467

J. Phyeiol. (1979), 291, pp. 467-481 With 10 textfigures Printed in Great Britain

SODIUM AND CALCIUM GATING CURRENTS IN AN APLYSIA NEURONE

BY D. J. ADAMS* AND P. W. GAGE From the School of Physiology and Pharmacology, University of New South Wales, Kensington, 2033, Awstralia

(Received 18 May 1978) SUMMARY

1. Currents generated by depolarizing and hyperpolarizing voltage pulses were recorded at temperatures of 4-12 0C in the voltage-clamped soma of R15 in aplysia abdominal ganglia exposed to solutions which supproed ionic currents. 2. Subtraction of linear capacitive and leakage current from current generated by in- ar transient voltage pulses to levels more positive than -20 mV revealed nn outward displacement currents at the onset of the clamp step (on-current) and transient inward displacement currents after the membrane potential returned to the holding potential (off-current). Only on-currents were studied. 3. Pulses to membrane potentials of -20 to 0 mV generated a displacement current with rapid onset and exponential decay. At membrane potentials more positive than 0 mV a second displacement current with a much slower onset and slower exponential decay was seen. Because the different threshold potentials for the two displacement currents were close to the different threshold potentials for Na and Ca ion currents, the two displacement currents were called Na and Ca 'gating' currents. 4. The amount of charge transfer during Ca gating currents increased sigmoidally with increasing depolarization, reaching a maximum at +30 to +40 mV. Half. maximum charge transfer occurred at + 15 mV. 5. Total charge movement during Ca gating currents was maximal with holding potentials of -30 to -40 mV. More positive or more negative holding potentials produced a decrease in charge movement. 6. The time course of the gating currents, but not the total charge displaced, was very sensitive to temperature. The time constant of decay of Ca gating currents had a Q10 of about 3, whereas the total amount of charge displaced had a Q10 of 1P2. 7. The charge transfer during both Na and Ca gating currents and the amplitude of Na and Ca (but not K) ionic currents were reduced in solutions containing 1 mmn-octanol. INTRODUCTION

Hodgkin & Huxley (1952) suggested that the voltage dependence of permeability changes in nerve membrane might arise from the effect of the electric field on the distribution or orientation of molecules with a charge or dipole moment (see also * Present address: Department of Physiology and Biophysics, University of Washington School of Medicine, SJ-40 Seattle, Washington 98195, U.S.A.

0022-3751/79/3910-0480 $01.50 © 1979 The Physiological Society

D. J. ADAMS AND P. W. GAGE iDanielli, 1941). It was not until about 20 years later that a small current with the characteristics of such a field-dependent charge movement was first recorded (Armstrong, & Bezanilla, 1973; Keynes & Rojas, 1973). This current has now been identified as an intramembrane 'displacement' current associated with 'gating' of sodium channels in squid giant axons (Armstrong & Bezanilla, 1973, 1974; Bezanilla & Armstrong, 1974; Keynes & Rojas, 1973, 1974), in myelinated amphibian nerve (Nonner, Rojas & Stampfli, 1975) and in Myxicola axons (Rudy, 1976). If voltagedependent shifts in charged groups within the membrane produce these gating currents and subsequent opening of ionic channels, then it seemed not unreasonable to anticipate that there might be a sodium gating current in an Aplysia neurone in which depolarization activates an increase in Na conductance. It also seemed possible that in the same neurone there might be a charge movement associated with the activation of Ca conductance (Adams & Gage, 1979a, b) which might be detectable as a non-linear displacement current. Indeed two displacement currents with characteristics consistent with Na and Ca gating currents have been discovered in Aplysia (Adams & Gage, 1976) and Helix neurones (Kostyuk, Krishtal & Pidoplichko, 1977). Preliminary reports of some of these observations have appeared elsewhere (Adams & Gage, 1976, 1977). 468

