CURRENTS ASSOCIATED WITH THE IONIC GATING STRUCTURES IN NERVE MEMBRANE* Clay M. Armstrong University of Rochester School of Medicine and Dentistry Rochester, New York 14642
Francisco Bezanilla Facultad de Cienciaslvniversidad de ChilelViAa del Mar, Chile; and Marine Biological Laboratory1 Woods Hole, Massachusetts 02543
INTRODUCTION The sodium (and potassium) channels of nerve membrane are controlled by charged gating structures that move in the membrane field, producing (at least in the case of the sodium channels) a small but measurable current, the gating current.'-' Similar currents have been observed by others and called displacement currents's or asymmetry currents? There is strong evidence that at least part of gating current is associated with the sodium channels, and this evidence is briefly reviewed below. Gating current of the potassium channels has not yet been identified. , ~ one would predict Gating current is a component of capacity c ~ r r e n t and that membrane capacity should be voltage-dependent and largest in the region where gating charge distribution is most sensitive to voltage.' We show here that membrane capacity of squid axons is voltage-dependent in the expected way. A marked frequency-dependence of membrane capacity has been demonstrated by Takashima and Schwann,' and we suggest that the frequency-dependent component is due to gating charge movement. Inactivation of the sodium conductance affects gating current amplitude,', O and we report further observations on this phenomenon. METHODS The essential elements of the technique for measuring gating current are the removal of permeant ions from both sides of the membrane, and subtraction of the linear portion of capacitative current, either by algebraically summing the current from exactly equal pulses of opposite sign, or by the more elaborate procedure described below. Gating current is small, and averaging is necessary to improve the signal-to-noise ratio. Details of the technique involved have been reported previo~sly,~ and we describe here only several improvements for increasing the speed and accuracy of recording. Apparatus
FIGURE 1 shows a block diagram of the system currently in use. All digital instruments are in a separate rack, which is connected to the remainder of
* This work was supported by Grant No. NS08951 from the United States Public Health Service. 265
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FIGURE 1. Block diagram of apparatus. See text for details.
the electrical equipment by a single signal and ground path. Other signals between the two parts of the apparatus are communicated by way of optical isolators, which are useful in preventing ground loops. The current signal recorded from the axon is digitalized and averaged, and then recorded on the magnetic tape unit of the computer. To increase the duration of the recorded sweep without sacrificing time resolution, the first 128 points in each sweep are taken (usually) at 5 microseconds per point, and the remaining 128 points at a slower rate (usually 50 microseconds per point). A major limitation on the time resolution of gating current measurements has been the necessity to blank out the first 20 to 50 microseconds following a step, to prevent saturation of the A / D converter. We have overcome this problem by using a technique based on a suggestion by Drs. David Landowne 2A) uses a transient generator (RC and Joel Brown. The technique (FIGURE circuitry and operational amplifiers) to produce a current similar to that coming from the nerve, but opposite in sign. The two currents (from the nerve and from the transient generator) are summed by the I-V converter, resulting in an output voltage that never exceeds the dynamic range of the following amplifiers or the A / D converter. Since the output of the transient generator is a linear function of the input voltage, the sum of its output for a complete cycle of a positive and a negative pulse (or, see below, four small pulses) is zero, so this device in no way alters the gating current signal that we record. Using this technique, blanking is unnecessary, and no time at all is lost following a voltage step: time resolution is limited only by the speed of the clamp, which can complete a voltage step in about 10 microseconds. A second advantage of this technique is that it cancels out a significant amount
GENERATOR
TRANSIENT GENERATOR
FIGURE 2A. A linear transient generator produces a current of time course similar to that of the axon, but of opposite sign. The two signals are summed by the I-V converter, reducing the amplitude of its output, and preventing saturation of the following amplifiers. See text.
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-J7 n n n n . ' l -70mv
'
-180 mv P/4 300 msrc FIGURE2B. P/4 pulse pattern. Current from the four small steps (amplitude P/4) is digitally subtracted from the current produced by the large step (amplitude P) to eliminate the linear portion of capacitative current.
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early outward peak of current, the gating current. Positive and negative pulses of 90 mV amplitude were applied from a holding potential of -70 mV (not P/4 pattern). The internal solution was 125 m M CsF sucrose. 8" C.
+
5 % No-SW
RESULTS When the Na concentration in the external medium is reduced to 5 % of its normal value, IAa(sodium current) is much reduced and is preceded by an outward peak of gating current (I6). IN^ can be eliminated by adding tetrodotoxin (TTX) to the external medium (FIGURE3). The early part of I, is not detectably affected by TTX,and its later part becomes visible as a current that decays smoothly and has largely subsided by the time of peak IN*.The absence of a TTX effect on Is suggests that the TTX receptor is far from the gating structures. Experiments with internal and external application of Zn++provide evidence 4 that Isa and Ig are related (for other evidence, see Reference 3). FIGURE shows IN*and Is recorded from an axon in 5% Na sea water when internally perfused with a solution containing 10 mM ZnCb IN. and Is decrease approximately in proportion to each other after addition of Zn++to the perfusion solution and both are almost completely abolished after a few minutes. During washout of Zn" from the axon interior, both currents recover at about the same rate, and recovery is essentially complete, except that after Zn", Na inactivation seems to be slowed or absent." 5: Externally applied Zn++has quite a different effect, as illustrated in FIGURE both Ix. and Is are slowed by a factor of two or three, but not eliminated. The lower traces show Is alone with and without external Zn++,and the slowing is quite evident. This is an interesting effect that requires further study, but the parallel slowing of and Is provides still more evidence that the two currents
FIGURE 4. Addition of 10 mM ZnC1, to the internal perfusion medium reversibly eliminates Lo and I,. Pulses of 80 mV amplitude were applied from a holding potential of -70 mV (not P / 4 pattern). 5% Na SW//133 Cs 5 Na. 8" C.
+
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FIGURE 5. The upper traces show the slowing of Is, and I, by external Zn". The higher amplitude trace is the control record, in 6 Ca + 30 Zn TMA//TMA Tris). For the lower amplitude trace, 27 mM ZnCL was added to the external solution. The bottom trace shows I, (without IN,,) in the presence of 30 rnM external ZnCl, (recorded in 6 Ca + 30 Zn + TMA//TMA + Tris). The middle trace is the control record (in 6 Ca++ TMA//TMA + Tris). 8 " C. P/4 pattern, with P = 100 mV, from a holding potential of -70 mV.
