Voltage Clamp Experiments on Axons with Potassium as the Only Internal and External Cation

EDUARDO ROJAS AND GERALD EHRENSTEIN Laboratory of Biophysics, N.I.N.D.B., N.I.H. , Bethesda. Maryland and Estacion de Biologia Marina, Montemar, Chile

Previous experiments on the squid giant axon in an extracellular solution contain- ing potassium as the only cation have dern- onstrated an anodal threshold phenome- non (Segal, '58) and a negative resistance region (Moore, '59) for potassium current. In order to determine whether the negative resistance is due to the membrane alone or whether some other substance must be present, it is desirable to measure current- voltage curves with both intracellular and extracellular solutions containing potas- sium as the only cation. Narahashi deter- inined that a certain portion of the current- voltage curve for the squid giant axon is linear when both intracellular and extra- cellular solutions are 538 mM potassium chloride (Narahashi, '63). In the present investigation, the voltage clamp technique was used to determine current-voltage curves for the squid giant axon with intra- cellular and extracellular solutions con- taining 600 mM potassium in the voltage range between about -140 and +I40 mV.

METHODS

Experiments were performed with the giant axon of the squid Dosidicus gigas at the Estacion de Biologia Marina, Monte- mar, Chile. Living squid were not avail- able. For this reason mantles which had been kept stored for a few hours in iced cold sea water were used.

The perfusion technique employed was similar to that of Tasaki ('63). A clean giant fiber with both ends tied was mounted on a chamber as illustrated in figure 1. An outlet glass tube, 400-500 c1 in diameter, was slowly introduced through one end for about 35 mm while the axo- plasm was removed by suction. Another glass tube, an inlet tube, 180-250 c1 in diameter, connected to the internal solu-

J. CELL. AND COMP. PHYSIOL., 66: 71-78.

tion reservoir, was then introduced through the opposite end. Next it was placed into the lumen of the first tube. After 1 or 2 ml of solution were perfused, internal elec- trodes were introduced to their final posi- tion. Then the outlet tube was withdrawn for about 20 mm. These operations were performed with micro-manipulators, not shown in the diagram, while action poten- tials were externally elicited and recorded. After positioning of the electrodes and withdrawal of the outlet tube, the current wire was used to pulse the axon and only internal recording was used. If the action potential was smaller than 94 mV, the axon was rejected. The axon chamber was perfused with cold sea water at about 10°C. The temperature was measured with a thermometer placed near the nerve fiber.

In most experiments, the following arti- ficial sea water was used:

430 mM NaCl 10 mM KCI 10 mM CaCL 50 mM M g C L

5 mM Tris The pH was adjusted to 7.6-8.0. The in- ternal perfusing solution was 600 mM KF. Its pH was adjusted to 7.35 by adding small amounts of 10 mM HCl. The rate of internal perfusion ranged from 4 to 40 microliters per minute. Since 20 mm of axon with an average diameter of about 0.8 mm was perfused, the internal solution was changed about one-half to five times per minute. The preparation was perfused 10 to 40 minutes in each experiment be- fore a voltage clamp run.

A capillary tube of about 80 p in diam- eter, with 3 M KC1 and with a clean 35 u platinum wire inside, connected to a calc- me1 cell was used as the internal voltage electrode. Another capillary tube filled with

71

72 EDUARDO ROJAS AND GERALD EHRENSTEIN

ERENCE ELECTRODE

INTERNAL

Fig. 1 procedure.

The combined suction perfusion technique and the point control voltage clamp

agar - 3 M KC1 and connected to a calo- mel cell was used as the external voltage electrode. A 100 CI platinum wire was used to supply current. It was insulated except for 15 mm that were carefully cleaned and platinized as described elsewhere (Moore and Cole, '63). All potentials were cor- rected for junction potentials determined at the beginning and at the end of each experiment.