METHODS The experiments were performed on the neurone R15 and occasionally on R14 (Frazier, Kandel, Kupferman, Waziri & Coggeshall, 1967). The cell membrane was voltage-clamped as described previously (Adams & Gage, 1979a), with particular attention being paid to maximizing the rate at which the voltage step was imposed on the membrane. To facilitate rapid voltage-clamping of the membrane, low resistance, bevelled voltage and current micro-electrodes with resistances of 1-2 MC were employed. A change in recorded membrane potential in response to a clamp step was normally complete within 100 Iesec. In order to reveal the non-linear component of the capacitive current the linear components were cancelled out by adding currents produced by a positive (depolarizing) step to that of a negative (hyperpolarizing) step of exactly equal amplitude. To improve the signal-to-noise ratio, usually ten to fifteen depolarizing steps and an equal number of hyperpolarizing steps were applied and the resultant currents averaged. The holding potential was usually moved to potentials beyond - 100 mV before applying the hyperpolarizing pulse so that possible charge movements associated with hyperpolarizing steps would be minimized. An alternative procedure employed in a number of experiments was the 'divided pulse technique' (Armstrong & Bezanilla, 1974). A positive (depolarizing) step of P mV was applied from a holding potential, VH, and the current during this step amplified x 1 and fed to a signal averager. After the step, V. was changed to a more negative potential (e.g. - 100 mV) and a negative (hyperpolarizing) step of - P/4 was applied, the resultant current amplified x 4 and fed to the signal averager. The advantage of this technique was that it avoided taking the membrane to extreme negative potentials at which rectification (Hagiwara & Saito, 1959; Marmor, 1971) or membrane breakdown (Rudolph & StAmpfli, 1959) may occur. Positive and negative command pulses were applied alternatively at a rate of 0*5-0-2 sec-1 except where mentioned otherwise. Examination of asymmetrical capacitive currents required the elimination of the ionic currents In most experiments, the external Na was replaced by Tris or choline ions which are essentially impermeant (Adams & Gage, 1979a), and tetrodotoxin (TTX, 1-3 x 10-5 M) was added to prevent any residual current flow through the Na channel. Tetraethylammonium (TEA) ions (50-100 mM) were added to the external solution to block the large outward K currents. In some experiments, 4-aminopyridine was also used to depress K currents. The inward Ca current was blocked either by replacing the external Ca2+ with Mg2+ ions or by the addition of Mn2+ ions (15-20 mm) (and occasionally both). This has been shown to abolish Ca current in this neurone (Adams & Gage, 1979a, b). Displacement currents were recorded only in neurones in which the leakage current in response to a 50 mV hyperpolarizing step was less than 20 nA and we saw no evidence of rectifica-

Na AND Ca GATING CURRENTS

469

tion of these leakage currents. It therefore seems reasonable to assume that there would be essentially no 'pedestal' of current remaining when leakage currents produced by positive and negative clamp pulses were subtracted. Experiments were done within the temperature range of 4-12 0C. The voltage-clamp arrangement was as described previously (Adams & Gage, 1979a). The output of the current monitor was amplified and sent to a PDP-8I minicomputer. A blanking pulse with a variable width (20-200 pasec) was sometimes used to blank the initial large portion of the capacitive current to avoid saturation of the preamplifiers. To digitize and average analogue signals, an AX08-laboratory peripheral (9-bit A/D converter) was used in conjunction with a LAB-8 Basic Averager Program. This allowed 500 data points to be averaged at rates from 30 to 100 Uasec per point but the trace length was restricted to 15-50 msec. The averaged current record was photographed, enlarged and traced. The total charge transfer associated with the asymmetrical displacement current was determined from the area under the curve measured with a planimeter. The base line for measurement of gating current was determined from the data points obtained preceding the step change in membrane potential. Only records in which the gating current appeared to return to this level were used for measuring the amount of charge. An accurate determination of the time course of the current record was obtained with a teletype printout of each data point. RESULTS

Effects of membrane potential on capacitive currents The voltage-dependent asymmetry of capacitive currents in response to depolarizing and hyperpolarizing clamp pulses of equal amplitude can be seen in Fig. 1A. The capacitive transients were recorded from a cell in Na-free ASW containing 10-5 MA

