolarizations, there was only a minor hyperpolarizing shift by 2.5 +/- 1.7 mV (n = 13) of the half-point of the peak conductance- voltage relationship with a slight steepening of the relationship from - 8.2 +/- 0.8 mV to -7.2 +/- 0.8 mV (n = 13). Increases in Gmax were dependent on the choice of cation used as a Na substitute intracellularly and ranged between 26 +/- 15% (Cs, n = 5) to 77 +/- 19% (TMA, n = 8). Associated with Ap-A toxin modification time to peak INa occurred later, but analysis of the time course INa at multiple potentials showed that the largest effects were on inactivation with only a small effect on activation. Consistent with little change in Na channel activation by Ap-A toxin, INa tail current relaxations at very negative potentials, where the dominant process of current relaxation is deactivation, were similar in control and after toxin modification. The time course of the development of inactivation after Ap-A toxin modification was dramatically prolonged at positive potentials where Na channels open. However, it was not prolonged after Ap-A toxin at negative potentials, where channels predominately inactivate directly from closed states. Steady state voltage-dependent availability (h infinity or steady state inactivation), which predominately reflects the voltage dependence of closed-closed transitions equilibrating with closed-inactivated transitions was shifted in the depolarizing direction by only 1.9 +/- 0.8 mV (n = 8) after toxin modification. The slope factor changed from 7.2 +/- 0.8 to 9.9 +/- 0.9 mV (n = 8), consistent with a prolongation of inactivation from the open state of Ap-A toxin modified channels at more depolarized potentials. We conclude that Ap-A selectively modifies Na channel inactivation from the open state with little effect on channel activation or on inactivation from closed state(s).     

Inhibition of sodium currents by local anesthetics in chloramine-T- treated squid axons. The role of channel activation

In order to test the requirement of Na channel inactivation for the action of local anesthetics, we investigated the inhibitory effects of quaternary and tertiary amine anesthetics on normally inactivating and noninactivating Na currents in squid axons under voltage clamp. Either the enzymatic mixture pronase, or chloramine-T (CT), a noncleaving, oxidizing reagent, was used to abolish Na channel inactivation. We found that both the local anesthetics QX-314 and etidocaine, when perfused internally at 1 mM, elicited a "tonic" (resting) block of Na currents, a "time-dependent" block that increased during single depolarizations, and a "use-dependent" (phasic) block that accumulated as a result of repetitive depolarizations. All three effects occurred in both control and CT-treated axons. As in previous reports, little time-dependent or phasic block by QX-314 appeared in pronase-treated axons, although tonic block remained. Time-dependent block was greatest and fastest at large depolarizations (Em greater than +60 mV) for both the control and CT-treated axons. The recovery kinetics from phasic block were the same in control and CT-modified axons. The voltage dependence of the steady state phasic block in CT-treated axons differed from that in the controls; an 8-10% reduction of the maximum phasic block and a steepening and shift of the voltage dependence in the hyperpolarizing direction resulted from CT treatment. The results show that these anesthetics can bind rapidly to open Na channels in a voltage-dependent manner, with no requirement for fast inactivation. We propose that the rapid phasic blocking reactions in nerve are consequences primarily of channel activation, mediated by binding of anesthetics to open channels, and that the voltage dependence of phasic block arises directly from that of channel activation.     

Patch clamp recordings from membranes which contain gap junction channels.

The septal membranes of the median and lateral giant axons of earthworm, which contain gap junctions, were exposed by cutting one segment of the cord. Patch recordings were obtained from the exposed cytoplasmic side of the septum. Seal resistances ranged from 2 to 15 G omega. The patch could be excised (detached) or left attached to the whole cell. Two types of channels were observed. One type was blocked by tetraethylammonium (TEA) or Cs+ and had a unitary conductance of 30-40 pS. It appears to be a K+ channel. The other channel type had a unitary conductance of 90-110 pS and was unaffected by TEA+ or Cs+. In the detached configuration the channel was shown to conduct Cs+, K+, Na+, TMA+, Cl- and TEA+ even in the presence of 2 mM Zn2+, 1 mM Ni2+, 1 mM Co2+, and 4 mM 4-aminopyridine. The conductance ratios relative to K+ were 1.0 for Cs+, 0.84 for Na+, 0.64 for TMA+, 0.52 for Cl- and 0.2 for TEA+. The channel appears to be voltage insensitive whether monitored in detached or attached recording mode. Both H+ and Ca2+ reduce the probability of opening. Thus, the 100 pS channel has many of the properties expected of a gap junction channel.     