Three platinum external electrodes (shown schematically in fig. 1) were placed on each side of the axon. The outer elec- trodes were grounded and the center elec- trodes were used for current measurement. Each electrode was 4 mm long and both center electrodes were separated from the adjacent outer electrodes by 1.5 mm. The internal voltage electrode was positioned so that its tip was in the center of the center electrode. The internal current elec- trode was positioned so that its non- insulated length corresponded to the length of the external electrodes. To obtain volt- age clamp, the potential difference between internal and external voltage electrodes was compared with the potential of a com- mand signal. Electronic feedback was employed to supply current through the internal current electrode until the two potentials were equal. The control system employed has been described by Moore and Cole ('63). In all experiments, axons were first clamped at their resting potentials and then a series of square voltage pulses of fixed duration and varying amplitude were applied.

RESULTS

After a few minutes of intracellular per- fusion with 600 mM potassium fluoride a first voltage-clamp run was obtained in artificial sea water. Next the external sea water was replaced by 600 mM potassium chIoride and a second voltage clamp run was obtained. A large volume of this solu- tion was used (250-500 ml) to insure the washout of other cations such as Ca++ and Mg++. At least ten minutes elapsed be- tween the start of solution flow and volt- age clamp. Finally, the external solution was replaced by sea water and a third voltage-clamp series was obtained.

Table 1 shows resting and action poten- tials before and after the external sea water was replaced by 600 mM potassium chloride. It can be seen that the resting potential recovered to about 95% and the action potential to about 85% of their con- trol values.

Figure 2 shows the voltage clamp mem- brane currents for this case. The number next to each curve indicates the absolute potential of the pulse. Records were not corrected for capacity and leakage cur- rents. There was a fast surge of current, hardly visible in the record, an early in- ward current and a delayed outward cur- rent. The present experimental records are very similar to those obtained from non-perf used axons.

Figure 3 shows the voltage clamp mem- brane currents obtained when the external solution was 600 mM potassium chloride.

EXPERIMENTS ON AXONS WITH POTASSIUM AS ONLY CATION 73

TABLE 1

Resting and action potentials of docidicus gigas axon inhacellularly perfused with 600 mM

potassium fluoride

Before After

R.P. A.P. R.P. A.P. Axon

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

- 47 - - 48 52 55 45 59 48 57 60 54 49 54 52.3

96 94 108 96 100 98 105 98 108 94 114 108 98 95 100 100.8 50.4

- 100 78

84 - - - 80 84 -

85.2

+ 4 2

+82--J

+102- 7 /c- 2 mA/cmZ

I I I I I I +l22-

0 2 4 6 8 msec

Fig. 2 Voltage clamp currents obtained when the axon is intracellularly perfused with 600 mM potassium fluoride and extracellularly perfused with sea water at about loo centigrade.

Figure 4 shows current-voltage relations for the early inward current (INa) and for the delayed outward current ( IK) before and after the axon was bathed with 600

mM potassium chloride. This figure also shows the current-voltage relation when the same concentration of potassium (600 mM) was on both sides of the excitable membrane. INa was measured at the time of peak and the other currents were meas- ured at 5 msec after the start of a pulse.

In figure 4, the steady-state current- voltage curve for external potassium chlo- ride is quite linear. However, there is a suggestion of an inflection at about -70 mV. If the steady state current-voltage curve were to be taken at 30 msec rather than at 5 msec, the inflection would be more pronounced. This small departure from linearity occurred in most, but not all, of the axons tested. Figure 4 also shows that there was fairly good recovery of the axon.

DISCUSSION

The ability of an axon to propagate action potentials is based on its amplifying

140

120

100.

80 .. 60 *

40

20

- 1 d

I -22 *

-154 1 I I I 1 LO 20 30 40 msec

Fig. 3 Voltage clamp currents obtained when the axon is intracellularly perfused with 600 mM potassium fluoride and extracellularly perfused with 600 mM potassium chloride. Resting poten- tial equal to zero. Temperature about 10’ centi- grade.