B

/

+300

/

+14 -12

,//11,1

-28

/

-100

VH-42

+60 mV

---------------

-56 -72

-98

I

X

-300 nA

Fig. 1. Non-linearity of capacitive currents. A, superimposed traces from photographs of capacitive currents recorded in Na-free ASW containing 10-6 M-TTX and 50 mMTEA. The currents were generated by rapid changes in membrane potential (rise time 100 #sec) from a holding potential (TVa) of -42 mV to the levels shown. Note that the capacitive current produced by depolarization to + 14 mV is contaminated, presumably by a Ca ionic current, in its later part. Temperature 7-5 0C. Calibration: vertical, 100 nA; horizontal, 2 msec. B, amplitudes of capacitive currents (recorded 200 Isec after their peak) plotted against membrane potential for three experiments similar to that illustrated in A, with holding potentials of -42 to -44 mV. Temperature 9-10-5 'C. Extrapolation of the linear regression line fitted to data obtained with hyperpolarizing pulses shows the asymmetry of capacitive currents upon depolarization beyond -25 mV due to some added non-linear current.

D. J. ADAMS AND P. W. GAGE 470 TTX and 50 mm-TEA in response to a series of single equal and opposite clamp pulses from a holding potential (VH) of -42 mV. The recorded membrane potential had reached a new steady level within 1 00tsec. When the amplitudes of capacitive currents measured in three similar experiments (200,a1sec after their peak) were plotted against clamp potential (Fig. 1 B) a discontinuity in their amplitude could be seen at approximately -25 mV (Fig. 1 B). A

'Na

/Ca

Fig. 2. Non-linear displacement currents preceding ionic currents in R15. Records were obtained by averaging currents in responses to ten positive pulses from -60 mV and ten negative pulses from -1 10 mV. A, an outward displacement current preceding the inward Na current (INa) generated by 58 mV clamp pulses in 3/4 Na-ASW (3/4, 0/0) containing 70 mM-TEA. Temperature 8 'C. B, outward displacement currents followed by an inward Ca ionic current (1ca) obtained in Na-free ASW containing 70 mm-TEA in response to 65 mV pulses. Note the presence of a second, slower outward current (marked with an arrow) preceding the inward Ca current. Temperature 10 0C. Calibrations: vertical, 100 nA; horizontal, 4 msec.

The amplitudes of inward capacitive currents obtained with hyperpolarizing pulses follow a straight line which is projected as a dashed line for comparison with the outward capacitive currents generated by depolarizing pulses. The slope of the continuous straight line through the experimental points at depolarizing potentials is greater than the slope of the projected (dashed) line. This additional, non-linear capacitive current was investigated by cancelling out the linear capacitive currents. For convenience these non-linear capacitive currents will be called 'displacement' currents. An averaged current recorded in ASW (0.75 Na) containing 70 mM-TEA is shown in Fig. 2 A. Sodium ions were left in the solution so that relative time courses of displacement and ionic current would be revealed. In this experiment were averaged currents generated by ten positive and ten negative pulses of 58 mV from holding potentials of -60 and - 110 mV respectively. A small outward displacement current can be seen to precede the inward Na+ ionic current (Fig. 2A).

Na AND Ca GATING CURRENTS 471 When sodium-free ASW was applied to the same cell and clamp pulses of 65 mV were delivered from the same holding potentials, an outward displacement current consisting of two components preceded an inward Ca ionic current (Fig. 2B). The removal of Na+ ions allowed the Ca current to be recorded free of Na current, and this together with the increased depolarization, revealed a second slower displacement current. This current (marked with an arrow in Fig. 2B) cannot have been residual leakage current because its amplitude is of the order of 20-30 nA. A 65 mV hyperpolarizing pulse in this cell generated a 'leakage' current of 21 nA. Any residual leakage current due to possible rectification of the leakage current would presumably have been smaller than 21 nA after the subtraction procedure.

The two displacement currents have different thresholds The displacement currents shown in Fig. 3 were recorded in a Na-free solution (Na replaced by choline) containing TTX (10-5 M) to eliminate the Na current, Mn ions (15 mM) to block the Ca current and TEA ions (50 mm) to block K current. When combinations of voltage steps and holding potentials were used so that the membrane was never depolarized beyond -40 mV, positive and negative currents generated by A

B

Fig. 3. Displacement currents generated at different levels of membrane potential in Na-free (choline substituted) ASW containing TTX (10-5 M), Mn (15 mM) and TEA (50 mM). Solutions contained the normal ( 11 mM) Ca. Temperature 6 0C. The holding potential was - 101 mV in A and -62 mV in B and C. The records were obtained by summing currents generated by fifteen positive and fifteen negative clamp pulses. A, average response to voltage steps of + 50 mV from a holding potential of - 101 mV. B, average displacement currents produced by voltage steps to -112 and -12 mV (± 50 mV) from a holding potential of -62 mY. C, average displacement currents produced by voltage steps to - 137 and + 13 mV (± 75 mV) from a holding potential of -62 mV. Calibrations: vertical, 50 nA; horizontal, 2 msec.

