Two components of outward currents were investigated under voltage clamp conditions in Tenebrio muscle fibres. The instantaneous current-voltage relation for the transient outward current showed outward rectification. The tail currents for the delayed outward currents were made up of either one or two exponential components. The activation process for the delayed current was analysed using positive tails that decayed with a simple exponential time course. The delayed current was half-activated at about + 35 mV. Two rate constants for activation are both monotonic functions of membrane potential. The reversal potential for the delayed current was only partially dependent on the external K-concentration. The role of the two outward currents in the production of the action potential was discussed.

The ionic currents in voltage-clamped mealworm muscle fibres have been separated into three components, two initial transient currents (inward and outward), followed by a late steady-state outward current in the preceding paper (Yamamoto, Fukami & Washio, 1981). The inward current was associated with the inward movement of Ca ions down its electrochemical gradient. The present work analyses the behaviour of the two outward current components using the voltage clamp technique.

The preparation and methods used are identical to those described in the previous paper (Yamamoto et al. 1981).

The transient outward current

As demonstrated in the preceding paper (Yamamoto et al. 1981), a transient outward current followed an inward current at about – 20 mV. The outward current reached a maximum, then inactivated with time. The amplitude of the current increased with larger depolarizations, up to a membrane potential of about + 5 mV. The amplitude of the current varied a great deal among fibres and appeared absent in some (see Fig. 1b in Yamamoto et al. 1978).

Fig. 1.

Families of membrane currents (lower traces) associated with step depolarizations (upper traces) from the holding membrane potential of –35 mV in normal saline (A), in calcium deficient saline (B) and in saline containing 15 mM-cobalt (C). A and B were taken from the same fibre. Note prominent humps in the current traces in A, which disappear in B and C. Calibration: 100 mV, 1μA for all records; 20 ms for A and B, and 21·4 ms for C.

Fig. 1.

Families of membrane currents (lower traces) associated with step depolarizations (upper traces) from the holding membrane potential of –35 mV in normal saline (A), in calcium deficient saline (B) and in saline containing 15 mM-cobalt (C). A and B were taken from the same fibre. Note prominent humps in the current traces in A, which disappear in B and C. Calibration: 100 mV, 1μA for all records; 20 ms for A and B, and 21·4 ms for C.

In order to estimate the reversal potential for the current, the direction of the tail-current was analysed when clamping back to different membrane potentials. The inward current was not blocked during analysis of the instantaneous I-V relation for the transient outward current because this caused the outward current to disappear (Fig. 1; and see also Figs. 3A and 5A). The transient outward current was absent in calcium deficient saline (Fig. 1B) or in saline containing 15 mM-cobalt (Fig. 1C). A conditioning pulse was used to depolarize the fibre to +10 mV for 5ms, the time necessary for the transient outward current to reach its peak. At first, the tail currents recorded were plotted semilogarithmically against time. They consisted of a capacitative surge followed by a slow component whose time constant was about 5 ms. The intercepts with the current axis of the slow component were measured as the instantaneous value of the tail current. Thus, Fig. 2 was obtained at seven different potentials, showing the relation between instantaneous tail current and absolute membrane potential. Unlike the K current of the squid axon (Hodgkin & Huxley, 1952a), the transient current rectifies considerably (see Binstock & Goldman, 1971; Meech & Standen, 1975). From the relationship, the reversal potential for the transient outward current was determined as – 46 mV, a slightly more negative value than that for the delayed outward current (cf. Fig. 7).

Fig. 2.

Instantaneous current-voltage graph. Tail current values were separated from capacitative surge by means of semilogarithmic plot.

Fig. 2.

Instantaneous current-voltage graph. Tail current values were separated from capacitative surge by means of semilogarithmic plot.

Fig. 3.

Analysis of current tail following depolarization lasting 30 ms in Co2+ saline. (A) A sample record of the current tail (upper trace) following depolarization to + 60 mV (lower trace). Dotted line in the current trace represents the background current level. (B) The 8emilogarithmic plot of the current trace in A. The plots were fitted by a straight line with intercept at 21 × 10−2A and time constant of 18 ms. (C) The semilogarithmic plot of the current tail following depolarization to −40 mV, obtained from another fibre. The measured current is plotted as filled circles. Fast component (open circles) with intercept 22 × 10−2A and time constant 17 ms was obtained by subtraction of extrapolated slow component from total current. The slow component has an intercept of 9·5 × 10−3 A and time constant of 96 ms. In all cases the tail currents were measured when clamping back to the holding potential of – 40 mV.