+
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are associated. (We thank Dr. Bertil Hille for initially informing us of the effects of external Zn++on IN..) The Time Course of Gating Current
The rising phase of gating current has not been observed before now because of the large size of the capacity current transient and the necessity to prevent saturation of the A / D converter by blanking, for up to 50 microseconds after the step. The technique described in the Methods section allows us to see all of the gating current, beginning at origin of the step. The lower traces in FIGURE5 were recorded using this technique and the P/4 pattern, which eliminates much of the rising phase seen when positive and negative pulses of equal size are applied from the holding p ~ t e n t i a l .It~ is interesting that a distinct rising phase remains nonetheless. Possible origins of this rising phase are discussed below. We have noted before that Is does not decay with the time course of a single exponentialD and this is evident in the middle trace of FIGURE 5, which has a clear slow component. Observation of the slower component is always complicated by the fact that the current trace never decays to an absolutely flat baseline: with our present techniques the current goes through a minimum and then slowly increases with time, perhaps as the result of slow alteration of an ionic permeability. Under these circumstances the definition of the baseline, which is, of course, an essential step in any fitting procedure, is rather an arbitrary matter. Our procedure is to fit a least squares straight line to the points between 1.3 and 2.6 msec after the step, and use this fitted line as baseline. The rationale is to select points before the baseline creep becomes serious, but well after the decay of the bulk of Is. FIGURE 6A is the original trace and FIGURE 6B is the same trace redrawn (by the computer) relative to the fitted baseline. A slow component is evident in both the original and the redrawn trace of FIGURE 6A and B, and it comes as no surprise that a single exponential fitted by a least squares method to the fall of the gating current (between the two cursors indicated by arrows in FIGURE6C) is not a good fit. A much better fit is obtained by using two exponentials determined by the proce-
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.i A
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400 k-c Origmel Record
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FIGURE 6. Determination of the baseline. (A) A least squares line was fitted to the points between the two cursors (arrows). The trace was then redrawn by the computer relative to this baseline in ( B ) . (C) The same trace, with an exponential curve fitted by a least squares method to the points between the arrows. The falling phase of the trace has a clear slow component, and is not well fitted by a single exponential. P/4 pattern, for a pulse to +50 mV from a holding potential of -70 mV. 6 Ca++ TMA//TMA + Tris. 8" C.
+
dure illustrated in FIGURE7. First a single exponential is fitted to the slow component (a least squares fit between the two cursors in FIGURE 7 A ) . This component is then subtracted from the original trace, and an exponential fitted to the remainder (FIGURE7B). The two component exponentials are then summed to give the curve in FIGURE7C, which is quite a good fit to the original curve. Reiteration of the process was unnecessary because the time constants of the two component exponentials are quite different, 69 and 382 microseconds. The fast component has a time constant substantially shorter than that of the single exponential fitted in FIGURE 6C. The slow component shown in FIGURE7A is surprisingly large, and it
FIGURE 7. Fitting of two exponentials to th-, falling phase. (A) An exponential was fitted to the trace between the arrows by a least squares method. (B) This exponential was subtracted from the original trace, and the resultant curve was then fitted with an exponential between the two arrows. ( C ) When summed, the two exponentials are an excellent fit for the falling phase of the current. See the legend of FIGURE 6 for conditions.
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would be of interest to know what fraction of the total charge movement it accounts for. Only a crude estimate can be given, because of ignorance regarding the early part of the time course of this component. If it is assumed to have the time course shown in FIGURE 7A, an assumption that we regard as unlikely to be close to the truth, the slow component accounts for more than half of the total charge movement. It seems clear from these observations that a sizeable slow component of gating current exists, but its significance and complete time course are, for the moment, totally obscure. Membrane Capacity and Gating Charge Movement
Membrane capacity was measured by integrating the current produced by a voltage step. For an ideal capacitor in parallel with a resistance, the integral for an applied step is the sum of a step (the capacitative component) and a ramp (a steady current flowing through the parallel resistance). If the capacitor is imperfect, capacity current is prolonged by the slow polarization of the dielectric. The result is a curve like the experimental trace shown in FIGURE 8: the slope of the integral (of membrane current) curve decreases progressively as capacity current subsides, until the curve becomes (in theory) a straight line. Gating current recorded from the same axon, for a step of the same size, is shown in the lower trace of FIGURE8. The integral curve ,.......".
FIGURE 8. The upper trace is the integral of membrane current for a step of Vm from -70 mV (the holding potential) to +50 mV. The lower trace is the gating current produced by the same step (relative to a . baseline determined as in FIGURE 6 ) . Tris SW + TTX//12SCs + 2 mM EDTA. 8°C.
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straightens out at about the same time that Ip subsides, which suggests that the slowly polarizing components of the dielectric are the structures that give rise to the gating current. This suggestion is well supported by a comparison of gating charge movement and membrane capacity. Total capacitative charge movement is obtained from a trace like the one in FIGURE 8 by extrapolating the straight-line part of the trace back to the origin of the step, and measuring the vertical distance (which has units of charge) between the extrapolated line and the integral curve just before the step. As with any method for measuring capacity, this one cannot distinguish between slow polarization of a component of the dielectric and a slow change in a resistance in parallel with a capacity. This problem is exactly analogous to the baseline problem noted above in connection with gating current measurements, and it manifests itself here as a slight curvature of the integral trace at long times, the portion of the trace that is ideally a straight line. We coped with this problem by fitting a least squares straight line to the points between 1.5 and 2.5 msec, the same limits that were used
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for fitting the baseline of gating current traces. Though somewhat arbitrary, this seems to us the fairest procedure for comparing gating charge movement and capacity measurements. The upper curve in FIGURE9 shows the differential capacitance of an axon, b Q / h V , for 10 mV intervals as a function V,. Capacity rises from about 1 pF/cm2 at -70 mV to a peak of about 1.35 puF/cm’ near -20 mV, and then decreases at more positive voltages. The part of membrane capacitance that is due to gating charge movement is proportional to the slope of the gating charge distribution curve, which is given for the same axon by the lower curve in FIGURE 9. The maximal slope of this curve occurs near -30 mV, not far from the voltage where membrane capacitance has its maximal value. At this voltage h Q / h V has a value of 300 nC cm-‘ V-’, or 0.3 cm-’. This is close to the difference between C,.(membrane capacity) at -70 mV (where there is relatively little charge movement) and Cm at
FIGURE9. The upper curve is the differential membrane capacity, AQ/AV, for 10-mV intervals. The lower curve (open circles) represents gating charge movement in units of electronic chargeslpm? for steps from a holding potential of -70 mV. Conditions are as in FIGURE 7.