D. J. ADAMS AND P. W. GAGE clamp pulses of equal amplitude were symmetrical and cancelled out. The average current shown in Fig. 3A was obtained in response to clamp steps of + 50 mV from a holding potential of - 101 mV and it can be seen that the capacitive and leakage currents must have been almost exactly equal and opposite as there was essentially no 'difference' current remaining. When the same voltage steps were applied from a holding potential of -62 mV, the capacitive currents did not cancel and displacement currents were revealed (Fig. 3B). The 'on' and 'off' displacement currents as shown in Fig. 3B were generated only by clamp pulses to a level more positive than -20 mV, the threshold for Na current activation in R1, (Adams & Gage, 1979a). Surprisingly, the decay of the on-current was more rapid than the decay of the offcurrent (Fig. 3B) but the wave form of the on-current (rapid onset with slower decay) was similar to that of displacement currents associated with the opening of Na channels in-myelinated and unmyelinated nerve fibres. When the amplitude of the voltage steps from the same holding potential (-62 mV) was increased to 75 mV so that the membrane was alternatively clamped to + 13 mV and to - 137 mV, the average 'on' and 'off' displacement currents contained a later, slower component (Fig. 3 C). The slower displacement current was smaller in amplitude than the earlier displacement current but lasted considerably longer. It appeared to last even longer when the pulse was turned off and in one experiment in which a longer trace was used, it was still present 5 msec after the end of the pulse. Because of its apparently long duration and the resultant complications from small residual K currents at times after 10 msec (4 'C) the time course of the 'off' displacement currents was not examined in these experiments and this paper contains descriptions of the 'on' displacement currents only. The slower displacement current produced by membrane depolarization was seen clearly only with clamp pulses to membrane potentials more positive than 0 mV, about the threshold for initiation of Ca ionic current. It is unlikely that these displacement currents were ionic currents for the following reasons. Although it was not possible to measure the areas of the slow off-currents, on and off displacement currents were in opposite directions (and it is not impossible that they might have had similar areas). It seems unlikely that ionic currents should have such characteristics over a wide range of clamp potentials. Ions which have concentration gradients which might produce outward currents are chloride and K+. The displacement currents were unchanged when either Cl- or K+ concentrations were changed in the extracellular solution. The displacement currents were unaffected by high concentrations of TEA or 4-aminopyridine which block the rapid transient outward K current (A-current) in other neurones (Kostyuk, Krishtal & Doroshenko, 1975b; Thompson, 1977). Furthermore, they could be recorded from holding potentials more positive than -50 mV under which conditions A-currents are inactivated (Connor & Stevens, 1971; Kostyuk, Krishtal & Doroshenko, 1975a). If the 'off' displacement currents were due to a transient increase in K of Cl conductance they should have been outward on repolarization to potentials more positive than the K or Cl equilibrium potential. As the 'off' displacement currents were always inward they could not be K or Cl ionic currents. It was therefore tentatively concluded that these currents were true displacement currents. As the potential at which the early, rapidly decaying component (Fig. 3B) is 472

Na AND Ca GATING CURRENTS 473 initiated ('threshold' potential) was the same as the threshold for Na ionic current, and the threshold potential for the later slower component was the same as that for Ca ionic current, the two displacement currents will be called Na and Ca 'gating' currents. It will be seen below that this nomenclature is not inconsistent with our results but it should be noted that there is no evidence that the whole of the charge movement producing these displacement currents is associated with the opening of channels for ions. Time course of the gating current The Na gating current could be generated alone with clamp pulses to levels between -20 and -5 mV. The Na gating current turned on rapidly and then decayed in an approximately exponential manner. The decays of the displacement currents in Fig. 3B could be fitted by single exponentials with a time constant of decay of 0-4 msec for the 'on-current' and of 0-7 msec for the 'off-current'. Unlike the 'rapid-on, exponential decay' time course of the Na gating current, the time course of the Ca gating current exhibited an initial slow rising phase followed by a slower, approximately exponential decay as can be seen in Fig. 3 C. Both outward on-currents in Aplysia neurones appeared to decay more rapidly than the off-currents but for the reasons given above, the off-currents were not analysed in detail. However, it can be seen that the charge movement during the fast component of the off-current in Fig. 3C is less than that in Fig. 3B.

