The larval muscle fibers of Drosophila show four outward K+ currents in addition to the inward Ca2+ current in voltage-clamp recordings. The Shaker (Sh) and the slowpoke (slo) mutations, respectively, eliminate the voltage-activated fast K+ current (IA) and the Ca2+-activated fast K+ current (ICF)-Quinidine specifically blocks the voltage-activated delayed K+ current (IK) at micromolar concentrations. We used Sh, slo and quinidine to remove specifically one or more K+ currents, so as to study physiological properties of these currents not previously characterized, and to examine their role in membrane excitability. A linear relationship was observed between the peak ICF and the peak Ica at different membrane potentials. ICF inactivated considerably during a 140ms pulse to +20 mV. Recovery from inactivation was not complete for up to 2 s at the holding potential of −50 mV, which is much slower than the recovery of Ca2+ current from inactivation. In addition to IA and ICF, two delayed K+ currents are also observed in these fibers, the voltage-activated IK and the Ca2+-activated Ics-Near the end of a 500 ms depolarizing pulse, both IA and ICF are inactivated. Ca2+-free and 20 mmol 1−1 Ca2+ saline were used to examine the tail currents of the remaining IK and Ics-The tail currents of Ics were slower than those of IK and reversed between −30 and −50mV in different fibers. We further studied the dose-dependence of the blockade of IK by quinidine, which did not indicate a simple one-to-one binding mechanism. Current-clamp recordings from normal, Sh, slo and the double-mutant Sh;slo fibers suggested that ICF plays a stronger role than IA in repolarization of the larval muscle membrane. Elimination of ICF facilitates the occurrence of action potentials. Further elimination of IK prolonged the action potentials to several hundred milliseconds.
The existence of mutations which affect specific ionic currents in Drosophila provides an opportunity to analyze the properties of individual currents and to study the roles of these currents in membrane excitability. The advantage of mutational analysis can be complemented with the use of pharmacological agents that specifically block certain currents. The larval body-wall muscles in Drosophila support four K+ currents which can be distinguished and separated from each other with the use of mutations and drugs. These currents include two voltage-activated K+ currents, the fast IA and the delayed IK, and two Ca2+-activated K+ currents, the fast ICF and the delayed Ics (Salkoff, 1983; Wu et al. 1983; Wu and Haugland, 1985; Gho and Mallart, 1986; Wei and Salkoff, 1986; Singh and Wu, 1989). Among these currents, IA and 1er are eliminated by the Shaker (Sh) (Salkoff and Wyman, 1981; Wu et al. 1983; Wu and Haugland, 1985) and the slowpoke (slo) (Elkins et al. 1986; Singh and Wu, 1989) mutations, respectively, and IK is blocked by quinidine at micromolar concentrations (Singh and Wu, 1989). We have used Sh, slo and quinidine to study properties of the K+ currents that have not been described previously, and to look at their role in membrane excitability in larval body-wall muscle fibers.
Different names have been used previously for the Ca2+-activated K+ currents in Drosophila. The fast current has been designated as IAcd (Salkoff, 1983), IAcd (Gho and Mallart, 1986; Wei and Salkoff, 1986) and Ic (Elkins et al. 1986), and the slow current as IKC (Wei and Salkoff, 1986) and Ic (Gho and Mallart, 1986). To avoid confusion and to preserve consistency with the conventional names used in other species we use the present nomenclature, i.e. ICF for the fast C current and Ics for the slow C current (Singh and Wu, 1989). Part of the results presented here have appeared in abstract form previously (Singh et al. 1986; Singh and Wu, 1987).
Materials and methods
The wild-type strain Canton-S was used as the normal control. An extreme allele of the Shaker (Kaplan and Trout, 1969) locus, ShKS133 (Wu and Haugland, 1985), was used for eliminating IA. Only one allele of slo (Elkins et al. 1986) is available and was used in these experiments. These two mutant alleles have been extensively characterized in larval muscle (Wu et al. 1983, 1989; Wu and Haugland, 1985; Singh and Wu, 1989; Haugland and Wu, 1990).
