Studies of the cardiac-like action potential in crayfish giant axons induced by platinized tungsten metal electrodes

This study arose from a chance observation made while using tungsten (W) wire as a substitute for platinum (Pt) as an intracellular current-passing electrode. Tungsten was expected to be a good alternative to Pt because of its superior mechanical properties and equivalent electrical properties.

When crayfish giant axons were cannulated with a platinized tungsten (Pt-W) wire, prepared similarly to platinized platinum electrodes, modification of the action potential (AP) occurred within 15 min. Whereas the normal action potential has a duration of approximately 1ms, action potentials with durations of up to 4 s were observed in axons exposed to the Pt-W wire. The rising phase of the AP was largely unaffected, while the falling phase developed a plateau similar to that seen in the cardiac action potential. It was not necessary for current to be passed through the wire for the effect to develop. Some component of the Pt-W wire or product of a chemical reaction between the Pt-W wire and axoplasm was evidently altering the membrane ionic permeability control mechanisms responsible for the production of the action potential.

Long-duration action potentials of cardiac muscle are due to a prolonged increase of Na⁺ and Ca²⁺ conductances combined with a low K⁺ conductance (Gettes & Reuter, 1974; Reuter, 1967; Weidmann, 1951). Elongated action potentials have been produced in nerve preparations only by the use of pharmacological or toxic agents which delay Na⁺ inactivation and/or K⁺ activation (Shrager, Macey & Strickholm, 1969; Shrager, Strickholm & Macey, 1969; Narahashi, 1974). The purpose of this study was to identify and characterize the ionic currents altered by the Pt-W wire, and how the effects are mediated, and to assess the potential of Pt-W as a probe of membrane electrophysiological function.

A preliminary account of this work has been presented in abstract form (Orr & Lieberman, 1983).

The medial giant axon of the ventral nerve cord of the crayfish Procambarus clarkii was dissected and isolated according to the method of Wallin (1967). The apparatus employed for membrane potential recording, space and current clamping (Fig. 1) was essentially as previously described (Lieberman & Lane, 1976) with the following modifications: the electrode used for monitoring membrane potential was a KCl-filled impaling glass microelectrode; the current-measuring circuit was a virtual ground system with an additional feedback circuit for voltage-clamping the guard electrodes and bath to a constant reference value; and two sets of external platinized Ag-AgCl plate electrodes were used. A set of external stimulating electrodes, placed near the suboesophageal ganglion, was used to generate propagated action potentials.

Microelectrode tips were bevelled in a swirling solution of 2·5 mol l⁻¹ KC1 containing a silica polishing powder (Corson, Goodman & Fein, 1979) to facilitate impalement of axons. Bevelling had the dual function of sharpening the electrode tip and lowering its electrical resistance. Best results were obtained with microelectrodes with resistances between 10 and 20 MΩ. The electrodes used to monitor the potential of the external solution consisted of a glass pipette filled with 2·5 mol l⁻¹ KC1 suspended in agar gel. Connection between the gel and the amplifier lead was made with a Ag-AgCl wire in 2·5 mol l⁻¹ KC1 solution. Resistances of the agar electrodes were between 2 and 10 KΩ.

The axial wire current electrode was a 25 μm diameter wire (Pt or W) with an uninsulated length of 6—8 mm. Electroplating of the wire with Pt was carried out in a platinizing solution containing 3% PtCl₃ and 0·025 % lead acetate in 30mmoll⁻¹ HC1. Plating was carried out at a current level of 1·5 mA for variable periods an Required to produce the appropriate type of electrode. Current-voltage (I—V) relationships were determined from the change in membrane potential for a 100-to 300-ms constant-current pulse applied across the membrane and recorded oscillo graphically using x-y plotting techniques. Membrane resistance, at the resting membrane potential, was estimated graphically by determining the slope of the I-V relationship at the zero current intercept. AP traces and membrane potentials (E_m) were permanently recorded using oscillographic methods.

Specific channel blockers were added to the external solution after the Pt-W wire had begun to take effect. In experiments in which plateau durations were studied, only large and obvious changes were considered to be significant. This was due to the dynamic nature of the Pt-W effect. Quantitative measurements and statistical analysis were therefore not reliable.