Permeation of Na+ through a delayed rectifier K+ channel in chick dorsal root ganglion neurons

In whole-cell patch clamp recordings from chick dorsal root ganglion neurons, removal of intracellular K+ resulted in the appearance of a large, voltage-dependent inward tail current (Icat). Icat was not Ca2+ dependent and was not blocked by Cd2+, but was blocked by Ba2+. The reversal potential for Icat shifted with the Nernst potential for [Na+]. The channel responsible for Icat had a cation permeability sequence of Na+ >> Li+ >> TMA+ > NMG+ (PX/PNa = 1:0.33:0.1:0) and was impermeable to Cl-. Addition of high intracellular concentrations of K+, Cs+, or Rb+ prevented the occurrence of Icat. Inhibition of Icat by intracellular K+ was voltage dependent, with an IC50 that ranged from 3.0-8.9 mM at membrane potentials between -50 and -110 mV. This voltage- dependent shift in IC50 (e-fold per 52 mV) is consistent with a single cation binding site approximately 50% of the distance into the membrane field. Icat displayed anomolous mole fraction behavior with respect to Na+ and K+; Icat was inhibited by 5 mM extracellular K+ in the presence of 160 mM Na+ and potentiated by equimolar substitution of 80 mM K+ for Na+. The percent inhibition produced by both extracellular and intracellular K+ at 5 mM was identical. Reversal potential measurements revealed that K+ was 65-105 times more permeant than Na+ through the Icat channel. Icat exhibited the same voltage and time dependence of inactivation, the same voltage dependence of activation, and the same macroscopic conductance as the delayed rectifier K+ current in these neurons. We conclude that Icat is a Na+ current that passes through a delayed rectifier K+ channel when intracellular K+ is reduced to below 30 mM. At intracellular K+ concentrations between 1 and 30 mM, PK/PNa remained constant while the conductance at -50 mV varied from 80 to 0% of maximum. These data suggest that the high selectivity of these channels for K+ over Na+ is due to the inability of Na+ to compete with K+ for an intracellular binding site, rather than a barrier that excludes Na+ from entry into the channel or a barrier such as a selectivity filter that prevents Na+ ions from passing through the channel.     

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.     

Slowing of sodium current inactivation by ruthenium red in snail neurons

The effects of ruthenium red (RuR) were tested on the membrane currents of internally perfused, voltage-clamped nerve cell bodies from the snail Limnea stagnalis. Bath application of nanomolar concentrations of RuR produces a prolonged Na current that decays approximately 40 times slower than the normal Na current in these cells. The relationship between the reversal potential for the prolonged Na current and the intracellular concentration of Na+ agrees well with the constant-field equation, assuming a small permeability for Cs+. Because a strong correlation was found between the magnitude of the normal Na current and that of the prolonged Na current, it is concluded that the prolonged Na current flows through the normal Na channels. This conclusion is supported by the similar selectivities, voltage dependencies, and tetrodotoxin (TTX) sensitivities of these two currents. This action of RuR to slow the inactivation of the Na channel was not observed at concentrations below 1 nM, but was complete at 10 nM. When the concentration of RuR is increased to 0.1 mM, the Ca current in these cells is blocked; but at this high concentration RuR also reduces the outward voltage-dependent currents and resting membrane resistance. Therefore, RuR is not a good Ca blocker because of its lack of specificity. However, its action of slowing Na current inactivation is very specific and could prove to be useful in studying the inactivation of the Na channel.     

Interaction of nonylguanidine with the sodium channel.

Alkyl and aromatic guanidines interact strongly with the tetrodotoxin (TTX)- receptor site in eel electroplaque membranes, showing competition with TTX. That these guanidines could be useful as highly reversible small molecular weight blockers of Na+ currents is therefore suggested. We have investigated the mechanisms of interaction of one of these derivatives, nonylguanidine, by studying its effects on Na+ currents in squid giant axons using voltage clamp techniques. Although nonylguanidine competed with TTX for binding to eel electroplaque membrane fragments (Ki = 1.8 X 10(-5) M), it reversibly blocked both inward and outward Na+ currents in intact axons only if applied to the interior. In axons with the Na+ inactivation removed by papain nonylguanidine produced a time-dependent block very similar to that reported for strychnine and pancuronium. The reduction of steady-state currents in these axons was also voltage-dependent, with increasing block observed with increasing step depolarization. These results suggest that nonylguanidine binds to a site accessible from the axoplasmic side of the channel, simulating Na+ inactivation in papain-treated axons and competing with the normal inactivation process in untreated axons. The competition between internal nonylguanidine and external TTX may result from perturbation by the positively charged nonylguanidine of the TTX-binding site from within the channel itself.     