Fig. 3.

Analysis of current tail following depolarization lasting 30 ms in Co2+ saline. (A) A sample record of the current tail (upper trace) following depolarization to + 60 mV (lower trace). Dotted line in the current trace represents the background current level. (B) The 8emilogarithmic plot of the current trace in A. The plots were fitted by a straight line with intercept at 21 × 10−2A and time constant of 18 ms. (C) The semilogarithmic plot of the current tail following depolarization to −40 mV, obtained from another fibre. The measured current is plotted as filled circles. Fast component (open circles) with intercept 22 × 10−2A and time constant 17 ms was obtained by subtraction of extrapolated slow component from total current. The slow component has an intercept of 9·5 × 10−3 A and time constant of 96 ms. In all cases the tail currents were measured when clamping back to the holding potential of – 40 mV.

Because of its rather capricious nature, further analysis of the transient outward current was not possible.

The delayed outward current

1. Semilogarithmic analysis of current tails

Semilogarithmic analysis of the current tails was performed in low K (3 MM) saline containing 15 mM-Co2+.. In such conditions, the delayed outward current is the only component activated and the tail that follows the end of a depolarization pulse develops in the positive direction at a holding potential that is usually around –40 mV, greatly facilitating analysis (Fig. 3 A). The duration of the depolarizing pulse was set to 30 ms, and it will be justified later (see Fig. 4). In each fibre, tail currents would be fitted by one or two exponentials. For the tail shown in Fig. 3 A, the exponential had a time constant (T) of 18 ms at a depolarization of 60 mV (Fig. 3B). Further data for this tail at different depolarizations, are given in Table 1. Fig. 3C shows a tail fitted by two exponentials. With a depolarization of +40 mV, the time constant for the fast component (16·7 ms) was close to that described in Fig. 3B, while the slow one was about six times as large, 96·4 ms. This slow component may reflect a slow potassium current similar to that in crayfish muscle (Hendek, Zachar & Zacharova, 1978). Alternatively, it is possible that the slow component may correspond to an accumulation of extracellular K+ ions (Brown, Clark & Noble, 1976). Further study must be done to resolve this problem.

Table 1.

Analysis of activation of the delayed current

Analysis of activation of the delayed current
Analysis of activation of the delayed current
Fig. 4.

Envelope of tail analysis. A, Superimposed records of membrane currents (upper traces) in response to +80 mV depolarization from a holding potential of – 40 mV (lower traces) with varying duration (10, 30, 60 and looms). Note the variation in amplitude of the tails. B, The amplitude of positive current tails (ordinate) as a function of duration of depolarizing pulse (abscissa). A and B were frorn different fibres.

Fig. 4.

Envelope of tail analysis. A, Superimposed records of membrane currents (upper traces) in response to +80 mV depolarization from a holding potential of – 40 mV (lower traces) with varying duration (10, 30, 60 and looms). Note the variation in amplitude of the tails. B, The amplitude of positive current tails (ordinate) as a function of duration of depolarizing pulse (abscissa). A and B were frorn different fibres.

2. Kinetic properties of the delayed current

Current decay tails are shown in Fig. 4 A recorded after depolarizations of constant magnitude but increasing duration. The peak amplitudes of these currents were then plotted against the duration of the preceding pulse (envelope of tails) (Fig. 4B). It can be seen that the envelopes for the delayed outward current have approximately exponential patterns and reach a steady-state amplitude after pulse durations of between 20 and 30 ms. With longer depolarizing pulses, however, the current tails declined in their size. Because it was demonstrated that the delayed current could be activated fully by a pulse of 20–30 ms in duration, 30 ms depolarizing pulses were used in subsequent experiment.

