STUDIES OF THE CARDIAC-LIKE ACTION POTENTIAL IN … · the action potential. Long-duration action...

jf. exp. Biol. 128, 1-17 (1987) \Printed in Great Britain © The Company of Biologists Limited 1987

STUDIES OF THE CARDIAC-LIKE ACTION POTENTIALIN CRAYFISH GIANT AXONS INDUCED BY PLATINIZED

TUNGSTEN METAL ELECTRODES

BY LISTON A. ORR AND EDWARD M. LIEBERMAN

Department of Physiology, School of Medicine, East Carolina University,Greenville, NC 27834, USA

Accepted 22 October 1986

SUMMARY

A lightly platinized tungsten (Pt-W) wire electrode, axially inserted into a crayfishgiant axon, causes the development of cardiac-like action potentials with durations ofup to 4 s. The plateau in membrane potential typically occurs within lOmin of thestart of action potential elongation. The effect occurs without passing currentthrough the Pt-W electrode and is temporally related to a dramatic decrease inintracellular pH (pH,). Such an effect cannot be induced by a decrease in pH,produced by equilibrating the axon with HCO3~-CO2 solution (pH6), an NH4C1rebound or direct intracellular injection of PO4

3~ buffer (pH45). Action potentialelongation is accompanied by a block of delayed rectification and the possibility thatinward rectification also develops cannot be ruled out. Plateau generation requiresNa+ and Caz+ inward currents as demonstrated by abolition of the plateau by[Na+]o or [Ca2+]o depletion or treatment with tetrodotoxin (TTX) or verapamil.The block of outward rectification by Pt-W requires external Na+ or Ca2+. Actionpotential elongation produced by 3,4-diaminopyridine is not sensitive to verapamiland the waveform is different from that produced by Pt-W. The data support thepossibility that different classes of excitable membranes have similar channelpopulations and that the functional differences between them reside in the inhibitoryor masking influences that are present in the microenvironments of the variousmembrane channels.

INTRODUCTION

This study arose from a chance observation made while using tungsten (W) wire asa substitute for platinum (Pt) as an intracellular current-passing electrode. Tungstenwas expected to be a good alternative to Pt because of its superior mechanicalproperties 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 actionpotential (AP) occurred within 15min. Whereas the normal action potential has aduration of approximately 1 ms, action potentials with durations of up to 4 s were

Key words: axons, crayfish, cardiac-like action potentials, tungsten electrodes, K+, Ca2+,channels.

2 L. A. ORR AND E. M. LIEBERMAN

observed in axons exposed to the Pt-W wire. The rising phase of the AP was largelyunaffected, while the falling phase developed a plateau similar to that seen in thecardiac action potential. It was not necessary for current to be passed through thewire for the effect to develop. Some component of the Pt-W wire or product of achemical reaction between the Pt-W wire and axoplasm was evidently altering themembrane ionic permeability control mechanisms responsible for the production ofthe action potential.

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

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

MATERIALS AND METHODS

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

Microelectrode tips were bevelled in a swirling solution of 2-5moll"1 KC1 con-taining a silica polishing powder (Corson, Goodman & Fein, 1979) to facilitateimpalement of axons. Bevelling had the dual function of sharpening the electrode tipand lowering its electrical resistance. Best results were obtained with microelectrodeswith resistances between 10 and 20 MQ. The electrodes used to monitor the potentialof the external solution consisted of a glass pipette filled with 2-5moll~1 KC1suspended in agar gel. Connection between the gel and the amplifier lead was madewith a Ag-AgCl wire in 2-5 mol I"1 KC1 solution. Resistances of the agar electrodeswere between 2 and 10 KQ.

The axial wire current electrode was a 25 /im diameter wire (Pt or W) with anuninsulated length of 6—8 mm. Electroplating of the wire with Pt was carried out in aplatinizing solution containing 3% PtCl3 and 0-025 % lead acetate in 30mmolP'HC1. Plating was carried out at a current level of l-5mA for variable periods a^

Cardiac-like action potentials in axons 3

Required to produce the appropriate type of electrode. Current-voltage (I-V)relationships were determined from the change in membrane potential for a 100- to300-ms constant-current pulse applied across the membrane and recorded oscillo-graphically using x—y plotting techniques. Membrane resistance, at the restingmembrane potential, was estimated graphically by determining the slope of the I-Vrelationship at the zero current intercept. AP traces and membrane potentials (Em)were permanently recorded using oscillographic methods.