A trapped intracellular cation modulates K+ channel recovery from slow inactivation

Upon depolarization, many voltage-gated potassium channels undergo a time-dependent decrease in conductance known as inactivation. Both entry of channels into an inactivated state and recovery from this state govern cellular excitability. In this study, we show that recovery from slow inactivation is regulated by intracellular permeant cations. When inactivated channels are hyperpolarized, closure of the activation gate traps a cation between the activation and inactivation gates. The identity of the trapped cation determines the rate of recovery, and the ability of cations to promote recovery follows the rank order K+ > NH4+ > Rb+ > Cs+ > Na+, TMA. The striking similarity between this rank order and that for single channel conductance suggests that these two processes share a common feature. We propose that the rate of recovery from slow inactivation is determined by the ability of entrapped cations to move into a binding site in the channel's selectivity filter, and refilling of this site is required for recovery.     

The permeability of the sodium channel in Myxicola to the alkali cations

Relative permeabilities to the alkali cations were determined, from the reversal potential (VRev), for the Na channel of internally perfused voltage-clamped Myxicola giant axons. PLi/PNa and PK/PNa are 0.94 and 0.076, respectively. Rb and Cs are not measurably permeant. VRev vs. the internal Na activity was well described by the constant field equation over a 300-fold range of internal Na concentrations. In agreement with findings on squid axons, the PK/PNa was found to increase when the K content of the internal perfusate was reduced (equivalent per equivalent substitution with TMA). Internal Rb and Cs also decreased the PK/PNa. The order of effectiveness of internal K, Rb, and Cs in increasing the Na selectivity of the Na channel was Cs greater than Rb greater than or equal to K. External Li increases the PK/PNa but this may be due to the formation of LiF internally. It may be that substances do not have to traverse the channel in order to affect the selectivity filter. Evidence is presented which suggests that the selectivity of the Na channel may be higher for Na in intact as compared to perfused giant axons. It was concluded that the channel selectivity properities do not reflect only some fixed structural features of the channel, but the selectivity filter has a labile organization.     

The K+ channel of sarcoplasmic reticulum. A new look at Cs+ block.

K+-selective ion channels from mammalian sarcoplasmic reticulum were inserted into planar phospholipid bilayers, and single-channel currents measured in solutions containing Cs+. Current through this channel can be observed in symmetrical solutions containing only Cs+ salts. At zero voltage, the Cs+ conductance is approximately 15-fold lower than the corresponding K+ conductance. The open channel rectifies strongly in symmetrical Cs+ solutions, and the Cs+ currents are independent of Cs+ concentration in the range 18-600 mM. Biionic (Cs+/K+) reversal potentials are only 10 mV, showing that Cs+ is nearly as permeant as K+, though much less conductive. Addition of Cs+ to symmetrical K+ solutions reduces current through the channel in a voltage-dependent way. The results can be explained by a free energy profile in which the channel's selectivity filter acts in two ways: to provide binding sites for the conducting ions and to serve as a major rate-determining structure. According to this picture, the main difference between high-conductance K+ and low-conductance Cs+ is that Cs+ binds to an asymmetrically positioned site approximately 20-fold more tightly than does K+.     

Removal of sodium inactivation and block of sodium channels by chloramine-T in crayfish and squid giant axons.

Modification of sodium channels by chloramine-T was examined in voltage clamped internally perfused crayfish and squid giant axons using the double sucrose gap and axial wire technique, respectively. Freshly prepared chloramine-T solution exerted two major actions on sodium channels: (a) an irreversible removal of the fast Na inactivation, and (b) a reversible block of the Na current. Both effects were observed when chloramine-T was applied internally or externally (5-10 mM) to axons. The first effect was studied in crayfish axons. We found that the removal of the fast Na inactivation did not depend on the states of the channel since the channel could be modified by chloramine-T at holding potential (from -80 to -100 mV) or at depolarized potential of -30 mV. After removal of fast Na inactivation, the slow inactivation mechanism was still present, and more channels could undergo slow inactivation. This result indicates that in crayfish axons the transition through the fast inactivated state is not a prerequisite for the slow inactivation to occur. During chloramine-T treatment, a distinct blocking phase occurred, which recovered upon washing out the drug. This second effect of chloramine-T was studied in detail in squid axons. After 24 h, chloramine-T solution lost its ability to remove fast inactivation but retained its blocking action. After removal of the fast Na inactivation, both fresh and aged chloramine-T solutions blocked the Na currents with a similar potency and in a voltage-dependent manner, being more pronounced at lower depolarizing potentials.(ABSTRACT TRUNCATED AT 250 WORDS)