-10 mV; i.e., the extra capacity at -10 mV can be largely explained as gating charge movement. Further experiments may improve the quantitation, but it seems clear from the experiment of FIGURE 9 that gating charge movement makes a significant contribution to C m in the range from -70 to +20 mV. Inactivation of Gating Charge Movement
In a previous report2~swe showed that a prepulse applied as in FIGURE 10 diminished 1, and IN* in proportion, and we gave evidence that the prepulse acted on I, by inactivating gNa (the sodium conductance). We have reexamined the effect of a prepulse using the P/4 procedure (see the inset of FIGURE 12 for the pulse pattern), and with this procedure the effect is still present, but somewhat less marked than with the old procedure. (Other experiments indicate that with the pattern of FIGURE 10, a positive prepulse before the negative test pulse enhances the inward current in the test pulse. The significance of this extra inward current is unknown.) An example of the effect
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FIGURE10. Pulse pattern used in previous experiments for demonstrating the effect of inactivation on Ia.
of a prepulse with the P/4 pattern is shown in FIGURE 11. The current trace of larger amplitude is the control, for which there was no prepulse. For the lower amplitude trace, the test pulse (to $20 mV) was preceded by a 10-msec prepulse (to +20 mV) followed by a 1-msec recovery interval at -70 mV. Reduction of gating current by a prepulse was studied quantitatively by integrating Is traces recorded after recovery intervals of various durations, yielding the results plotted in FIGURE12. Charge movement in the test pulse FIGURE11. The effect of a prepulse on I,. The lower amplitude trace shows I, for a step to +20 mV after a prepulse (see the inset of FIGURE 12 for the pulse pattern). The other trace is the control, without prepulse. The prepulse was to +20 mV for 10 msec, and there was a 1-msec recovery interval between pre- and test pulse. Tris SW + TTX//125Cs, at 8 ° C .
5.
50 pa/crn2
. ?
00 psec 6
is strongly dependent on the recovery interval. After recovery for 0.5 msec, charge movement is about 40% of the control movement, but it almost equals control movement after a recovery interval of 12 msec. The prepulse apparently immobilizes more than half of the gating charge, trapping part of it for a period of milliseconds. Ultimately, of course, the gating charge must return to its “closed” position, and be free to move during a test pulse, for the effect of a prepulse is not irreversible. It could be argued that 0.5 msec is
FIGURE12. Gating charge movement during the test pulse as a function of the interval between conditioning and test pulse. Gating charge movement has been normalized relative to that after a very long recovery interval. The inset shows the pulse pattern. 20 Ca 22 Na + Tris TTX//125 CsF, 8 ” C.
+
+
8-c
,
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too short an interval to allow even the rapid component of return current to subside. With this in mind we examined the inward gating current tail at the end of the prepulse, and found that it could be fitted fairly well by an exponential with a time constant of 95 psec. The 0.5-msec recovery interval was thus more than five time constants for this component. The remainder of the gating charge returned over an interval of more than 12 msec (the time required for complete recovery), producing a current that was much too small to detect.
DISCUSSION There is now a strong case for associating gating current with opening and closing of the Na channels.', Briefly, the evidence is that Ig has an appropriate time course; at least a part of it appears to inactivate as does gNn; and both IN^ and I, are blocked reversibly by prolonged depolarization or internal perfusion with Znf+. To this list can now be added another correlation, the slowing of both IN. and I. by external Zn++ion. I, and IN* are clearly related, but regarding the precise manner of their interrelationship there are perhaps more questions than answers at present. A first question is the exact time course of the gating current associated with the opening of the Na channels. The records presented above have a distinct rising phase that lasts for 25-50 psec. We have shown that much of the rising phase for steps of equal amplitude from the holding potential arises from inward current during the negative step.' In the records illustrated here, however, this charge movement has been reduced if not eliminated by using the P/4 pattern: the subtraction pulses cover a very negative voltage range where there is relatively little charge movement. There are three possible origins for the rising phase seen with this procedure. (1) There is enough current even in the negative range near -170 mV to account for it. (2) It is due to a slower-than-expected response of the voltage clamp, which completes a step in 50 p e c rather than 10 psec. This seems unlikely to us, but not impossible. (3) The rising phase is a feature of the gating current associated with the opening of the N a channels. Physically this would mean that the first steps involved in the opening of the channels involve relatively little charge movement. At present we are unable to decide clearly among these possiblities. Another question concerns the significance of the slower component of gating current demonstrated in FIGURES 6 and 7.A slow component of current has been noted by us previously," but has not been mentioned by Keynes and Rojas'. or Meves.6 This is probably the result of a difference in method. We see this component clearly only when using the P/4 pulse pattern, and in records where the current is followed by a long and relatively flat baseline. It remains to be seen whether the presence of this component will be confirmed by others when they adopt a similar procedure. The origin of the slow component, which accounts for a very significant fraction of total charge movement, is almost totally obscure at present, and it could be associated with any, or none, of the three gating functions, Na activation, Na inactivation, o r K activation. At present we are inclined to believe it is associated with Na activation, but the evidence is not strong enough to merit a summary.
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Inactivation affects gating current, and appears to immobilize a large fraction of the gating charge. This is suggested by FIGURES 11 and 12, which show that a positive prepulse, which should inactivate IN*, diminishes the gating charge movement during a following test pulse. After a prepulse, only a fraction of gating charge moves back to “closed” position quickly, as can be directly confirmed by integrating the gating current tail at the end of the prepulse. The remainder of the charge apparently moves back over the course of a number of milliseconds, producing a current that is too small to detect with present methods. The evidence that a prepulse affects Ig by inactivating g N n is: (i) recovery of I, following the prepulse is similar in time course to the recovery of gX.; and (ii) pronase, which destroys Na inactivation, removes the effect of a prepulse on both Ic and gNn. In spite of this rather clear evidence, an effect of inactivation on I: has been disputed by In our view, confusion has arisen mainly from a failure to distinguish between the inactivation process described by Hodgkin and Huxley” and slow inactivation, which is quite a different phenomenon. If pulses are applied to a fiber held at a relatively positive potential, net charge movement is inward, as has been reported by Keynes and Rojas.” This current has been interpreted as normal gating current, and taken as evidence that I, does not inactivate. We confirm the existence of this current, which we observed in our original experiments, but we do not believe that it is gating charge moving in the normal mode, for the following reason. A fiber held at +56 mV for two minutes shows inward current when pulses are applied to it, but when pulsed after a sudden change of the holding potential from f 5 6 to -70 mV, there is initially neither gating current nor IN^. Iy and IN^ then recover with parallel time courses, with a half time of about 3 0 sec.’ The origin of the inward current seen at positive holding potentials is thus not at all clear, since the fiber can produce IN. neither at +56 mV, nor, for many seconds, at -70 mV after return from +56 mV. There is no evidence to link the inward current at positive holding potentials or the N a activation mechanism. Information regarding the inactivation process described by Hodgkin and Huxley can be obtained only by means of relatively short depolarizations (not exceeding 50 or 100 msec) applied to an axon held at a potential in the normal range (60 mV or more negative). The only published experiments of this type are our own, and these yield the results described above. As noted by Chandler and Meves,I8 internal perfusion with Cs+ slows or removes Na inactivation. We find that after prolonged exposure to Cs+, inactivation of IxI is markedly slowed, and inactivation of Ie becomes more difficult to demonstrate. The removal of inactivation by Cs’ is fortunate in a sense, for it made simpler the demonstration of equal on and off charge movement (i.e., equality of outward charge movement during opening of the channels and inward movement during closing of the channels), a point important in demonstrating that gating current is capacitative. We stress, however, that in fresh axons, on and off charge movements are equal only for pulses of short duration. We have shown here that membrane capacity is voltage-dependent, with a maximal value of roughly 1.35 pF/cmz at about -10 mV, and a value of roughly 1 pF/cm* at -60 mV. The difference in capacitance at the two voltages is at least in large part due to gating charge movement. This large contribution of the gating structures to membrane capacitance provides an
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answer to a frequently asked question: Why do not membrane molecules other than the gating structures contribute to capacitative current? Some contribution from nongating molecules cannot be excluded, but it is clear that a large increase in capacitance from this source would slow action potential propagation and be functionally disadvantageous.