Specific channel blockers were added to the external solution after the Pt-W wirehad 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 thedynamic nature of the Pt-W effect. Quantitative measurements and statisticalanalysis were therefore not reliable.

Changes in the ionic composition of the crayfish physiological solution (vanHarreveld, 1936), used to study the ionic requirements for the development of the

Fig. 1. Diagram of the current and space clamp system. Membrane potential (Em) wasmonitored with an impaling glass microelectrode (Elrec) referred differentially to a secondglass electrode placed in the bath. The axial wire stimulating electrode (El,) was a glass-insulated 25 (lm diameter platinized tungsten or platinum wire with an exposed length of7-8 mm. Current was passed between El, and a double set of platinized Ag/AgCl2 plates(placed on either side of the axon) serving as guards (Eg) and a centre plate (Elc) withwhich membrane current (Im) was monitored. The two Elc plates represented approx-imately 20 % of the total surface area of the Elg and Elc surface area. The virtual groundcurrent monitor utilized a voltage feedback system to maintain the bath and the electrodesat a uniform voltage (Elref) to ensure a constant partition of current between the plateelectrodes, based on their relative surface area. Current through El, was generated by anoperational amplifier voltage to constant current converter (I—> V) driven by a TextronixTMS06 pulse generator.


Pt-W effect, were accomplished by substituting Tris-HCl for NaCl, KC1 orOsmolarity was maintained with Tris buffer (pH 7-4). For Cl-free solutions all saltswere made with isethionate as the anion. Ca2+ was slightly increased to compensatefor the decrease in Ca2+ activity that occurs with isethionate (Lieberman, 1979).

RESULTS

Preliminary observations on the elongated action potential

Changes in the time course of the crayfish giant axon action potential occurredwithin 15 min of cannulation with the Pt-W wire. The initial changes involved aprolonging of the AP falling phase, a slight slowing of the rising phase and a smallreduction in AP amplitude (Fig. 2A). The falling phase continued to slow, devel-oping 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 Em were often seen near the end of longer-duration actionpotentials (Fig. 2C).

The membrane potential showed changes that were typical of those shown inFig. 2D. There was a hyperpolarization of about 5 mV prior to the beginning of APelongation. Concurrent with plateau formation there was a slow depolarization thatcontinued until the axon lost its excitability. Holding the Em at 80 mV allowed theaxon to remain excitable for 20 min to 1 h until membrane resistance fell precipi-tously, 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 thelevel and duration of the current used to electroplate the W wire with Pt. Any largedeviation from the 5 s at 1-SmA protocol resulted in a wire which had too thick orthin a coat of Pt to produce the effect. Apparently, both metals must be exposed inapproximately equal amounts in order for the maximal effect to occur. Plateauamplitudes (measured from the resting membrane potential) ranged between 20 and80 mV and averaged 65—70 mV. Each Pt-W wire had an effective lifespan, relative toaction potential elongation, of up to 3 weeks. Electrodes could often be 'recharged' byan additional plating with Pt.

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

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

The Pt-W effect was found to be reversible upon removal of the wire from the axonduring the early stages of AP elongation. If the AP duration was less than 50 ms

Cardiac-like action potentials in axons

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Fig. 2. The time-dependent development of the elongated action potential followingcannulation of the axon with a platini2ed tungsten wire. In A-C action potentials weregenerated at a distance from the position of the Pt-W wire and propagated through thecannulated region. Action potentials generated by space and current-clamp pulses werenot different from propagated action potentials. (A) Superimposed action potentialtraces showing the initial change in action potential kinetics. A small slowing of the risingphase and small reduction in amplitude occur. (B) Superimposed traces demonstratingthe characteristic plateau formation over a 10-min period. Note change in time scale.(C) A typical long-duration action potential. The oscillations at the end of the plateaucommonly occurred in long-duration plateaus and appeared to be small, 5-10 msduration, action potentials. (D) Strip chart recording of the membrane potential follow-ing the cannulation of the axon with the Pt-W wire. The small depolarization near thebeginning of the trace marks the time the axon was cannulated.