Changes in the ionic composition of the crayfish physiological solution (van Harreveld, 1936), used to study the ionic requirements for the development of the Pt-W effect, were accomplished by substituting Tris-HCl for NaCl, KC1 or CaCl₂, Osmolarity was maintained with Tris buffer (pH 7·4). For Cl-free solutions all salts were made with isethionate as the anion. Ca²⁺ was slightly increased to compensate for the decrease in Ca²⁺ activity that occurs with isethionate (Lieberman, 1979).

Changes in the time course of the crayfish giant axon action potential occurred within 15 min of cannulation with the Pt-W wire. The initial changes involved a prolonging of the AP falling phase, a slight slowing of the rising phase and a small reduction in AP amplitude (Fig. 2A). The falling phase continued to slow, developing into a plateau typically within 10 min of the start of AP elongation (Fig. 2B). AP durations increased rapidly from this point, reaching durations of 20 ms to 4 s. Oscillations in the E_m were often seen near the end of longer-duration action potentials (Fig. 2C).

The membrane potential showed changes that were typical of those shown in Fig. 2D. There was a hyperpolarization of about 5 mV prior to the beginning of AP elongation. Concurrent with plateau formation there was a slow depolarization that continued until the axon lost its excitability. Holding the E_m at 80 mV allowed the axon to remain excitable for 20 min to 1 h until membrane resistance fell precipitously, resulting in a complete and irreversible loss of excitability.

The ability of the Pt-W wires to generate the elongated AP was very sensitive to the level and duration of the current used to electroplate the W wire with Pt. Any large deviation from the 5 s at 1·5 mA protocol resulted in a wire which had too thick or thin a coat of Pt to produce the effect. Apparently, both metals must be exposed in approximately equal amounts in order for the maximal effect to occur. Plateau amplitudes (measured from the resting membrane potential) ranged between 20 and 80 mV and averaged 65–70 mV. Each Pt-W wire had an effective lifespan, relative to action potential elongation, of up to 3 weeks. Electrodes could often be ‘recharged’ by an additional plating with Pt.

The rate of increase of AP duration during the formation of a plateau was found to be affected by three major factors. Significantly increasing the rate of AP production temporarily decreased the duration of an elongating plateau. This was followed by continued elongation at a slower rate. Similar results were obtained by increasing the flow rate of the external bathing solution. Larger axons (with greater volume of dilution) took longer to exhibit the effect.

In the normal crayfish axon, a single, long-duration depolarizing current pulse normally produced a single AP. Occasionally several APs were produced by a single pulse in a Pt-W-altered axon. The repetitive firing response was seen both before and during AP elongation.

The Pt-W effect was found to be reversible upon removal of the wire from the axon during the early stages of AP elongation. If the AP duration was less than 50 ms removal of the wire usually caused the duration to return to normal within 1 min. Removal of the wire after the AP duration had reached 100 ms usually had no effect on reversal of the AP elongation and membrane depolarization.

It was found that hyperpolarizing or depolarizing the axon by passing a continuous current through the axial wire caused a decrease in duration and plateau amplitude of an elongated AP. It appeared that the steady-state resting potential of the axon was the optimal potential for the Pt-W effect.

A series of experiments was conducted to determine which component or combination of components of the Pt-W wire was responsible for the effect. Axons were cannulated with a plain W wire for up to 2·5 h with no change in AP kinetics. Injection of a K⁺ isethionate solution (artificial axoplasm) containing 1 mmol l⁻¹ Na₂WO₄ into the axon also had no effect. The influence of lead in the platinizing solution was investigated by cannulating axons with a W wire which had been plated in a 30 mmol l⁻¹ HC1 solution containing 0·025 % lead acetate as the sole solute. No change in AP kinetics was observed. Pt wires and platinized Pt wires have been used for decades with no unusual effects, suggesting that the combination of both W and Pt was necessary for the action potential elongation to occur. This idea was tested with a W wire electroplated in a 30 mmol l⁻¹ HC1 solution containing 3% PtCl₄ but no lead acetate. When cannulated into an axon, this wire was successful in producing elongated action potentials.

Pt-W amalgams are used extensively in chemical processes as inhomogeneous catalysts and as such may generate H⁺ or free radicals. The possibility that the electrode generated H⁺ was tested by injecting the pH indicator, phenol red, into the axoplasm in sufficient quantity to dye the axoplasm clearly red (pH >7). On cannulating the axon with a Pt-W electrode the axoplasm slowly turned yellow (pH < 6·6) over a period of 5–10 min. The action potential began to elongate as the colour change became visible. On occasion, the action potential of a Pt-W-treated axon alternated between a slightly elongated (5–10 ms) and a fully elongated AP (>50ms). In one extraordinary experiment this occurred in an axon injected with phenol red. As the action potential oscillated between long and short durations the colour oscillated between yellow (pH < 6·6) and red (pH >7), respectively, providing clear evidence of a relationship between pH, and the elongation of the action potential.