The information obtained by semilogarithmic analysis of the maximum outward current decay tails (e.g. Fig. 3B) was used to plot activation curves for the delayed current (Noble & Tsien, 1968, 1969; Brown, Giles & Noble, 1977). The exponentially decaying currents recorded immediately after return to the holding potential from various voltage steps were plotted against the stepped membrane potential. For the fibre whose tail current is shown in Fig. 3 A, activation was obtained up to 180 mV from the holding potential (Fig. 5 A, Table 1). It was half-activated at about + 35 mV (N= 0·5) and fully activated at about +100 mV (N= 1). In five fibres, full activation of this current was achieved at a mean potential of + 112 mV, S.E. 19·3 mV, range + 50 to +180 mV. Rate constants αn and βn were calculated from the measured values of N and T−1 (Table 1), using the equations (Hodgkin & Huxley, 1952b)

Fig. 5.

Voltage dependence of kinetics of the delayed outward current. (A) Voltage dependence of fractional activation (N) in the steady state measured as the peak current change from background on return to holding potential (– 40 mV). Ordinates: peak current and n. Abscissa: membrane potential. (B) Open circles show measured values of τ−1. Triangles and filled circles are calculated value for αn and βn, respectively, τ values were obtained from measurements of the slopes of the lines determined by the method explained in Fig. 2. Curves were drawn by eye.

Fig. 5.

Voltage dependence of kinetics of the delayed outward current. (A) Voltage dependence of fractional activation (N) in the steady state measured as the peak current change from background on return to holding potential (– 40 mV). Ordinates: peak current and n. Abscissa: membrane potential. (B) Open circles show measured values of τ−1. Triangles and filled circles are calculated value for αn and βn, respectively, τ values were obtained from measurements of the slopes of the lines determined by the method explained in Fig. 2. Curves were drawn by eye.

τ−1, αn and βn are also plotted in Fig. 5 B. It can be seen that both rate coefficients, αn and βn, are monotonic functions of membrane potential. They cross each other at + 33 mV.

3. Ionic nature of the delayed current

To elucidate the ionic dependence of the delayed current, the effects of changing external K concentrations on the reversal potential were examined. The reversal potential of the outward current was determined by double steps in Co2+ saline (Fig. 6 A). The membrane was first depolarized by 80 mV and then clamped to different potentials when the outward current had reached its maximum. In the illustrated experiment (Fig. 6B) the reversal potential was about — 80 mV in the Co2+ saline containing 3 mM-K. Similar experiments were repeated at three external K concentrations, 3, 12 and 30 mM, using different muscle fibres. The results are illustrated in Fig. 7. The reversal potential for the delayed current shifted in a positive direction as the external K concentration was raised. The relationship between potential and log [K+]0 at K concentrations higher than 12 mM was approximated by the Nernst equation:

Fig. 6.

(A) Determination of the reversal potential of the delayed current by double pulses. Upper traces show membrane potential (holding potential is – 50 mV) and lower traces membrane current. Records were taken in Co2+ saline containing 12 mM-K. (B) Instantaneous current-voltage relationship obtained from the same fibre as A in Co2+ saline containing 3 mM-K. The reversal potential was – 80 mV.

Fig. 6.

(A) Determination of the reversal potential of the delayed current by double pulses. Upper traces show membrane potential (holding potential is – 50 mV) and lower traces membrane current. Records were taken in Co2+ saline containing 12 mM-K. (B) Instantaneous current-voltage relationship obtained from the same fibre as A in Co2+ saline containing 3 mM-K. The reversal potential was – 80 mV.

but the absolute value of potential was far more negative than that expected from the equation (broken line in Fig. 7 was drawn according to equation (4). [K+], was taken as 70 mM, the value determined by Belton & Grundfest (1962)). At concentrations below 12 mM, the reversal potential is apparently less sensitive to changes in |K+]0.

The effect of changing the external K concentration on the current-voltage relationship for the delayed current was also studied in single muscle fibres. Unexpectedly, the current magnitude was not decreased by a ten-fold elevation of external K concentration, from 3 to 30 mM, despite a decrease in driving force.

4. Conductances for the delayed current

The instantaneous I-V relation (Fig. 6B) showed an ohmic nature at potentials positive to the holding potential. Thus the conductance for the delayed current was calculated as
where V is the absolute membrane potential, Vrev is the measured reversal potential and Id is the maximum outward current for each depolarization. Fig. 8 shows the dependence of the conductance upon the membrane potential.
Fig. 7.