REFERENCES C. M. & F. BEZANILLA. 1973. Currents related to movement of 1. ARMSTRONG, the gating particles of the sodium channel. Nature 242: 459. F. & C. M. ARMSTRONG. 1974. Gating currents of the sodium chan2. BEZANILLA, nels: three ways to block them. Science 183: 753. C. M. & F. BEZANILLA. 1974. Charge movement associated with 3. ARMSTRONG, the opening and closing of the activation gates of the Na channels. J. Gen. Physiol. 63: 533-552. 4. KEYNES,R. D. & E. ROJAS. 1973. Characteristics of the sodium gating current in squid giant axons. J. Physiol. 233: 28P. 5. KEYNES,R. D. & E. ROJAS. 1974. Kinetics and steady state properties of the charged system controlling sodium conductance in the squid giant axon. J. Physiol. 239 393. 6. MEVES,H. 1974. The effect of holding potential on the asymmetry currents in squid giant axons. J. Physiol. 243: 847. 7. CHANDLER, W.K. & M. F. SCmwDER. Personal communication. 8. TAKASFTIMA, S. & H. P. SCHWA". 1974. Passive electrical properties of squid axon membrane. J. Membt. Biol. 17: 51. F. & C. M. ARMSTRONG. 1975. Kinetic properties and inactivation of 9. BEZANILLA, the gating currents of sodium channels in squid axon. Phil. Trans. R. SOC. Lond., Ser. B. 270: 449. 10. Fox, J. M., E. ROJAS& R. SWMPFLI. 1974. Blocking of sodium and potassium conductance by internal application of Zn++in the node of Ranvier. Pflugers Arch. 351: 271. A. L. & A. F. HUXLEY.1952. A quantitative description of membrane 1 1 . HODGKIN, current and its application to conduction and excitation in nerve. J. Physiol. 117: 500. 12. KEYNES,R. D., E. ROJAS& B. RUDY.1974. Dmonstration of first-order voltagedependent transition of the sodium activation gates. J. Physiol. 239: 1OOP. W.K. & H. Meves. 1970. Evidence for two types of sodium conduc13. CHANDLER, tance in axons perfused with sodium fluoride solution. J. Physiol. 211: 653.
DISCUSSION DR. RACKER:Could you describe the pronase preparations you used and tell us whether you have established what is removed from the membrane after pronase treatment? I would also like to ask Dr. Mueller whether this complicates his explanation of inactivation. DR. BEZANILLA:The pronase preparation used is not highly purified and some components may be inactive in the perfusion solution, which contains
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fluoride. We don’t know what part of the enzyme is really acting, but we do know that the effect is due to the enzymatic activity because the reaction is strongly temperature-dependent. Furthermore, boiling destroys the activity. DR. MUELLER:Our model is not inconsistent with the pronase data. If the association energy of a nonconducting oligomer is raised by protease activity, this could result in a similar lack of inactivation. The gating currents can be accounted for in similar manner. Of course, we do not claim that the same system is operative in nerve membrane. DR. URRY: Could you give a value for the gating charge per channel? DR. BEZANILLA:In order to answer that question I would have to make an assumption about channel density, so instead of giving you that value, I shall give you another and you make a calculation according to the assumption that you want to make. Therefore I am going to tell you that there are twelve hundred electronic charges per pm2. DR. LEE: Did you see any effect of pronase treatment on the potassium channel? DR. BEZANILLA:No. DR. LEE: Are the gating currents dependent on metabolic energy? DR. BEZANILLA:No. The gating currents are seen regardless of the ionic electrochemical gradients across the membrane. Also, we generally use fluoride inside and provide no chemical source of metabolic energy. DR. MILLER:How convinced are you that the rising phase of your gating current is real? DR. BEZANILLA: In our older procedure we added currents resulting from hyperpolarizing and depolarizing pulses of equal size, from the holding potential. We proposed that the currents for each step, negative and positive, rose instantaneously and then fell, but amplitudes and time constants were different for the two pulses and hence their sum showed a rising phase. In other words, there is some gating charge movement during the negative steps, and this adds onto and distorts the positive step gating current, which is the current we wish to see. In the new procedure we take one pulse from the very negative region of membrane potential, where the charge movement is extremely small, and we still see some rising phase in the averaged current. So the question remains: does the current associated with the opening of channels have a rising phase? DR. FINKELSTEIN: Can 1 pursue that point a moment? I thought in your earlier publication you had shown that by proper setting of your pulses you could completely eliminate the rising phase. DR. BEZANILLA: In those experiments we had a blanking period of 20-50 microseconds and there was therefore some uncertainty with regard to the rising phase. In our present method we have improved the time resolution and we see a rising phase. DR. MAURO: Dr. Bezanilla would you kindly comment on the temperaturedependence of the gating currents. DR. BEZANILLA: The temperature-dependence is quite strong with a Qlo of the order of 2.5 or 2.7. However, we have to be careful in deciding whether this overall QIOhas any meaning because now that we see more than one component we should analyze them separately.