The possibility of action potential elongation due solely to lowered pH_i was tested by the creation of an acidic axoplasm using either the NH₄ rebound method or addition of CO₂ to a physiological HCO₃⁻solution (Boron & DeWeer, 1976; Moody, 1980). These methods did not produce AP elongation. Attempts were made to produce an elongated AP by injecting potassium phosphate solutions (pH 4·5) directly into the axon in sufficient quantity to replace 50 % or more of the axoplasm. Phenol red was included in the solution to monitor the acidity of the axoplasm. Action potential duration increased slightly (2 ms) with no change in indicator colour. Although the evidence showed that a decreased pH_i could not be the sole mechanism, it is possible that such a decrease is a necessary condition for action potential elongation. To further investigate the role of pH_i, 20 mmol l⁻¹ NH₄C1 was added to the superfusate to counter the Pt-W-induced decrease in pH_i. This procedure caused previously formed plateaus progressively to shorten and disappear. With the removal of the NH₄C1, to generate an acidic axoplasm, the plateau reformed with durations in excess of those originally seen.

A comparison of the I-V relationship for a normal axon with that for a Pt-W-treated axon, after AP elongation had occurred, reveals an obvious difference in the depolarized potential region (Fig. 3). The control axon exhibits outward rectification whereas the Pt-W-treated axon exhibits an apparent inward rectification. The large change in E_m indicated by the dashed line represents the voltage shift of the AP plateau. Plateau formation could be explained by three possible mechanisms, alone or in combination: (1) a large, sustained inward current (i.e. a Na⁺ current with a delayed inactivation or a sustained Ca²⁺ current); (2) a decrease in outward K⁺ current; (3) a decrease in total K⁺ conductance, relative to rest, similar to that seen in cardiac muscle (Gettes & Reuter, 1974), in skeletal muscle (Adrian, 1969) and in oocytes (Hagiwara & Yoshii, 1979). Evidence for these possibilities was sought by injecting a train of small, negative, constant-current pulses across the membrane as an elongated AP was generated (Wiedmann, 1951). A typical result from this type of experiment is shown in Fig. 4, where the voltage deflections are over 200% larger near the end of the plateau than those seen at the rest potential, suggesting an increase in membrane resistance (R_m) during the plateau (presence of an inward rectifying channel). The apparent increase of R_m was variable from axon to axon, ranging from almost no increase to as much as 300 %. An increase in R_m was usually not seen in plateaus with durations less than 50 ms. The largest increases occurred in plateaus with durations greater than 100 ms.

Barium (0·1–1·0 mmol l⁻¹), which is known to block inwardly rectifying channels in hyperpolarized membranes (Standen & Stanfield, 1978), was used to test for the presence of an inward rectifier unmasked by the action of the Pt-W wire. Ba²⁺ did not alter the E_m or increase the amplitude of membrane potential responses to hyperpolarizing square current pulses, as would be expected if conducting, inwardly rectifying channels were being blocked.

When the Na⁺ concentration in the saline was reduced to 5 mmol l⁻¹, the AP amplitude of a normal axon was reduced by approximately one-third. After the axon had been cannulated with a Pt-W wire, there was slight slowing of both the rising and falling phases of the AP but no plateau was formed. Upon replacement of the control concentration of Na⁺ (190mmoll⁻¹), AP amplitude increased to normal and was accompanied by the rapid formation of a plateau.

Similar results were obtained with external solutions containing 25, 52, 73 and 97·5 mmol l⁻¹ Na⁺. The effects of 25 and 73 mmol l⁻¹ are shown in Fig. 5. With increasing external [Na⁺] the action potential reached greater final durations with a maximum observed duration of 15 ms at 97·5 mmol l⁻¹ Na⁺. Addition of control [Na⁺]_o rapidly increased the plateaus to 100 ms or greater. I-V plots from Pt-W-altered axons in 5, 25 and 52 mmol l⁻¹ Na⁺ exhibited normal outward rectification. Axons in 73 and 97·5 mmol U¹ Na⁺ exhibited typical Pt-W-induced ‘inward’ rectification.