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 effecton reversal of the AP elongation and membrane depolarization.

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

Components of the Pt-W wire responsible for the elongated action potential

A series of experiments was conducted to determine which component or com-ftbination 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 1~Na2WO4 into the axon also had no effect. The influence of lead in the platinizingsolution was investigated by cannulating axons with a W wire which had been platedin a 30 mmol I"1 HC1 solution containing 0-025 % lead acetate as the sole solute. Nochange in AP kinetics was observed. Pt wires and platinized Pt wires have been usedfor decades with no unusual effects, suggesting that the combination of both W andPt was necessary for the action potential elongation to occur. This idea was testedwith a W wire electroplated in a 30 mmol P 1 HC1 solution containing 3% PtCL^ butno lead acetate. When cannulated into an axon, this wire was successful in producingelongated action potentials.

Pt-W amalgams are used extensively in chemical processes as inhomogeneouscatalysts and as such may generate H+ or free radicals. The possibility that theelectrode generated H+ was tested by injecting the pH indicator, phenol red, into theaxoplasm in sufficient quantity to dye the axoplasm clearly red (pH>7) . Oncannulating 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 thecolour change became visible. On occasion, the action potential of a Pt-W-treatedaxon alternated between a slightly elongated (5—10 ms) and a fully elongated AP(>50ms). In one extraordinary experiment this occurred in an axon injected withphenol red. As the action potential oscillated between long and short durations thecolour oscillated between yellow (pH<6-6) and red (pH>7) , respectively, pro-viding clear evidence of a relationship between pH, and the elongation of the actionpotential.

The possibility of action potential elongation due solely to lowered pH; was testedby the creation of an acidic axoplasm using either the NH4 rebound method oraddition of CO2 to a physiological HCO3~ solution (Boron & DeWeer, 1976; Moody,1980). These methods did not produce AP elongation. Attempts were made toproduce an elongated AP by injecting potassium phosphate solutions (pH4-5)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 indicatorcolour. Although the evidence showed that a decreased pH; could not be the solemechanism, it is possible that such a decrease is a necessary condition for actionpotential elongation. To further investigate the role of pH;, 20 mmol I"1 NH4C1 wasadded to the superfusate to counter the Pt-W-induced decrease in pHj. Thisprocedure caused previously formed plateaus progressively to shorten and disappear.With the removal of the NH4C1, to generate an acidic axoplasm, the plateau reformedwith durations in excess of those originally seen.

Effect of Pt-W on membrane resistance

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 thefl

Cardiac-like action potentials in axons

depolarized potential region (Fig. 3). The control axon exhibits outward rectifi-cation whereas the Pt-W-treated axon exhibits an apparent inward rectification. Thelarge change in Em indicated by the dashed line represents the voltage shift of the APplateau. Plateau formation could be explained by three possible mechanisms, aloneor in combination: (1) a large, sustained inward current (i.e. a Na+ current with adelayed inactivation or a sustained Ca + current); (2) a decrease in outward K+

current; (3) a decrease in total K+ conductance, relative to rest, similar to that seenin cardiac muscle (Gettes & Reuter, 1974), in skeletal muscle (Adrian, 1969) and inoocytes (Hagiwara & Yoshii, 1979). Evidence for these possibilities was sought byinjecting a train of small, negative, constant-current pulses across the membrane asan elongated AP was generated (Wiedmann, 1951). A typical result from this type ofexperiment is shown in Fig. 4, where the voltage deflections are over 200% largernear the end of the plateau than those seen at the rest potential, suggesting anincrease in membrane resistance (Rm) during the plateau (presence of an inwardrectifying channel). The apparent increase of Rm was variable from axon to axon,ranging from almost no increase to as much as 300 %. An increase in Rm was usuallynot seen in plateaus with durations less than 50 ms. The largest increases occurred inplateaus with durations greater than 100 ms.