Francisco Bezanilla Facultad de Cienciaslvniversidad de ChilelViAa del Mar, Chile; and Marine Biological Laboratory1 Woods Hole, Massachusetts 02543
INTRODUCTION The sodium (and potassium) channels of nerve membrane are controlled by charged gating structures that move in the membrane field, producing (at least in the case of the sodium channels) a small but measurable current, the gating current.'-' Similar currents have been observed by others and called displacement currents's or asymmetry currents? There is strong evidence that at least part of gating current is associated with the sodium channels, and this evidence is briefly reviewed below. Gating current of the potassium channels has not yet been identified. , ~ one would predict Gating current is a component of capacity c ~ r r e n t and that membrane capacity should be voltage-dependent and largest in the region where gating charge distribution is most sensitive to voltage.' We show here that membrane capacity of squid axons is voltage-dependent in the expected way. A marked frequency-dependence of membrane capacity has been demonstrated by Takashima and Schwann,' and we suggest that the frequency-dependent component is due to gating charge movement. Inactivation of the sodium conductance affects gating current amplitude,', O and we report further observations on this phenomenon. METHODS The essential elements of the technique for measuring gating current are the removal of permeant ions from both sides of the membrane, and subtraction of the linear portion of capacitative current, either by algebraically summing the current from exactly equal pulses of opposite sign, or by the more elaborate procedure described below. Gating current is small, and averaging is necessary to improve the signal-to-noise ratio. Details of the technique involved have been reported previo~sly,~ and we describe here only several improvements for increasing the speed and accuracy of recording. Apparatus
FIGURE 1 shows a block diagram of the system currently in use. All digital instruments are in a separate rack, which is connected to the remainder of
* This work was supported by Grant No. NS08951 from the United States Public Health Service. 265
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Annals New York Academy of Sciences
FIGURE 1. Block diagram of apparatus. See text for details.
the electrical equipment by a single signal and ground path. Other signals between the two parts of the apparatus are communicated by way of optical isolators, which are useful in preventing ground loops. The current signal recorded from the axon is digitalized and averaged, and then recorded on the magnetic tape unit of the computer. To increase the duration of the recorded sweep without sacrificing time resolution, the first 128 points in each sweep are taken (usually) at 5 microseconds per point, and the remaining 128 points at a slower rate (usually 50 microseconds per point). A major limitation on the time resolution of gating current measurements has been the necessity to blank out the first 20 to 50 microseconds following a step, to prevent saturation of the A / D converter. We have overcome this problem by using a technique based on a suggestion by Drs. David Landowne 2A) uses a transient generator (RC and Joel Brown. The technique (FIGURE circuitry and operational amplifiers) to produce a current similar to that coming from the nerve, but opposite in sign. The two currents (from the nerve and from the transient generator) are summed by the I-V converter, resulting in an output voltage that never exceeds the dynamic range of the following amplifiers or the A / D converter. Since the output of the transient generator is a linear function of the input voltage, the sum of its output for a complete cycle of a positive and a negative pulse (or, see below, four small pulses) is zero, so this device in no way alters the gating current signal that we record. Using this technique, blanking is unnecessary, and no time at all is lost following a voltage step: time resolution is limited only by the speed of the clamp, which can complete a voltage step in about 10 microseconds. A second advantage of this technique is that it cancels out a significant amount
GENERATOR
TRANSIENT GENERATOR
FIGURE 2A. A linear transient generator produces a current of time course similar to that of the axon, but of opposite sign. The two signals are summed by the I-V converter, reducing the amplitude of its output, and preventing saturation of the following amplifiers. See text.
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-J7 n n n n . ' l -70mv
'
-180 mv P/4 300 msrc FIGURE2B. P/4 pulse pattern. Current from the four small steps (amplitude P/4) is digitally subtracted from the current produced by the large step (amplitude P) to eliminate the linear portion of capacitative current.
?-.--
+w-
.
/-@*'
>. %-@"
.&*'-
early outward peak of current, the gating current. Positive and negative pulses of 90 mV amplitude were applied from a holding potential of -70 mV (not P/4 pattern). The internal solution was 125 m M CsF sucrose. 8" C.
+
5 % No-SW
RESULTS When the Na concentration in the external medium is reduced to 5 % of its normal value, IAa(sodium current) is much reduced and is preceded by an outward peak of gating current (I6). IN^ can be eliminated by adding tetrodotoxin (TTX) to the external medium (FIGURE3). The early part of I, is not detectably affected by TTX,and its later part becomes visible as a current that decays smoothly and has largely subsided by the time of peak IN*.The absence of a TTX effect on Is suggests that the TTX receptor is far from the gating structures. Experiments with internal and external application of Zn++provide evidence 4 that Isa and Ig are related (for other evidence, see Reference 3). FIGURE shows IN*and Is recorded from an axon in 5% Na sea water when internally perfused with a solution containing 10 mM ZnCb IN. and Is decrease approximately in proportion to each other after addition of Zn++to the perfusion solution and both are almost completely abolished after a few minutes. During washout of Zn" from the axon interior, both currents recover at about the same rate, and recovery is essentially complete, except that after Zn", Na inactivation seems to be slowed or absent." 5: Externally applied Zn++has quite a different effect, as illustrated in FIGURE both Ix. and Is are slowed by a factor of two or three, but not eliminated. The lower traces show Is alone with and without external Zn++,and the slowing is quite evident. This is an interesting effect that requires further study, but the parallel slowing of and Is provides still more evidence that the two currents
FIGURE 4. Addition of 10 mM ZnC1, to the internal perfusion medium reversibly eliminates Lo and I,. Pulses of 80 mV amplitude were applied from a holding potential of -70 mV (not P / 4 pattern). 5% Na SW//133 Cs 5 Na. 8" C.
+
Armstrong & Bezanilla:
FIGURE 5. The upper traces show the slowing of Is, and I, by external Zn". The higher amplitude trace is the control record, in 6 Ca + 30 Zn TMA//TMA Tris). For the lower amplitude trace, 27 mM ZnCL was added to the external solution. The bottom trace shows I, (without IN,,) in the presence of 30 rnM external ZnCl, (recorded in 6 Ca + 30 Zn + TMA//TMA + Tris). The middle trace is the control record (in 6 Ca++ TMA//TMA + Tris). 8 " C. P/4 pattern, with P = 100 mV, from a holding potential of -70 mV.
+
+
+
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Sodium Gating Currents
;\
I ; . i. I
\
. . . . . . . . . . 2w
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......................... . . . . . . .........
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Jolaxd
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4 W '
.............................................. +*: L... ......................