Addition of 100 mmol l⁻¹ TTX into the bathing solution after a plateau had been formed reduced the plateau duration. The action potential was then abolished. A current pulse injection resulting in an E_m deflection of the same amplitude and duration as a normal AP would not initiate a plateau. An I-V plot from an axon exposed to TTX was produced immediately after the axon had been cannulated with a Pt-W wire and exhibited normal outward rectification. The outward rectification seen during depolarizing steps began to decrease as the Pt-W wire took effect. After 30 min the curve became completely linear, but did not go on to rectify in an apparently inward manner (Fig. 6).

Experiments similar to those carried out in low [Na⁺]_o were performed in one-quarter and one-half control [Ca²⁺]_o. Plateaus did not develop in Pt-W-altered axons bathed in one-quarter control [Ca²⁺]_o and action potentials were only slightly elongated in one-half control [Ca²⁺]₀. Raising [Ca²⁺]_o to the control level (13-5 mmol l⁻¹) caused the plateau duration and amplitude to increase dramatically. External [Ca²⁺] affected the plateau size in a titratable manner and like [Na⁺]_o was also required for the blocking of outward rectification.

When the Ca²⁺ channel blocker verapamil (10⁻⁰ mol l⁻¹) was included in the bathing solution, action potentials had a maximum duration of 10ms and no plateaus. Plateaus formed in the absence of verapamil were abolished in its presence. I-V plots (data not shown) demonstrated an absence of normal outward rectification but no apparent inward rectification. The result was a straight, ohmic I-V plot similar to that seen with the Pt-W effect plus TTX.

Superfusion with the Ca²⁺ channel blocker La³⁺ at concentrations of 1 and 5 mmol l⁻¹, abolished plateaus and prevented the apparent inward rectification in a manner similar to verapamil.

In initial experiments to determine the role of K⁺, the external K⁺ concentration was raised while using a holding current to maintain the E_m at the potential expected in control [K⁺]_o. High [K⁺]_o resulted in a decrease of AP duration of Pt-W-treated axons. Maintaining the E_m at a constant level ensured that the reduction in AP duration was not due to effects of depolarization on voltage-sensitive channels.

In four times control [K⁺]_o (21·6 mmol l⁻¹), R_m of an axon cannulated with a platinized Pt wire fell to one-third of its level in 5·4 mmol l⁻¹ [K⁺]_o (2136 Ωcm²vs 683 Ωcm²) (Fig. 7A). When the same procedure was carried out in a Pt-W-altered axon, a significant drop in R_m at 21·6 mmol l⁻¹ [K⁺]_o was not seen (1175 Ωcm²vs 1100 Ωcm²), suggesting that steady-state leakage channels normally opened by high [K⁺]_o are prevented from doing so in the altered membrane (Fig. 7B). High [K⁺]_o abolished the Pt-W-induced ‘inward-going’ rectification, significantly reduced the plateau and allowed the voltage-sensitive outwardly rectifying channels to operate normally (open under depolarization).

Several agents known to cause AP elongation in a number of nerve preparations were employed to compare their effects on the action potential of the crayfish giant axon with the response described for Pt-W. External application of either 1 mmol l⁻¹ tetraethylammonium (TEA) or 2 mmol l⁻¹ Ba²⁺ did not affect the kinetics of the normal AP. AP plateaus with durations of up to 350ms were produced by external application of 0·5 mmol l⁻¹ 3,4-diaminopyridine (DAP) (Fig. 8). Addition of 10⁻⁵ mol l⁻¹ verapamil or 0·5 mmol l⁻¹ Mn²⁺ to the DAP-containing solution did not modify the effect, indicating that there is no significant Ca²⁺ component of the DAP-induced plateau, in contrast to the Pt-W-altered APs.

The duration of action potentials of the crayfish medial giant axon is increased from <lms to >50 ms by an intracellular Pt-W wire electrode. This effect is accompanied by a decrease in axonal pH_i, but cannot be produced by such a reduction induced by various other treatments. The mechanism for the AP elongation can be explained by a decrease in outward (delayed) rectification coincident with an increase in inward Ca²⁺ current. Comparison of I—V plots before and after the Pt-W wire has taken effect (Fig. 3) reveals a block of delayed rectification. The AP plateau is inhibited by low [Ca²⁺]_o and Ca²⁺ channel blockers in a preparation where Ca²⁺ influx normally plays an insignificant role in AP kinetics of the crayfish axon (Yamagishi & Grundfest, 1971). Although the apparent increase in membrane resistance (inward rectification) (Fig. 4) could as well be explained by changes in inward currents (Ca²⁺ and/or Na⁺) induced by the pulses used to estimate membrane resistance, true inward rectification cannot be ruled out without voltage-clamp studies of currents flowing during the plateau (Goldman & Morad, 1977).