Barium (O'l—lOmmoll"1), which is known to block inwardly rectifying channelsin hyperpolarized membranes (Standen & Stanfield, 1978), was used to test for thepresence of an inward rectifier unmasked by the action of the Pt-W wire. Ba2+ did notalter the Em or increase the amplitude of membrane potential responses to hyper-polarizing square current pulses, as would be expected if conducting, inwardlyrectifying channels were being blocked.

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Fig. 3. Effect of the Pt-W electrode on current-voltage relationships from a single axon.An I—V relationship obtained with a platinized Pt electrode (Pt-Pt, closed circles) isplotted together with that obtained from an axon treated with a platinized W electrode(Pt-W, open circles) for comparison.


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Fig. 4. Oscilloscope tracing of an action potential with superimposed current pulsesdemonstrating an apparent change in membrane resistance during the plateau. Note theapproximately 200 % increase in pulse height at the end of the plateau relative to rest.

Influence of [Na+]0 and the Na+ channel blocker, tetrodotoxin, on plateaudevelopment

When the Na+ concentration in the saline was reduced to Smmoll"1, the APamplitude of a normal axon was reduced by approximately one-third. After the axonhad been cannulated with a Pt-W wire, there was slight slowing of both the rising andfalling phases of the AP but no plateau was formed. Upon replacement of the controlconcentration of Na+ (190mmoir'), AP amplitude increased to normal and wasaccompanied by the rapid formation of a plateau.

Similar results were obtained with external solutions containing 25, 52, 73 and97-5mmoll~' Na+. The effects of 25 and 73mmoll~' are shown in Fig. 5. Withincreasing external [Na+] the action potential reached greater final durations with amaximum observed duration of 15 ms at 97-5mmoll~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 52mmoll~1 Na+ exhibited normal outward rectification.Axons in 73 and 97-5 mmoU"1 Na+ exhibited typical Pt-W-induced 'inward'rectification.

Addition of lOOmmoll"1 TTX into the bathing solution after a plateau had beenformed reduced the plateau duration. The action potential was then abolished. Acurrent pulse injection resulting in an Em deflection of the same amplitude andduration as a normal AP would not initiate a plateau. An I-V plot from an axonexposed to TTX was produced immediately after the axon had been cannulated witha Pt-W wire and exhibited normal outward rectification. The outward rectificationseen during depolarizing steps began to decrease as the Pt-W wire took effect. After30 min the curve became completely linear, but did not go on to rectify in anapparently inward manner (Fig. 6).

Influence of [Ca2+]0 and the Ca2+ channel blockers verapamil and La3+

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 slightlyelongated in one-half control [Ca2+]0. Raising [Ca2+]o to the control level (13-5mmol I 1 ) caused the plateau duration and amplitude to increase dramatically.External [Ca2+] affected the plateau size in a titratable manner and like [Na+]o wasalso required for the blocking of outward rectification.

When the Ca2+ channel blocker verapamil (lO^moll"1) was included in thebathing solution, action potentials had a maximum duration of 10ms and noplateaus. Plateaus formed in the absence of verapamil were abolished in its presence.I-V plots (data not shown) demonstrated an absence of normal outward rectificationbut no apparent inward rectification. The result was a straight, ohmic I-V plotsimilar to that seen with the Pt-W effect plus TTX.

Superfusion with the Ca2+ channel blocker La3+ at concentrations of 1 and5 mmol P 1 , abolished plateaus and prevented the apparent inward rectification in amanner similar to verapamil.

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Fig. 5. Effect of low [Na+]o on the development of the Pt-W effect. (A) The maximumduration AP developed in 25 mmol I"1 [Na+]o was approximately 2 ms. (B) Within 2 minof the re-admission of 190 mmol I"1 [Na ] o the plateau elongated to 100 or moremilliseconds. (C) In 73 mmol I"1 [Na ] o plateau development is present but held to5-7 ms (see shorter AP in D). (D) Following the readmission of 190 mmol 1~' [Na+]o theplateau further develops to greater than 25 ms within seconds.