;\ . . . w . . . . ........................................... ..................
are associated. (We thank Dr. Bertil Hille for initially informing us of the effects of external Zn++on IN..) The Time Course of Gating Current
The rising phase of gating current has not been observed before now because of the large size of the capacity current transient and the necessity to prevent saturation of the A / D converter by blanking, for up to 50 microseconds after the step. The technique described in the Methods section allows us to see all of the gating current, beginning at origin of the step. The lower traces in FIGURE5 were recorded using this technique and the P/4 pattern, which eliminates much of the rising phase seen when positive and negative pulses of equal size are applied from the holding p ~ t e n t i a l .It~ is interesting that a distinct rising phase remains nonetheless. Possible origins of this rising phase are discussed below. We have noted before that Is does not decay with the time course of a single exponentialD and this is evident in the middle trace of FIGURE 5, which has a clear slow component. Observation of the slower component is always complicated by the fact that the current trace never decays to an absolutely flat baseline: with our present techniques the current goes through a minimum and then slowly increases with time, perhaps as the result of slow alteration of an ionic permeability. Under these circumstances the definition of the baseline, which is, of course, an essential step in any fitting procedure, is rather an arbitrary matter. Our procedure is to fit a least squares straight line to the points between 1.3 and 2.6 msec after the step, and use this fitted line as baseline. The rationale is to select points before the baseline creep becomes serious, but well after the decay of the bulk of Is. FIGURE 6A is the original trace and FIGURE 6B is the same trace redrawn (by the computer) relative to the fitted baseline. A slow component is evident in both the original and the redrawn trace of FIGURE 6A and B, and it comes as no surprise that a single exponential fitted by a least squares method to the fall of the gating current (between the two cursors indicated by arrows in FIGURE6C) is not a good fit. A much better fit is obtained by using two exponentials determined by the proce-
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Annals New York Academy of Sciences
.i A
L
400 k-c Origmel Record
. ........................................... ' c;. ... . . . . . . .!. . . . . . . . . .I. . .
FIGURE 6. Determination of the baseline. (A) A least squares line was fitted to the points between the two cursors (arrows). The trace was then redrawn by the computer relative to this baseline in ( B ) . (C) The same trace, with an exponential curve fitted by a least squares method to the points between the arrows. The falling phase of the trace has a clear slow component, and is not well fitted by a single exponential. P/4 pattern, for a pulse to +50 mV from a holding potential of -70 mV. 6 Ca++ TMA//TMA + Tris. 8" C.
+
dure illustrated in FIGURE7. First a single exponential is fitted to the slow component (a least squares fit between the two cursors in FIGURE 7 A ) . This component is then subtracted from the original trace, and an exponential fitted to the remainder (FIGURE7B). The two component exponentials are then summed to give the curve in FIGURE7C, which is quite a good fit to the original curve. Reiteration of the process was unnecessary because the time constants of the two component exponentials are quite different, 69 and 382 microseconds. The fast component has a time constant substantially shorter than that of the single exponential fitted in FIGURE 6C. The slow component shown in FIGURE7A is surprisingly large, and it
FIGURE 7. Fitting of two exponentials to th-, falling phase. (A) An exponential was fitted to the trace between the arrows by a least squares method. (B) This exponential was subtracted from the original trace, and the resultant curve was then fitted with an exponential between the two arrows. ( C ) When summed, the two exponentials are an excellent fit for the falling phase of the current. See the legend of FIGURE 6 for conditions.
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Sodium Gating Currents
would be of interest to know what fraction of the total charge movement it accounts for. Only a crude estimate can be given, because of ignorance regarding the early part of the time course of this component. If it is assumed to have the time course shown in FIGURE 7A, an assumption that we regard as unlikely to be close to the truth, the slow component accounts for more than half of the total charge movement. It seems clear from these observations that a sizeable slow component of gating current exists, but its significance and complete time course are, for the moment, totally obscure. Membrane Capacity and Gating Charge Movement
Membrane capacity was measured by integrating the current produced by a voltage step. For an ideal capacitor in parallel with a resistance, the integral for an applied step is the sum of a step (the capacitative component) and a ramp (a steady current flowing through the parallel resistance). If the capacitor is imperfect, capacity current is prolonged by the slow polarization of the dielectric. The result is a curve like the experimental trace shown in FIGURE 8: the slope of the integral (of membrane current) curve decreases progressively as capacity current subsides, until the curve becomes (in theory) a straight line. Gating current recorded from the same axon, for a step of the same size, is shown in the lower trace of FIGURE8. The integral curve ,.......".
FIGURE 8. The upper trace is the integral of membrane current for a step of Vm from -70 mV (the holding potential) to +50 mV. The lower trace is the gating current produced by the same step (relative to a . baseline determined as in FIGURE 6 ) . Tris SW + TTX//12SCs + 2 mM EDTA. 8°C.
-'.
IO3nuu/Cd
3
.
.
I
.
- .
.
.
a
1oou3ald
I
......................... 200 Il*c
straightens out at about the same time that Ip subsides, which suggests that the slowly polarizing components of the dielectric are the structures that give rise to the gating current. This suggestion is well supported by a comparison of gating charge movement and membrane capacity. Total capacitative charge movement is obtained from a trace like the one in FIGURE 8 by extrapolating the straight-line part of the trace back to the origin of the step, and measuring the vertical distance (which has units of charge) between the extrapolated line and the integral curve just before the step. As with any method for measuring capacity, this one cannot distinguish between slow polarization of a component of the dielectric and a slow change in a resistance in parallel with a capacity. This problem is exactly analogous to the baseline problem noted above in connection with gating current measurements, and it manifests itself here as a slight curvature of the integral trace at long times, the portion of the trace that is ideally a straight line. We coped with this problem by fitting a least squares straight line to the points between 1.5 and 2.5 msec, the same limits that were used
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Annals New York Academy of Sciences
for fitting the baseline of gating current traces. Though somewhat arbitrary, this seems to us the fairest procedure for comparing gating charge movement and capacity measurements. The upper curve in FIGURE9 shows the differential capacitance of an axon, b Q / h V , for 10 mV intervals as a function V,. Capacity rises from about 1 pF/cm2 at -70 mV to a peak of about 1.35 puF/cm’ near -20 mV, and then decreases at more positive voltages. The part of membrane capacitance that is due to gating charge movement is proportional to the slope of the gating charge distribution curve, which is given for the same axon by the lower curve in FIGURE 9. The maximal slope of this curve occurs near -30 mV, not far from the voltage where membrane capacitance has its maximal value. At this voltage h Q / h V has a value of 300 nC cm-‘ V-’, or 0.3 cm-’. This is close to the difference between C,.(membrane capacity) at -70 mV (where there is relatively little charge movement) and Cm at
FIGURE9. The upper curve is the differential membrane capacity, AQ/AV, for 10-mV intervals. The lower curve (open circles) represents gating charge movement in units of electronic chargeslpm? for steps from a holding potential of -70 mV. Conditions are as in FIGURE 7.