In initial studies to investigate the active component of the wire, it was found that plain tungsten, sodium tungstate, platinum or lead acetate had no effect on the action potential. The only effective combination was lightly platinized tungsten metal. The effectiveness of the Pt-W wire was not dependent on the passage of current through it. Several characteristics of the Pt-W-induced effect seemed to indicate that the effective agent was released into the axoplasm from the Pt-W wire to react with the axonal membrane. The AP elongation took time to develop after cannulation of the axon, and axons with greater diameters (greater volume of dilution) took longer to exhibit the effect. The reversibility of the effect in its early stages of development indicates that the reaction product is of a rather labile nature or rapidly buffered. This would also explain the results of an experiment in which the injection of Pt-W-treated axoplasm into an axon had no effect on the AP. A constant source of the product seems necessary to reach an effective concentration. After a time, the membrane becomes permanently altered; removal of the wire would not cause a reversal of the effect.

These observations led to the consideration of ionic hydrogen as a possible active agent. Phenol red studies revealed that the Pt-W wire decreased pH_i with a time course coincident with the initiation of AP elongation. While a decreased pH_i alone did not result in AP elongation, it was found to be a necessary component of the effect. The NH₄C1 rebound experiment provides evidence for this conclusion. This method for altering pH_i produces an initial alkalinization of the axoplasm on addition of NH4Cl to the superfusate. During this period, the Pt-W-induced plateaus are abolished. The plateaus are reformed rapidly on re-acidification of the axoplasm by removal of the external NH₄Cl Recent studies in squid axons have shown that passage of large, long-duration currents through wire electrodes generated H⁺ (Mullins, Requena & Whittenburg, 1985) which in turn was related to a large influx of Ca²⁺ (J. Requena & L. J. Mullins, personal communication). In addition, decrease of pH_i in squid giant axons has been found to decrease outward K⁺ current (Wanke, Carbone & Testa, 1979; Carbone, Prir & Wanke, 1981). Other than the necessary decrease in pH_i, it is not known what other products of the Pt-W/axoplasm reaction are involved in the AP elongation.

The block of outward rectification can account for the AP elongation caused by DAP (Fig. 8) (Kirsch & Narahashi, 1978) and part of the elongation caused by Pt-W. The decrease in outward rectification produced by Pt-W was found to be dependent on [Ca²⁺]_o. Apparent inward rectification is abolished by both verapamil treatment or [Ca²⁺]_o depletion. Abolition of the action potential can be a result of the increase in periaxonal K⁺ generated by the long depolarization represented by the plateau (Shrager, Starkus, Lo & Peracchia, 1983; Frankenhauser & Hodgkin, 1963), which serves to unblock the delayed rectifier (Dubois & Bergman, 1977), the increase in [Ca²⁺]_i serving to enhance K⁺ efflux and limit further Ca²⁺ influx (Eckert, Tillotson & Brehm, 1981; Eckert & Ewald, 1982).

In untreated crayfish axons, a [K⁺]_o-induced depolarization causes R_m to decrease at normal rest E_m (Lieberman, 1979) suggesting that high [K⁺]_o opens the voltagesensitive K⁺ channels and maintains them in a conducting condition even when the potential is returned to the level expected in control [K⁺]_o by an applied current (Fig. 7). In Pt-W-altered crayfish axons, an increase of [K⁺]_o causes almost no change in R_m at resting and hyperpolarized levels although voltage sensitivity of the outward rectifier is re-established. Under conditions where high [K⁺]_o would be expected to open outward rectifying channels (Dubois & Bergman, 1977), they remain closed under the influence of Pt-W.

In cardiac muscle, a slowly activated Ca²⁺ current plays a major role in producing and maintaining the action potential plateau (Rougier et al. 1969; Cranefield, Aronson & Wit, 1974). It is likely that one effect of the Pt-W product is to increase Ca²⁺ influx during AP generation to the point that it makes a significant contribution to plateau formation. Moody (1980) found that internal acidification of crayfish slow muscle fibres caused a decrease in outward rectification and an increased voltage contribution of Ca²⁺ influx, leading to all-or-none Ca²⁺ action potentials. TEA also permitted the generation of all-or-none Ca²⁺ action potentials, suggesting that inward Ca²⁺ current, normally present but shunted by the voltage-sensitive outward K⁺ current, was now able to modify the membrane potential. In the Pt-W-altered axon, the increased inward Ca²⁺ current contributes to the generation of the AP plateau with a similar decrease in outward rectification.