Effect of membrane potential on gating currents The rate of decay of Na gating currents generated in response to depolarizing clamp pulses up to 0 mV appeared to be voltage-sensitive, increasing with increased depolarization. Similarly, the rate of decay of the Na gating current during the clamp pulse, in both squid axons and the node of Ranvier, increases with increasing depolarization(Keynes & Rojas, 1974; Meves, 1974; Nonner et al, 1975). The rise time and rate of decay of Ca gating current were also voltage-sensitive. For example, in one experiment, averaged gating currents were recorded in Na-free ASW containing 10-5 -TTX, 20 mM-MnCl2 and 70 mM-TEA, with depolarizing clamp pulses to potentials of + 12, + 23 and + 36 mV. These are shown in Fig. 4. The time-to-peak of the Ca gating current decreased and the rate of decay increased with increasing depolarization. The amplitude of both Na and Ca gating currents also varied with clamp potential. Although the amplitude of the Na gating current increased with increasing depolarization, it was difficult to measure accurately the total amount of charge moved at potentials more depolarized than O mV as the time course of decay of the current was obscured by the rising phase of the Ca gating current. The amount of charge displaced during Ca gating currents clearly increased with increasing depolarization as can be seen in Fig. 4. Accurate measurement of this area over a range of voltage pulses was difficult because a slow outward current (presumably residual K current in spite of the TEA) was often seen at potentials more positive than +20 to +30 mV (see e.g. +36 mV, Fig. 4). This was much less prominent at lower temperatures (see, for example Fig. 8). Furthermore, because of the relatively brief depolarizing pulses that were used, it had to be assumed that the

D. J. ADAMS AND P. W. GAGE

474

Fig. 4. A, averaged Na and Ca gating currents obtained with voltage steps of 74, 85 and 98 mV from holding potentials of -62 and - 105 mV; i.e. the gating currents were activated at potentials (V.) of + 12, + 23 and + 36 mV. Currents were recorded in a Na-free (choline substituted) ASW containing 10-5 M-TTX, 20 mM-Mn and 70 mM-TEA. Temperature 5 'C. Calibration: vertical, 50 nA; horizontal, 2 msec. 1*0

U x

E

05

a

-20

-10

0

20 30 10 Membrane potential (mV)

40

50

60

Fig. 5. Voltage dependence of Ca gating charge movement. Total charge displaced at each potential (Qv) expressed as a fraction of the maximum charge displacement (Q...). The gating currents, recorded in Na-free ASW containing 3 x 10-6 M-TTX, 20 mM-Mn and 70 mM-TEA (three experiments denoted by three separate sets of symbols) were generated from holding potentials of -60 mV (squares), -50 mV (triangles) and -46 mV (circles). Temperature 6-10 'C. The continuous line was drawn by eye through the points. The dashed line shows the voltage dependence of Ca conductance (Adams & Gage, 1979b) for comparison.

Na AND Ca GATING CURRENTS 475 gating current was decaying to the pre-pulse current level. In most cases, this seemed reasonable (see, for example, Fig. 4, + 12 and +23 mV; Fig. 7; Fig. 8, 4-7 'C; Fig. 1 0). Finally, the early part of the Ca gating current had to be obtained by drawing a line back to the time of the beginning of the pulse. The exact magnitude of the errors introduced by these uncertainties and assumptions is not known but in our opinion, would not have been greater than 20% at potentials more positive than + 20 mV and less at more negative potentials. It is probably worth repeating that we do no think that these currents were sitting on a pedestal of non-linear leakage current for reasons already outlined. With these possible sources of error in mind, measurements of charge movement in response to a range of depolarizing pulses in three experiments are presented in Fig. 5. Total charge movement at a potential V (Qv), expressed as a fraction of maximum charge movement at any potential (Qmax), is plotted against clamp potential. The amount of charge transferred increased sigmoidally with increasing depolarization, but did not appear to increase further at potentials more positive than about +35 mV. The dashed curve, included for comparison, shows the voltage dependence of Ca conductance in R15 (Fig. 11, Adams & Gage, 1979b). Half of the maximum Ca gating charge was transferred at about + 15 mV which is close to the half point of + 12 mV for Ca conductance. Although errors in measurement may have affected the curve shown in Fig. 5, it is clear that the voltage dependence of the charge movement is closer to that of Ca conductance than that of Na conductance (Adams & Gage, 1979b). This supports the assumption that the slow displacement current is a Ca gating current.