74 EDUARDO ROJAS AND GERALD EHRENSTEIN

3’0 r

Fig. 4 Current-voltage curves obtained in the following situations. 0-0-0 I External sea water at the beginning A-n-A of the experiment. .-.--. IExternal sea water at the end of A-A-A J the experiment. B - M - ~ External potassium chloride.

fluoride. Temperature about 10°C. Intracellular perfusion with 600 mM potassium

property-the negative slope in the SO- dium current-voltage curve, usually re- ferred to as the negative resistance. The negative resistance can also be described as voltage-dependent permeability, where the variation of permeability with voltage is sufficiently large. Thus, the sodium neg- ative resistance corresponds to the voltage- dependent permeability described by the sodium “m” parameter of Hodgkin and Huxley ( ’52) . The Hodgkin-Huxley potas- sium “n” parameter is qualitatively similar to the sodium “m” parameter, suggesting the possibility of a potassium negative re- sistance in the presence of high external potassium concentration. Previous experi- ments on the squid giant axon bathed in a solution where potassium was the only cation have demonstrated this negative re- sistance. Although this potassium negative resistance is not responsible for the normal

action potential, it is qualitatively similar to the sodium negative resistance that is responsible. Hence, information concern- ing the nature of the potassium negative resistance may also be relevant to the ac- tion potential mechanism.

Figure 4 shows that the steady state current-voltage curve for equal concentra- tions of potassium inside and outside is ap- proximately linear. The small departure from linearity sometimes observed may represent a small residual voltage-depend- ent permeability. In any event, the nega- tive resistance region present for an unper- fused axon with the same external solu- tion is eliminated or greatly reduced when the axon is perfused with potassium fluo- ride. This implies one of the following:

(a) There is a species difference. Our experiments were performed on Dosidicus gigas, whereas the non-perfusion experi- ments with high external potassium con- centration were performed on Loligo pealli.

(b) The fluoride in the perfusion solu- tion was responsible for eliminating the negative resistance.

(c) The perfusion washed out some substance in the axoplasm that is neces- sary for the negative resistance.

We cannot with certainty decide among these possibilities. However, (a) is un- likely because voltage clamp data, when available for both species, are quite simi- lar. An example of this is the similarity between figure 2 and the comparable curves for Loligo pealli. Also, figure 2 shows that fluoride does not eliminate the sodium negative resistance. Thus, if the sodium and potassium negative resistances are due to similar phenomena, (b) is un- likely. The most likely explanation is that some substance in the axoplasm is neces- sary for the potassium negative resistance.

There is a possibility that the potassium negative resistance has been shifted rather than eliminated. Gilbert and Ehrenstein have shown that the minimum of the negative resistance region for an unper- fused axon in an external solution with high external potassium concentration is shifted in the hyperpolarizing direction when the external calcium concentration is decreased (Gilbert and Ehrenstein, ’65). They observed a shift from -40 mV to -55 mV when the calcium concentration

EXPERIMENTS ON AXONS WITH POTASSIUM AS ONLY CATION 75

of the external solution was decreased from 10 mM to zero. It is possible that a decrease in internal calcium concentra- tion, such as would be caused by perfu- sion, might shift the negative resistance further. However, the magnitude of the shift would have to be at least 80 mV to take it beyond the region of our measure- ments.

We have tacitly assumed that the com- position of the solution at the surface of the membrane is the same as that in the perfusing solution, despite the presence of a layer of axoplasm between the mem- brane and the perfusing solution. Tasaki and Luxoro ('64) have shown that this layer does not constitute a significant bar- rier for diffusing ions. In support of this view, we have observed the following:

( a ) When potassium was replaced by sodium in the perfusing solution there was an almost instantaneous decrease in the action potential.

(b) When radioactive NaZ2, Rbs6, or ClSE was placed in the perfusing solution, the surrounding sea water became radioactive in a few minutes. About five minutes later, the rate of outflux of radioactive ions reached a steady value.

These observations indicate that the ionic composition at the inside surface of the membrane was the same as in the perfusing solution.

LITERATURE CITED Gilbert, D. L., and G. Ehrenstein 1965 Effect

of calcium and magnesium on voltage clamped squid axons immersed in isosmotic potassium chloride. Abst. 23 Internat. Congr. Physiol. Sci., Tokyo.

Hodgkin, A. L., and A. F. Huxley 1952 A quantitative description of membrane current and its application to conduction and excita- tion in nerve. J. Physiol., 117: 500.