-10 mV; i.e., the extra capacity at -10 mV can be largely explained as gating charge movement. Further experiments may improve the quantitation, but it seems clear from the experiment of FIGURE 9 that gating charge movement makes a significant contribution to C m in the range from -70 to +20 mV. Inactivation of Gating Charge Movement
In a previous report2~swe showed that a prepulse applied as in FIGURE 10 diminished 1, and IN* in proportion, and we gave evidence that the prepulse acted on I, by inactivating gNa (the sodium conductance). We have reexamined the effect of a prepulse using the P/4 procedure (see the inset of FIGURE 12 for the pulse pattern), and with this procedure the effect is still present, but somewhat less marked than with the old procedure. (Other experiments indicate that with the pattern of FIGURE 10, a positive prepulse before the negative test pulse enhances the inward current in the test pulse. The significance of this extra inward current is unknown.) An example of the effect
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FIGURE10. Pulse pattern used in previous experiments for demonstrating the effect of inactivation on Ia.
of a prepulse with the P/4 pattern is shown in FIGURE 11. The current trace of larger amplitude is the control, for which there was no prepulse. For the lower amplitude trace, the test pulse (to $20 mV) was preceded by a 10-msec prepulse (to +20 mV) followed by a 1-msec recovery interval at -70 mV. Reduction of gating current by a prepulse was studied quantitatively by integrating Is traces recorded after recovery intervals of various durations, yielding the results plotted in FIGURE12. Charge movement in the test pulse FIGURE11. The effect of a prepulse on I,. The lower amplitude trace shows I, for a step to +20 mV after a prepulse (see the inset of FIGURE 12 for the pulse pattern). The other trace is the control, without prepulse. The prepulse was to +20 mV for 10 msec, and there was a 1-msec recovery interval between pre- and test pulse. Tris SW + TTX//125Cs, at 8 ° C .
5.
50 pa/crn2
. ?
00 psec 6
is strongly dependent on the recovery interval. After recovery for 0.5 msec, charge movement is about 40% of the control movement, but it almost equals control movement after a recovery interval of 12 msec. The prepulse apparently immobilizes more than half of the gating charge, trapping part of it for a period of milliseconds. Ultimately, of course, the gating charge must return to its “closed” position, and be free to move during a test pulse, for the effect of a prepulse is not irreversible. It could be argued that 0.5 msec is
FIGURE12. Gating charge movement during the test pulse as a function of the interval between conditioning and test pulse. Gating charge movement has been normalized relative to that after a very long recovery interval. The inset shows the pulse pattern. 20 Ca 22 Na + Tris TTX//125 CsF, 8 ” C.
+
+
8-c
,
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Annals New York Academy of Sciences
too short an interval to allow even the rapid component of return current to subside. With this in mind we examined the inward gating current tail at the end of the prepulse, and found that it could be fitted fairly well by an exponential with a time constant of 95 psec. The 0.5-msec recovery interval was thus more than five time constants for this component. The remainder of the gating charge returned over an interval of more than 12 msec (the time required for complete recovery), producing a current that was much too small to detect.
DISCUSSION There is now a strong case for associating gating current with opening and closing of the Na channels.', Briefly, the evidence is that Ig has an appropriate time course; at least a part of it appears to inactivate as does gNn; and both IN^ and I, are blocked reversibly by prolonged depolarization or internal perfusion with Znf+. To this list can now be added another correlation, the slowing of both IN. and I. by external Zn++ion. I, and IN* are clearly related, but regarding the precise manner of their interrelationship there are perhaps more questions than answers at present. A first question is the exact time course of the gating current associated with the opening of the Na channels. The records presented above have a distinct rising phase that lasts for 25-50 psec. We have shown that much of the rising phase for steps of equal amplitude from the holding potential arises from inward current during the negative step.' In the records illustrated here, however, this charge movement has been reduced if not eliminated by using the P/4 pattern: the subtraction pulses cover a very negative voltage range where there is relatively little charge movement. There are three possible origins for the rising phase seen with this procedure. (1) There is enough current even in the negative range near -170 mV to account for it. (2) It is due to a slower-than-expected response of the voltage clamp, which completes a step in 50 p e c rather than 10 psec. This seems unlikely to us, but not impossible. (3) The rising phase is a feature of the gating current associated with the opening of the N a channels. Physically this would mean that the first steps involved in the opening of the channels involve relatively little charge movement. At present we are unable to decide clearly among these possiblities. Another question concerns the significance of the slower component of gating current demonstrated in FIGURES 6 and 7.A slow component of current has been noted by us previously," but has not been mentioned by Keynes and Rojas'. or Meves.6 This is probably the result of a difference in method. We see this component clearly only when using the P/4 pulse pattern, and in records where the current is followed by a long and relatively flat baseline. It remains to be seen whether the presence of this component will be confirmed by others when they adopt a similar procedure. The origin of the slow component, which accounts for a very significant fraction of total charge movement, is almost totally obscure at present, and it could be associated with any, or none, of the three gating functions, Na activation, Na inactivation, o r K activation. At present we are inclined to believe it is associated with Na activation, but the evidence is not strong enough to merit a summary.