Effect of holding potential on gating currents Meves (1974) has reported that the effect of holding potential on the Na gating current in squid axons develops slowly (over several minutes). Therefore when assessing the effect of holding potential on gating currents in R15, the membrane potential was shifted to a new holding potential for at least 3-5 min before currents were elicited with depolarizing pulses. The effect of a wide range of holding potentials on both Ca gating and ionic currents is illustrated in Fig. 6. Averaged gating and ionic currents were obtained in Na-free ASW (choline substitution) containing 5 mM-Mn2+ and 70 mM-TEA. More Mn was not added in order that small Ca currents could be recorded together with the gating currents. Depolarizing steps from holding potentials of - 110, - 80, - 55, and -22 mV to a command potential of + 15 mV were used to elicit the currents. Although the decays of the Ca gating currents shown in Fig. 6 are obscured by activation of inward Ca current, it appears that both the slow displacement current and the Ca ionic current became smaller at holding potentials more hyperpolarized than -55 mV. It is unlikely that the gating current was smaller at - 110 mV than at -55 mV because of changes in the Ca current, as the Ca current is smaller at - 110 mV than at -55 mV (Adams & Gage, 1979b). Although a blanking pulse of 200 ,usec was used in this experiment, it can be seen that the Na gating current was absent from the records shown in Fig. 6. A similar observation was made in two further experiments in which Na was replaced by choline and TTX was not added. However, when Na was replaced by Tris the early displacement current remained, suggesting that in the absence of TTX, choline may interact in some way with a site involved in the gating of the Na channel.

D. J. ADAMS AND P. W. GAGE

476

-110mV

-80 mV

-55 mV

-22 mV

--------

_

=

Fig. 6. Effect of holding potential on Ca gating and ionic currents. Averaged gating and ionic currents were obtained in a Na-free (choline substituted) ASW containing 5 mM-Mn2+ and 70 mM-TEA when the membrane potential was displaced from the holding potentials indicated to a potential of + 15 mV. Clamp pulses in the negative direction were equal in amplitude to the positive pulses from each holding potential, but were generated from a holding potential of - 110 mV. A blanking pulse of 200 /ssec was employed at the onset of each voltage step. Temperature 8 'C. Note the reduced amplitude of both displacement and ionic currents upon hyperpolarization from -55 mV. Calibration: vertical, 100 nA; horizontal, 2 msec.

-82 mV ___

_

_

-60 mV

Fig. 7. The effect of holding potential on charge movement. Na and Ca gating currents were recorded in Na-free (choline substituted) ASW containing 10-5 M-TTX, 20 mM-Mn and 70 mM-TEA. The gating currents were generated by pulses from holding potentials of -82 and -60 mV to a command potential of +20 mV. Holding potential for negative pulses was - 105 mV. Temperature 4-8 'C. Calibration: vertical, 50 nA; horizontal, 2 msec.

Na AND Ca GATING CURRENTS

477

In order to confirm the depression of Ca gating charge movement at hyperpolarized potentials without the complications of ionic currents, experiments were done with solutions containing higher Mn concentrations so that the Ca ionic current was completely suppressed. Gating currents recorded in one of these experiments are shown in Fig. 7. Gating currents were generated by pulses to + 20 mV from holding potentials of -82 and -60 mV. It can be seen that the amplitude of the Ca gating current was significantly smaller when the holding potential was -82 mV. The amplitude of Ca gating currents was also depressed at holding potentials more positive than -40 mV (see Fig. 6). At a holding potential of 0 mV (held for more than 2 sec) Na and Ca currents could still be seen but were markedly depressed in amplitude. The steady-state inactivation of both the Ca current and charge displacement caused by hyperpolarization or depolarization (with respect to the resting membrane potential) further supports the assumption that the slow displacement current is associated with activation of the Ca conductance.