Influence of [K+Jo

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

In four times control [K+]o (Zl^mmoll"1), Rm of an axon cannulated with aplatinized Pt wire fell to one-third of its level in 5-4mmoir' [K+]o (2136 Qcm2 vs683 Qcm2) (Fig. 7A). When the same procedure was carried out in a Pt-W-alteredaxon, a significant drop in Rm at 21-6mmoir' [K+]o was not seen (1175 Qcm2 vs1100 Qcm2), 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 theplateau and allowed the voltage-sensitive outwardly rectifying channels to operatenormally (open under depolarization).

Comparisons with other agents that cause action potential elongation

Several agents known to cause AP elongation in a number of nerve preparationswere employed to compare their effects on the action potential of the crayfish giant

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Fig. 6. The effect of tetrodotoxin (TTX) on a Pt-W-altered axon. Three superimposedI—V plots from TTX-treated axons showing the reduction of outward rectification duringthe onset of the Pt-W effect. Closed circles; I-V curve measured immediately aftercannulation of the axon with the Pt-W electrode. Open circles; I-V curve obtainedapproximately 5 min after cannulation. The ohmic I-V plot (triangles) from TTX-treated axon was obtained 30 min after cannulation with Pt-W wire. No further changewas seen with additional exposure. TTX prevented the development of the apparentinward rectification typical of the prolonged action potential. A developed plateau wasreduced in duration by TTX even though the action potential remained near normalamplitude for several minutes following the initial reduction in duration.


axon with the response described for Pt-W. External application of either 1 mmol 1 'tetraethylammonium (TEA) or 2 mmol I"1 Baz+ did not affect the kinetics of thenormal AP. AP plateaus with durations of up to 350ms were produced by externalapplication of 0-5 mmolP1 3,4-diaminopyridine (DAP) (Fig. 8). Addition of

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Fig. 7. The effect of [K + ] o on the I -V relationships of control and Pt-W-treated axons.(A) Axon cannulated with platinized platinum electrode. High [K + ] o decreases themembrane resistance in the hyperpolarizing direction as well as decreasing outward-goingrectification. At Em = — 85 mV the axon treated with high [ K + ] o (open circles) has amuch lower resistance than before K + treatment (closed circles). (B) A typical I -Vrelationship for a Pt-W-treated axon is shown ( • ) . The I -V curve of an axon treated withhigh [ K + ] o (open circles) compared with an axon in control [K + ] (closed circles)demonstrates that Pt-W protects the voltage-sensitive outward K + channels from [ K + ] o .The hyperpolarizing segment of the I-V relationship is unchanged by [ K + ] o . Thedevelopment of the 'inward' rectification is abolished but opening of the outward K +

channels is allowed in the depolarizing range of voltage by excess [K + ] o .

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Fig. 8. The effect of 3,4-diaminopyridine (DAP) on the duration and waveform of thecrayfish action potential. (A) A normal propagated action potential. (B) The full effect ofDAP is illustrated. In comparison with the effect of Pt-W, the DAP-modified actionpotential has a greater initial rise time, much more repetitive activity on the plateau,especially early, and a much faster decay of the plateau suggestive of a passive dischargerather than a plateau due to a maintained inward current.


10~5moll~' verapamil or 0-5mmolP' Mn2+ to the DAP-containing solution did notmodify the effect, indicating that there is no significant Ca2+ component of the DAP-induced plateau, in contrast to the Pt-W-altered APs.