Moore, J. W. 1959 Excitation of the squid axon membrane in isosmotic potassium chloride. Nature, 183: 265.

Moore, J. W., and K. S. Cole 1963 Voltage clamp techniques. Physical Techniques in Bio- logical Research, VI: 263.

Narahashi, T. 1963 Dependence of resting and action potentials on internal potassium in perfused squid giant axons. J. Physiol., 169: 91.

Segal, J. R. 1958 An anodal threshold phenom- enon in the squid giant axon. Nature, 182: 1370.

Tasaki, I. 1963 Permeability of squid axon membrane to various ions. J. Gen. Physiol., 46: 755.

Tasaki, I., and M. Luxoro 1964 Intracellular perfusion of Chilean giant squid axons. Sci- ence, 145: 1313.

Open Discussion EHRENPREIS : Rosenberg and I showed

some years ago that bathing the squid giant axon in a whole series of enzymes was totally without effect on the action potential with one exception, namely the cobra venom preparation. We showed the active principal of cobra venom to be phos- pholipase. At very low concentrations of cobra venom, one sees in a very short time, 25 minutes or so, a complete and irrever- sible abolition of the action potential. We have since applied cobra venom to other tissues and these are almost uniformly destroyed. In smooth muscle and eel elec- troplaques, the electrical responses are abolished very rapidly. However, I think there is evidence from a pharmacological standpoint to show that there is asym- metry in the membrane. For example, under controlled conditions, using the

cobra preparation, one could achieve a reversible blockade of the action potential by merely bathing the squid axon in d- tubocurarine. These have to be very con- trolled conditions of time and concentra- tion. So it is obvious that d-tubocurarine can act on the outside of the active mem- brane. However, as you probably know, the injection of d-tubocurarine within the axon is without effect. I cite the classical experiment of del Castillo and Katz who showed that either acetylcholine or d-tubo- curarine applied by electrophoretic jet is effective on the external part of the post synaptic membrane, but if the pipette is placed a little bit below the surface there is no effect of these compounds. I think there is evidence that there is indeed a profound asymmetry of the junctional

76 EDUARDO ROJAS AND GERALD EHRENSTEIN

membrane in so far as drug effects are concerned.

ROJAS: I have to add to the previous points that I mentioned another type of evidence that was obtained through an ex- periment done by Werner Loewenstein on epithelial cell junctions in which the char- acteristic of this junction was analyzed. If we assume the same internal environ- ment in cell no. 1 as adjacent cell no. 2, the results are almost exactly like our ex- periment, in which we try to place on both sides of the membrane the same cations at the same concentration. In Loewenstein's experiment, the current voltage curve was linear.

HUNEEUS-COX: Did you do these meas- urements with sodium inside and outside?

ROJAS: I am glad that you asked this queetion. These experiments were inter- rupted by an earthquake and all of our equipment fell down, but we made a few determinations of the current voltage re- lationship with sodium. I have to admit that this was a very difficult experiment, because we had to place sodium fluoride inside the axons and sodium is a very bad cation. If we place sodium fluoride out- side, it precipitates divalent cations. The situation, in general, makes the task very hard. However, we did two experiments, one of which I can try to remember and describe to you briefly. In this experiment at the beginning we obtained the current in mA/cm2 for both the inward sodium current, and the potassium current. Dur- ing the test with equal sodium on both sides, we got a linear sort of relationship between the current and the voltage. At the end of our experiment our control value for the height of the action poten- tial was 70 millivolts and the resting poten- tial recovered to 45 millivolts with pre- vious initial control values of 50 mv for resting potential and 105 mv for action potential. Now the slope of this curve was 1/7.5 the slope for the potassium experi- ment. That's all I can tell you about the sodium case.