Effect of external K+ concentrations on reversal potential of the delayed current. Vertical bars represent the standard error of the mean. Number of fibre* sampled were: 3 mM-K, 18; 13 mM-K, 9; and 30 mM-K, 15. All measurements were made in Co2+ saline. Dotted line shows an extrapolation of the experimental data. Broken line is a Nemst relationship drawn by assuming that the membrane behaves as a K-electrode.

Fig. 7.

Effect of external K+ concentrations on reversal potential of the delayed current. Vertical bars represent the standard error of the mean. Number of fibre* sampled were: 3 mM-K, 18; 13 mM-K, 9; and 30 mM-K, 15. All measurements were made in Co2+ saline. Dotted line shows an extrapolation of the experimental data. Broken line is a Nemst relationship drawn by assuming that the membrane behaves as a K-electrode.

Fig. 8.

Semi-logarithmic plot of the steady state conductance for the delayed outward current

Fig. 8.

Semi-logarithmic plot of the steady state conductance for the delayed outward current

It seems likely that the ‘hump’ present in the response of a voltage-clamped mealworm larva muscle fibre under step depolarization represents the early transient outward current designated in crustacean muscles (Mounier & Vassort, 1975b; Hendek & Zachar, 1977). It is impossible to record the outward current responsible for the hump separately from the Ca inward current because any treatment to event Ca entry into the cell inhibits its development (Fig. 1). This outward component seems to be analogous to the Ca-activated potassium outward current known to be present in many excitable membranes, especially in the membrane of molluscan neuronal somata (Meech, 1978). TEA-sensitivity of the hump (Fig. 7 in the preceding paper, Yamamoto et al. 1981) also makes it similar to the Ca-dependent K current of molluscan neurones (Meech & Standen, 1975).

The transient outward current is also unveiled when the inward current is attenuated by prepulses, particularly in the hyperpolarizing direction (Figs. 8, 9 of the preceding paper, Yamamoto et al. 1981). A similar result has been obtained in a variety of preparations (see Meech, 1978).

Calcium-mediated outward current can be expected to balance the effects of calcium activation itself. In mealworm muscle, there is a wide range of variation in the electrical excitability under normal conditions; namely both prolonged calcium spikes and non-regenerative responses occur in different muscle fibres of the same preparation (Yamamoto & Washio, 1979). Such a variety of electrogenicity, as occurs in many arthropod muscles (Atwood, Hoyle & Smyth, 1965; Hoyle, 1974) could result from the various degrees of activation of the transient outward current.

Characteristics of the delayed current were similar to those of ‘intermediate-type’ crab muscle fibres (Mounier & Vassort, 1975 a).

As shown in Fig. 4, for depolarizing pulses longer than 30 ms a decrease in the positive tail amplitude occurs which is usually accompanied by a reduction in the magnitude of the delayed outward current during pulses. This can be attributed to either inactivation or shift of the equilibrium potential due to the large outward currents.

As to the ionic basis for the delayed current, potassium involvement was strongly suggested by the dependence of reversal potential on the external K concentration (Fig. 7). However, to get a good fit for the experimental plots, the line drawn to the Nernst equation using the measured value of external K concentration (Belton & Grundfest, 1962), must be shifted in the negative direction by about 20 mV. Furthermore, the slope of the potential-log [K+]0 relation was significantly different from the theoretical slope below 12 mM. This is likely due in part to a small amount of K loading in the extracellular space immediately adjacent to the membrane (Frankenhaeuser & Hodgkin, 1956) because of the currents flowing during the command pulse. However, this is also the sort of behaviour to be expected if some ions other than K carry a fraction of the delayed current. Whatever ions are involved, it appears correct that the K channel is responsible for the delayed outward current because of its sensitivity to TEA.

The action potentials of the mealworm muscle fibres are accompanied by positive after potentials whose amplitude increased with decreasing external K concentration (Belton & Grundfest, 1962). We have also observed that cathodal break provoked a prominent undershoot in unclamped fibre bathed in low-K saline containing Coa+ (in which the initial Ca spike was blocked). Under voltage clamp, only the delayed outward current could be detected in such fibres as expected. This suggests that the delayed current contributes to the repolarization and after potentials following an action potential under normal conditions.

The authors wish to thank Professor G. Hoyle for reading the manuscript an making valuable suggestions. Thanks are also due to Miss Junko Kaneko for her technical assistance.

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