DISCUSSION

The duration of action potentials of the crayfish medial giant axon is increasedfrom < l m s to >50ms by an intracellular Pt-W wire electrode. This effect isaccompanied by a decrease in axonal pH;, but cannot be produced by such areduction induced by various other treatments. The mechanism for the APelongation can be explained by a decrease in outward (delayed) rectification co-incident with an increase in inward Ca2+ current. Comparison of I—V plots beforeand after the Pt-W wire has taken effect (Fig. 3) reveals a block of delayed recti-fication. The AP plateau is inhibited by low [Ca2+]o and Ca2+ channel blockers in apreparation where Ca2+ influx normally plays an insignificant role in AP kinetics ofthe crayfish axon (Yamagishi & Grundfest, 1971). Although the apparent increase inmembrane resistance (inward rectification) (Fig. 4) could as well be explained bychanges in inward currents (Ca2+ and/or Na+) induced by the pulses used toestimate membrane resistance, true inward rectification cannot be ruled out withoutvoltage-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 thatplain tungsten, sodium tungstate, platinum or lead acetate had no effect on the actionpotential. The only effective combination was lightly platinized tungsten metal. Theeffectiveness of the Pt-W wire was not dependent on the passage of current throughit. Several characteristics of the Pt-W-induced effect seemed to indicate that theeffective agent was released into the axoplasm from the Pt-W wire to react with theaxonal membrane. The AP elongation took time to develop after cannulation ofthe axon, and axons with greater diameters (greater volume of dilution) took longerto exhibit the effect. The reversibility of the effect in its early stages of developmentindicates 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 ofPt-W-treated axoplasm into an axon had no effect on the AP. A constant source of theproduct seems necessary to reach an effective concentration. After a time, themembrane becomes permanently altered; removal of the wire would not cause areversal of the effect.

These observations led to the consideration of ionic hydrogen as a possible activeagent. Phenol red studies revealed that the Pt-W wire decreased pH; with a timecourse coincident with the initiation of AP elongation. While a decreased pH; alonedid not result in AP elongation, it was found to be a necessary component of theeffect. The NH4C1 rebound experiment provides evidence for this conclusion. Thismethod for altering pH; produces an initial alkalinization of the axoplasm on additionof NH4C1 to the superfusate. During this period, the Pt-W-induced plateaus areabolished. The plateaus are reformed rapidly on re-acidification of the axoplasm by


removal of the external NH4C1. Recent studies in squid axons have shown thatpassage of large, long-duration currents through wire electrodes generated H+

(Mullins, Requena & Whittenburg, 1985) which in turn was related to a large influxof Ca + (J. Requena & L. J. Mullins, personal communication). In addition,decrease of pH; in squid giant axons has been found to decrease outward K+ current(Wanke, Carbone & Testa, 1979; Carbone, Prir & Wanke, 1981). Other than thenecessary decrease in pH;, it is not known what other products of the Pt-W/axoplasmreaction are involved in the AP elongation.

The block of outward rectification can account for the AP elongation caused byDAP (Fig. 8) (Kirsch & Narahashi, 1978) and part of the elongation caused byPt-W. The decrease in outward rectification produced by Pt-W was found to bedependent on [Ca2+]o. Apparent inward rectification is abolished by both verapamiltreatment or [Ca2+]o depletion. Abolition of the action potential can be a result of theincrease in periaxonal K+ generated by the long depolarization represented by theplateau (Shrager, Starkus, Lo & Peracchia, 1983; Frankenhauser & Hodgkin, 1963),which serves to unblock the delayed rectifier (Dubois & Bergman, 1977), theincrease in [Ca2+]; 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 Rm to decreaseat normal rest Em (Lieberman, 1979) suggesting that high [K+]o opens the voltage-sensitive K+ channels and maintains them in a conducting condition even when thepotential 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 nochange in Rm at resting and hyperpolarized levels although voltage sensitivity of theoutward rectifier is re-established. Under conditions where high [K+]o would beexpected to open outward rectifying channels (Dubois & Bergman, 1977), theyremain closed under the influence of Pt-W.

In cardiac muscle, a slowly activated Ca2+ current plays a major role in producingand 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 increaseCa2+ influx during AP generation to the point that it makes a significant contributionto plateau formation. Moody (1980) found that internal acidification of crayfish slowmuscle fibres caused a decrease in outward rectification and an increased voltagecontribution of Ca2+ influx, leading to all-or-none Ca2+ action potentials. TEA alsopermitted the generation of all-or-none Ca2+ action potentials, suggesting thatinward Ca + current, normally present but shunted by the voltage-sensitive outwardK+ current, was now able to modify the membrane potential. In the Pt-W-alteredaxon, the increased inward Ca2+ current contributes to the generation of the APplateau with a similar decrease in outward rectification.