Effect of temperature on the gating currents If the displacement currents recorded in R15 do indeed arise from charge movement within the membrane, then a change in temperature might be expected to alter only the rate and not the total transfer of charge. The effect of temperature on the gating currents was studied in a temperature range of 4-12 TC in four experiments. There was a change in the time course of both gating currents when temperature was changed. For example, when temperature was increased from 4-7 to 10-5 TC in one of these experiments the time course of gating currents became markedly changed (Fig. 8). The time-to-peak of Ca gating currents was shorter and the rate of decay of

Fig. 8. Effects of temperature on gating currents. Na and Ca gating currents recorded at 4*7 00 (top trace) and 10-5 00 (bottom trace). The Na-free (Tris substitution) ASW contained 10-1 m-TTX, 20 mm-Mn and 50 mm-TEA. Holding potential - 60 mV for positive pulses, - 105 mV for negative pulses. Pulses of 85 mY were used to activate currents. Calibrations: Vertical, 50 nA; horizontal, 2 msec.

4D. J. ADAMS AND P. W. GAGE both currents was faster at higher temperatures. Although measurement of charge transfer was made difficult by the appearance of the later outward current at 10-5 'C, the measured areas came out much the same at the two temperatures. Measurements of the time constant of decay of Ca gating currents from four experiments gave Q10 values of about 3 (3 3, 3 0, 2 8 and 2.5). A similarly high Q10 has been reported for the time course of Na gating currents in squid axons (Keynes & Rojas, 1974) and myelinated nerve fibres (Nonner et al. 1975). In contrast to the high Q10 obtained for the rate of charge transfer, the total amount of charge displaced was relatively temperature insensitive. The average Q10 of the maximum charge displaced during the Ca gating current in the four experiments was 1t2. 478

Effect of octanol on Na and Ca ionic and gating currents If excitability depends on conformational changes in channel proteins within the membrane, then octanol which has been shown to increase the fluidity of membranes (Grisham & Barnett, 1973) might modify the membrane of Aplysia neurones and thereby affect the ionic and gating currents. Membrane currents generated by depolarizing pulses in the neurone R14 before (A) and after (B) a 10 min exposure to 1 mM-n-octanol are shown in Fig. 9. N-octanol selectively depressed both Na and Ca currents by approximately 60% but the amplitude of the delayed outward K current was unaffected. There was no significant change in the resting membrane potential. The effect of octanol on the inward currents could be reversed by prolonged perfusion (more than 1 hr) with normal ASW. The results obtained are in agreement with the selective depression of the Na conductance in squid axons by n-octanol reported by Armstrong & Binstock (1964) (also see Fig. 15 in review by Bunker & Vandum, 1965). To determine if this action of n-octanol were due to an effect of octanol on gating of the Na and Ca channels, gating currents were recorded in the presence of n-octanol. Records obtained at various times during exposure to n-octanol are shown in Fig. 10. Normal gating currents were seen before exposure to octanol (A). After 15 min in the A

B

|,~

-

Fig. 9. Membrane currents generated by depolarizing clamp steps before (A) and after 10 min exposure to 1 mM-n-octanol (B). Both Na and Ca currents were markedly depressed by octanol but the outward K currents were not affected. Holding potential -48 mV; temperature 12 'C. Calibrations: vertical, 50 mV for voltage traces, 500 nA for current traces; horizontal, 10 msec.