Although a prolonged inward current (Ca²⁺ or Na⁺) is a sufficient explanation for the apparent inward rectification (Fig. 3) and the increased R_m during the plateau (Fig. 4) induced by Pt-W, the contribution of an inward rectifying channel cannot be ruled out except with voltage-clamp studies of the currents flowing during the plateau. External Ba²⁺, did not result in an increased membrane resistance at resting or hyperpolarized potentials, as might be expected if inwardly rectifying channel were present in the membrane. However, inward rectifiers ‘created’ by the action of Pt-W would not necessarily be Ba²⁺-sensitive.

An aspect of the Pt-W effect not seen in the action potential of cardiac muscle is the slow but continuous depolarization that usually begins soon after plateau formation. The most likely explanation for this involves the influence of relative chloride permeabilities in nerve and muscle membrane. Crayfish axon membrane has a relatively low chloride permeability (Lieberman & Nosek, 1976; Strickholm & Clark, 1977) compared to muscle membrane (Adrian, 1969). A K⁺ permeability which decreases during depolarization (inward rectification) would be a liability in muscle fibres if it were not damped by a high chloride permeability, because it would otherwise lead to an unstable resting potential. The Pt-W-altered axon contains K⁺ channels which fail to increase their permeability during depolarization. Removing chloride from the external solution of Pt-W-altered axons has no effect on AP kinetics or on the rate of depolarization, indicating that the chloride permeability of the Pt-W-altered axon remains relatively small and thus provides a plausible explanation for the Pt-W-induced depolarization.

Tungsten wire serves as a good substitute for Pt in the construction of axial wire electrodes for quantitative electrophysiological studies of axons. We are using these electrodes regularly in this laboratory on crayfish giant axons with appropriate precautions to prevent their reactivity with axoplasm, as described in this study. The primary advantage of tungsten is its mechanical strength, as compared to Pt of the same diameter, allowing the use of smaller wire for studies on axons with diameters as small as 100 μm.

The technique used by Nussbaumer (1981) to etch tungsten overcomes the necessity to platinize the tungsten wire, thus avoiding problems of electrode reactivity. If platinization is desirable a low-current, long-term platinization procedure will provide an even, full coverage of tungsten preventing its reactivity with axoplasm.

As described in this study, the Pt-W electrode may serve as a useful tool to generate H⁺ in a controlled manner for studies of H⁺ transport, its effect on electrical properties of membranes and relationships to biochemical structure. The advantage of the electrode is that it can be used in intact axons and avoids the problem of external membrane surface exposure to agents used to change pH; such as CO₂ or NH₄⁺ or to problems associated with replacement of the axoplasm, in whole or in part, with artificial solutions.

Finally, the events related to the alteration of a crayfish axon by Pt-W, which causes conductance changes resembling those expected in cardiac cells, are schematically represented in Fig. 9 and may provide some insights into the relationship between different types of excitable membranes. It is unlikely that the Pt-W product creates channels de novo considering the speed of onset of the Pt-W effect. All channels responsible for the Pt-W effect are therefore assumed to be channels already present in the membrane but structurally modified or chemically inhibited to be non-functional. An agent which alters ionic currents in one membrane type so that they resemble ionic currents in another suggests that excitable membranes possess similar ionic channels. Evidence exists that unitary Ca²⁺ currents in nerve of three different species have similar kinetics (Brown, Camerer, Kunze & Lux, 1982). The difference between different classes of excitable membranes (i.e. nerve vs muscle) could be due to modifications or ‘masking’ influences on the membrane channels. The products of the reaction of Pt-W with axoplasm may add or remove such a masking influence from a particular channel type, so that it responds in a manner characteristic of a different class of membrane.

The authors are appreciative of the technical assistance of J. Pascarella, S. Hassan and Dr A. M. Butt during the course of this work and the secretarial assistance of Brenda Elks and Denise Wilson in preparing the manuscript. This work was supported in part by a grant from Sigma Xi (to LAO) and a grant from the Army Research Office DAAG 29–82-K-0182 (to EML).