Although a prolonged inward current (Ca2+ or Na+) is a sufficient explanation forthe apparent inward rectification (Fig. 3) and the increased Rm during the plateau(Fig. 4) induced by Pt-W, the contribution of an inward rectifying channel cannot beruled out except with voltage-clamp studies of the currents flowing during theplateau. 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 ofPt-W would not necessarily be Ba2+-sensitive.

An aspect of the Pt-W effect not seen in the action potential of cardiac muscle is theslow but continuous depolarization that usually begins soon after plateau formation.The most likely explanation for this involves the influence of relative chloridepermeabilities in nerve and muscle membrane. Crayfish axon membrane has arelatively low chloride permeability (Lieberman & Nosek, 1976; Strickholm &Clark, 1977) compared to muscle membrane (Adrian, 1969). A K+ permeabilitywhich decreases during depolarization (inward rectification) would be a liability inmuscle fibres if it were not damped by a high chloride permeability, because it wouldotherwise lead to an unstable resting potential. The Pt-W-altered axon contains K+

channels which fail to increase their permeability during depolarization. Removingchloride from the external solution of Pt-W-altered axons has no effect on AP kineticsor on the rate of depolarization, indicating that the chloride permeability of thePt-W-altered axon remains relatively small and thus provides a plausible explanationfor the Pt-W-induced depolarization.

Tungsten wire serves as a good substitute for Pt in the construction of axial wireelectrodes for quantitative electrophysiological studies of axons. We are using theseelectrodes regularly in this laboratory on crayfish giant axons with appropriateprecautions to prevent their reactivity with axoplasm, as described in this study. Theprimary advantage of tungsten is its mechanical strength, as compared to Pt of thesame diameter, allowing the use of smaller wire for studies on axons with diametersas small as 100 fim.

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

As described in this study, the Pt-W electrode may serve as a useful tool togenerate H+ in a controlled manner for studies of H+ transport, its effect onelectrical properties of membranes and relationships to biochemical structure. Theadvantage of the electrode is that it can be used in intact axons and avoids theproblem of external membrane surface exposure to agents used to change pH; such asCO2 or NH4"1" or to problems associated with replacement of the axoplasm, in wholeor in part, with artificial solutions.

Finally, the events related to the alteration of a crayfish axon by Pt-W, whichcauses conductance changes resembling those expected in cardiac cells, areschematically represented in Fig. 9 and may provide some insights into the relation-ship between different types of excitable membranes. It is unlikely that the Pt-Wproduct 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 channelsalready present in the membrane but structurally modified or chemically inhibited tobe non-functional. An agent which alters ionic currents in one membrane type so that


[Na+]0+[Ca

Pt-W.H ++

X?

gK£

TTX

gNa+

Verapamil

Fig. 9. The mode of action of Pt-W leading to action potential elongation. It is uncertainwhat the immediate product of the Pt-W and axoplasm reaction is (X?) in addition to H + .The product appears to reduce the voltage sensitivity of the outward rectifier (gKvt) in amanner dependent on [Na+] and [Ca2+] in the external solution. Depletion of either ionreduces the block while tetrodotoxin (TTX), verapamil or La3+ prevents the APelongation but not the block of the outward rectifier. In order for the background steady-state K+ conductance (gKM) to be reduced both Na+ and Ca2+ fluxes are required. ThePt-W/axoplasm product appears to prevent opening of a proportion of the so-calledsteady-state 'leak* channels carrying K+ outwardly under certain conditions (highexternal [K+]). Whether the development of the inward (anomalous) rectifier occurs andis important to the development of the plateau is unresolved at this time.

they resemble ionic currents in another suggests that excitable membranes possesssimilar ionic channels. Evidence exists that unitary Ca2+ currents in nerve of threedifferent species have similar kinetics (Brown, Camerer, Kunze & Lux, 1982). Thedifference 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 amasking influence from a particular channel type, so that it responds in a mannercharacteristic of a different class of membrane.

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

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