Na AND Ca GATING CURRENTS 479 presence of octanol (B) the amplitudes of both Na and Ca gating currents were markedly depressed. After exposure to octanol for a furthter 20-30 min (C) gating currents were depressed even more. The time-dependent decrease of the inward Na and Ca currents caused by octanol is thus accompanied by a decrease in the amount of charge movement during the gating currents. The depression by octanol of the Na and Ca currents again supports A

B

C

Fig. 10. Effect of n-octanol on gating currents obtained by averaging fifteen positive pulses of 85 mV from a holding potential of -60 mV and fifteen negative 85 mV pulses from a holding potential of - 10 mY. Results were obtained in a Na-free (Tris substitution) ASW containing 10-4M-TTX, 15 mM-Mn and 70 mM-TEA. A, control (normal ASW). B, gating currents recorded 15 min after adding 1 mM-n-octanol to the perfusion solutions. C, after 40 min exposure to octanol. Temperature 4-6 'C. Calibrations: vertical, 50 nA, horizontal, 2 msec.

the identification of displacement currents as gating currents. The reduction of the total charge displaced indicates that octanol depresses the ionic currents by inhibiting gating charge movement. DISCUSSION

Depolarization of the surface membrane of the soma of R15 generates capacitive and ionic currents. When the ionic currents are suppressed subtraction of symmetrical capacitive currents and leakage currents reveals non-linear 'displacement' currents which are thought to arise from charge movements within the membrane rather than ion movements across the membrane for the reasons given above. It is proposed that the, fast displacement current is associated with initiation of Na conductance changes and the slow displacement current with initiation of Ca conductance changes. Evidence for this is: (1) the two displacement currents are elicited at different 'threshold' potentials; the fast displacement current is initiated at potentials corresponding to those at which Na conductance is activated (about

D. J. ADAMS AND P. W. GAGE 480 -20 mV) while the slow displacement current first appears at membrane potentials about 20 mV more positive, coinciding with the potential level at which Ca conductance is activated (Adams & Gage, 1979b); (2) the voltage dependence of the slow charge movement is similar to the voltage dependence of Ca conductance; (3) unlike Na conductance, Ca conductance is depressed at holding potentials more negative than the resting membrane potential and this is accompanied by a decrease in the amount of charge transferred during the slow displacement current; (4) pharmacological evidence that the displacement currents are associated with Na and Ca but not K conductance changes comes from the selective effect of octanol on the currents; octanol depresses Na and Ca currents without significantly affecting K currents; octanol also reduces the amount of charge displaced during both displacement currents; (5) the characteristics of K conductance changes are different from the characteristics of the slow displacement current; the threshold potential and the potential for half-activation of the K conductance are not the same as for the slow displacement current; furthermore, shifting the holding potential to more negative potentials reduces inactivation of the K conductance whereas the amplitude of the slow displacement current is depressed. It therefore seems reasonable to propose that the fast displacement current is a 'Na gating current' and the slow displacement current is a 'Ca gating current'. Unlike the asymmetrical displacement currents recorded in squid and amphibian myelinated nerve, the slow displacement current in Aplysia neurones has a distinct, relatively slow rising phase followed by an exponential decay. The slow rising phase of the Ca gating current is thought to be real and not due to technical problems (Adams & Gage, 1978). The existence of a rising phase means that there is relatively little charge movement associated with the initial steps involved in opening the channel and requires a model containing a sequence of reaction steps for opening the channel such as that proposed by Bezanilla & Armstrong (1976) for Na gating currents. An asymmetrical displacement current associated with Ca conductances has been described recently in internally dialysed Helix neurones (Kostyuk et al. 1977). As with the Ca gating current described here, the charge displacement in Helix neurones was voltage-dependent and inactivated at positive potentials. However, in contrast to the Ca gating current in Aplysia neurones, the displacement currents in Helix neurones appeared to have no significant rising phase. This apparent difference could be due to the higher temperatures (20-22 °C) at which displacement currents were recorded in Helix neurones by Kostyuk et al. (1977), taking into account the high temperature sensitivity that we have found for the rising phase, or there may be real differences in the gating of Ca channels in the two different nerve cells. In conclusion, displacement currents generated by membrane depolarization in R15 appear to be associated with the opening of Na and Ca channels. Elucidation of their characteristics may provide clues for investigating the Ca currents thought to be important in transmitter secretion and muscle contraction. The work was supported by a grant from the Australian Research Grants Committee. We are grateful to R. Malbon, P. Holtz and C. Prescott for technical assistance.

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REFERENCES

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Sodium and calcium gating currents in an Aplysia neurone.

467 J. Phyeiol. (1979), 291, pp. 467-481 With 10 textfigures Printed in Great Britain SODIUM AND CALCIUM GATING CURRENTS IN AN APLYSIA NEURONE BY D...
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