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Inactivation affects gating current, and appears to immobilize a large fraction of the gating charge. This is suggested by FIGURES 11 and 12, which show that a positive prepulse, which should inactivate IN*, diminishes the gating charge movement during a following test pulse. After a prepulse, only a fraction of gating charge moves back to “closed” position quickly, as can be directly confirmed by integrating the gating current tail at the end of the prepulse. The remainder of the charge apparently moves back over the course of a number of milliseconds, producing a current that is too small to detect with present methods. The evidence that a prepulse affects Ig by inactivating g N n is: (i) recovery of I, following the prepulse is similar in time course to the recovery of gX.; and (ii) pronase, which destroys Na inactivation, removes the effect of a prepulse on both Ic and gNn. In spite of this rather clear evidence, an effect of inactivation on I: has been disputed by In our view, confusion has arisen mainly from a failure to distinguish between the inactivation process described by Hodgkin and Huxley” and slow inactivation, which is quite a different phenomenon. If pulses are applied to a fiber held at a relatively positive potential, net charge movement is inward, as has been reported by Keynes and Rojas.” This current has been interpreted as normal gating current, and taken as evidence that I, does not inactivate. We confirm the existence of this current, which we observed in our original experiments, but we do not believe that it is gating charge moving in the normal mode, for the following reason. A fiber held at +56 mV for two minutes shows inward current when pulses are applied to it, but when pulsed after a sudden change of the holding potential from f 5 6 to -70 mV, there is initially neither gating current nor IN^. Iy and IN^ then recover with parallel time courses, with a half time of about 3 0 sec.’ The origin of the inward current seen at positive holding potentials is thus not at all clear, since the fiber can produce IN. neither at +56 mV, nor, for many seconds, at -70 mV after return from +56 mV. There is no evidence to link the inward current at positive holding potentials or the N a activation mechanism. Information regarding the inactivation process described by Hodgkin and Huxley can be obtained only by means of relatively short depolarizations (not exceeding 50 or 100 msec) applied to an axon held at a potential in the normal range (60 mV or more negative). The only published experiments of this type are our own, and these yield the results described above. As noted by Chandler and Meves,I8 internal perfusion with Cs+ slows or removes Na inactivation. We find that after prolonged exposure to Cs+, inactivation of IxI is markedly slowed, and inactivation of Ie becomes more difficult to demonstrate. The removal of inactivation by Cs’ is fortunate in a sense, for it made simpler the demonstration of equal on and off charge movement (i.e., equality of outward charge movement during opening of the channels and inward movement during closing of the channels), a point important in demonstrating that gating current is capacitative. We stress, however, that in fresh axons, on and off charge movements are equal only for pulses of short duration. We have shown here that membrane capacity is voltage-dependent, with a maximal value of roughly 1.35 pF/cmz at about -10 mV, and a value of roughly 1 pF/cm* at -60 mV. The difference in capacitance at the two voltages is at least in large part due to gating charge movement. This large contribution of the gating structures to membrane capacitance provides an
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answer to a frequently asked question: Why do not membrane molecules other than the gating structures contribute to capacitative current? Some contribution from nongating molecules cannot be excluded, but it is clear that a large increase in capacitance from this source would slow action potential propagation and be functionally disadvantageous.
REFERENCES C. M. & F. BEZANILLA. 1973. Currents related to movement of 1. ARMSTRONG, the gating particles of the sodium channel. Nature 242: 459. F. & C. M. ARMSTRONG. 1974. Gating currents of the sodium chan2. BEZANILLA, nels: three ways to block them. Science 183: 753. C. M. & F. BEZANILLA. 1974. Charge movement associated with 3. ARMSTRONG, the opening and closing of the activation gates of the Na channels. J. Gen. Physiol. 63: 533-552. 4. KEYNES,R. D. & E. ROJAS. 1973. Characteristics of the sodium gating current in squid giant axons. J. Physiol. 233: 28P. 5. KEYNES,R. D. & E. ROJAS. 1974. Kinetics and steady state properties of the charged system controlling sodium conductance in the squid giant axon. J. Physiol. 239 393. 6. MEVES,H. 1974. The effect of holding potential on the asymmetry currents in squid giant axons. J. Physiol. 243: 847. 7. CHANDLER, W.K. & M. F. SCmwDER. Personal communication. 8. TAKASFTIMA, S. & H. P. SCHWA". 1974. Passive electrical properties of squid axon membrane. J. Membt. Biol. 17: 51. F. & C. M. ARMSTRONG. 1975. Kinetic properties and inactivation of 9. BEZANILLA, the gating currents of sodium channels in squid axon. Phil. Trans. R. SOC. Lond., Ser. B. 270: 449. 10. Fox, J. M., E. ROJAS& R. SWMPFLI. 1974. Blocking of sodium and potassium conductance by internal application of Zn++in the node of Ranvier. Pflugers Arch. 351: 271. A. L. & A. F. HUXLEY.1952. A quantitative description of membrane 1 1 . HODGKIN, current and its application to conduction and excitation in nerve. J. Physiol. 117: 500. 12. KEYNES,R. D., E. ROJAS& B. RUDY.1974. Dmonstration of first-order voltagedependent transition of the sodium activation gates. J. Physiol. 239: 1OOP. W.K. & H. Meves. 1970. Evidence for two types of sodium conduc13. CHANDLER, tance in axons perfused with sodium fluoride solution. J. Physiol. 211: 653.
DISCUSSION DR. RACKER:Could you describe the pronase preparations you used and tell us whether you have established what is removed from the membrane after pronase treatment? I would also like to ask Dr. Mueller whether this complicates his explanation of inactivation. DR. BEZANILLA:The pronase preparation used is not highly purified and some components may be inactive in the perfusion solution, which contains
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fluoride. We don’t know what part of the enzyme is really acting, but we do know that the effect is due to the enzymatic activity because the reaction is strongly temperature-dependent. Furthermore, boiling destroys the activity. DR. MUELLER:Our model is not inconsistent with the pronase data. If the association energy of a nonconducting oligomer is raised by protease activity, this could result in a similar lack of inactivation. The gating currents can be accounted for in similar manner. Of course, we do not claim that the same system is operative in nerve membrane. DR. URRY: Could you give a value for the gating charge per channel? DR. BEZANILLA:In order to answer that question I would have to make an assumption about channel density, so instead of giving you that value, I shall give you another and you make a calculation according to the assumption that you want to make. Therefore I am going to tell you that there are twelve hundred electronic charges per pm2. DR. LEE: Did you see any effect of pronase treatment on the potassium channel? DR. BEZANILLA:No. DR. LEE: Are the gating currents dependent on metabolic energy? DR. BEZANILLA:No. The gating currents are seen regardless of the ionic electrochemical gradients across the membrane. Also, we generally use fluoride inside and provide no chemical source of metabolic energy. DR. MILLER:How convinced are you that the rising phase of your gating current is real? DR. BEZANILLA: In our older procedure we added currents resulting from hyperpolarizing and depolarizing pulses of equal size, from the holding potential. We proposed that the currents for each step, negative and positive, rose instantaneously and then fell, but amplitudes and time constants were different for the two pulses and hence their sum showed a rising phase. In other words, there is some gating charge movement during the negative steps, and this adds onto and distorts the positive step gating current, which is the current we wish to see. In the new procedure we take one pulse from the very negative region of membrane potential, where the charge movement is extremely small, and we still see some rising phase in the averaged current. So the question remains: does the current associated with the opening of channels have a rising phase? DR. FINKELSTEIN: Can 1 pursue that point a moment? I thought in your earlier publication you had shown that by proper setting of your pulses you could completely eliminate the rising phase. DR. BEZANILLA: In those experiments we had a blanking period of 20-50 microseconds and there was therefore some uncertainty with regard to the rising phase. In our present method we have improved the time resolution and we see a rising phase. DR. MAURO: Dr. Bezanilla would you kindly comment on the temperaturedependence of the gating currents. DR. BEZANILLA: The temperature-dependence is quite strong with a Qlo of the order of 2.5 or 2.7. However, we have to be careful in deciding whether this overall QIOhas any meaning because now that we see more than one component we should analyze them separately.