GRUNDFEST : There are two things which are rather peculiar. One is the fact that in squid axons which are exposed to high KC1 one has reversible effects. When one exposes lobster axons, which are per- meable to chloride, to potassium chloride

they no longer produce spikes, although the resting potential might come back. This is also true of various kinds of crusta- cean muscle fibers which again are per- meable to chloride. Another thing which is peculiar, or perhaps not so peculiar, be- cause I think I can find an explanation, is that in your last slide which showed that upon exposure to 600 millimoles of potas- sium fluoride, the membrane resistance, or the conductance decreased about one-sixth of the maximum potassium conductance. Now that I would call potassium inactiva- tion, that is blocking of potassium chan- nels, which can be done, for example, by substituting cesium or rubidium for the potassium. We have demonstrated, for example, in eel electroplaques, that this block of the potassium channels is com- pletely independent of the calcium.

ROJAS: I would like to add two points. The potassium experiment was done with potassium chloride outside and the sodium experiment was done with potassium fluo- ride outside. The second thing is that the slope of this curve in the potassium case is not one-sixth of the slope in normal sea water, but is about one-half.

GRUNDFEST: What were the figures for the conductance that you gave?

ROJAS: The resistance of the axon in potassium chloride is 133 ohms cm', and the other one is 75 ohms cm2.

GRUNDFEST : Thank you for rectifying my misunderstanding. Nevertheless, it is worth noting that the slope resistance in KCI was about double that in Na. K-inac- tivation in eel electroplaques raises the membrane resistance 2 to 10 fold, depend- ing upon experimental conditions.

ADELMAN: Did you try lower equal con- centrations on both sides of the mem- brane, such as 100 millimolar potassium fluoride or even 10 mM potassium fluoride?

ROJAS: No, we have not. ADELMAN: I think it would be interest-

ing to see whether or not the conductance is a function of the ionic strength.

ROJAS: Yes, I would say so, but we haven't done the experiment yet.

MOORE: In the high potassium experi- ment I see from your records you were using the resting potential as the potential about which you made your pulses.

EXPERIMENTS ON AXONS WITH POTASSIUM AS ONLY CATION 77

ROJAS : Yes, the membrane potential was held at the resting potential, in this case 6 mV.

MOORE: I think then the difference be- tween that result and others that have been found simply arises from the experi- mental conditions. I think that if you do the experiment where you hold at the low resting potential, there is no question that you get a straight line, but you are also changing another experimental variable. If you start from the same holding potential as that which you used for the experiment with the normal medium, you will find that you will get very slowly developing currents which vary with time. If you then start from this higher potential and sweep very slowly rather like Trautwein has done at a few millivolts per second, you will also trace out a curve. I think you also showed in your records that if you went in a de- polarizing direction, the currents were essential invarient with time, whereas in the hyperpolarizing direction they were de- creasing. When you made your current- voltage plots from the currents at very early times you obtained a straight line. When you change the time for which you take the current to make this plot, the shape will vary. Is that correct?

ROJAS: Yes. First I would like to call on Dr. Ehrenstein, who has done an ex- periment with Dr. Gilbert, in which they measured the current voltage relationship in the presence of high external potas- sium in unperfused axons. I should say first that I plot the current voltage curve by taking points from the current curves at 1 msec and at 10 msec. I did this on

purpose, because although the currents have a time variation, the current voltage curve is almost linear at all times.

EHRENSTEIN: I think that the differ- ence between the two sets of curves was not a matter of the length of the pulses. Gilbert and I, two years ago, did some ex- periments in which we held the nerve at the resting potential and obtained long- time steady state results, 10 msec at least, and found, as Moore has previously found, a negative resistance. When we went a little further on that point and changed the calcium concentration, we found that the negative resistance remained, although there were other changes in the actual curve. In the curves that Rojas and I ob- tained, the voltage conditions were exactly the same; we did measure currents at a long time as well. There were two differ- ences. One was that the axons were from Chilean squid. The main difference, I believe, was that we were perfusing. Inci- dently, we were also perfusing with fluo- ride, according to Tasaki’s previous experi- ence, and so perhaps some of the fluoride helped to get rid of any small impurities of calcium. I believe that this indicates that the reason for the difference in these re- sults compared to the negative resistance in previous results is just due to the ab- sence of calcium or perhaps some other substance usually present in axoplasm.

ROJAS: Let me add something to that. We have been doing some experiments here in Woods Hole with Taylor and Bin- stock and it appears that calcium is neces- sary for the negative resistance to be pres- ent in high external potassium.