The body-wall muscle preparation of mature third-instar larvae (Jan and Jan, 1976) was used for recordings, as described previously (Wu and Haugland, 1985; Singh and Wu, 1989). Two-microelectrode voltage-clamp recordings were made with a holding potential of −50 or −80 mV, as specified in the figure legends. Both the voltage-clamp and the current-clamp recordings were made at 4°C. Recording saline contained (in mmol 1−1) 128 NaCl; 35.5 sucrose; 2 KC1 and 5 Hepes, and was buffered at pH7.1. In addition, the Ca2+-free saline had 0.5mmoll−1 EGTAand MgCl2 and was adjusted to 20 mmol 1−1 for improving membrane stability (Wu and Haugland, 1985). Unless otherwise specified, the saline with Ca2+ contained 20 mmol 1−1 CaCl2, 4 mmol 1−1 MgCl2 and no EGTA. Quinidine and 4-aminopyridine (4-AP) were obtained from Sigma Chemical Co. (St Louis, MO).
The muscle fibers used in these experiments are supercontracting muscles (Osborne, 1967). The contractions induced during recordings, especially during strong depolarizations, damage the fiber quickly. Although it is possible to make refiable recordings from a fiber, it is difficult to take a single fiber through repeated experiments using different experimental paradigms or recording solutions. Therefore, all individual experiments described here were performed on separate fibers.
Voltage or current pulses were generated at intervals of 10 s by a Master-8-cp pulse generator (A.M.P.I., Jerusalem, Israel). The voltage-clamp circuit was equipped with a high-voltage head stage (model HVHD, Almost Perfect Electronics, Basel, Switzerland). The current electrode was shielded up to a few millimeters from the tip with a coaxial double shield. The inner shield was driven at the potential of the electrode and the outer shield was grounded. The voltage electrode was filled with KC1 and the current electrode with a mixture of KC1 and potassium citrate. The electrodes were pulled to a resistance between 5 and 15 MΩ. The current signal was recorded through the virtual ground using the amplifier M701 (WPI, New Haven, CT). The current and the voltage signals were pulse-coded using a Neurocorder unit DR-384 (Neuro Data Instruments Corp., New York, NY), and recorded using a video cassette recorder. The data were digitized and subsequently analyzed on an Apple-Macintosh II computer. Current densities in voltage-clamp recordings were obtained by normalizing the current with respect to the fiber capacitance (Haugland, 1987; Haugland and Wu, 1990). Leakage current was subtracted digitally and the initial 3 ms of recordings which contain capacitive transients have been omitted in the figures for the sake of clarity, as described previously (Singh and Wu, 1989).
Early membrane currents
Voltage-clamp recordings made in 1.8 mmol 1−1 Ca2+, the concentration generally used in Drosophila saline, showed considerable variation in the Ca2+-dependent currents. This problem was overcome by increasing the external Ca2+ concentration. Saline with 20 mmol 1−1 Ca2+ was therefore used (Singh and Wu, 1989) for voltage-clamp measurements of Ca2+-dependent currents in this paper.
Early membrane currents recorded from different genotypes are shown in Fig. 1. The fast current peak seen in the normal fibers (Fig. 1A) consists of two outward currents, the voltage-activated (IA) and the Ca2+-activated (ICF) K+ currents, and an inward Ca2+-current, Ica-As shown previously (Gho and Mallart, 1986; Singh and Wu, 1989), the outward currents mask the inward current which is therefore not apparent in the figure. Lack of ICF in the slo mutation (Elkins et al. 1986; Singh and Wu, 1989) revealed the sum of IA and lea, in which the outward peak was reduced but the inward current was still masked (Fig. 1B). In a similar way, the Sh mutant that lacks IA (Salkoff and Wyman, 1981; Wu et al. 1983; Wu and Haugland, 1985) showed the sum of ICF and Ica (Fig. 1C). The inward Ca2+ current can be seen during the short interval before it is masked by the outward ICF-Since these fibers have both IA and ICF, the total outward current is stronger than in Sh or in slo. Elimination of both the outward currents (IA and ICF) in the Sh;slo double mutant (Fig. 1D) unmasks the inward Ica
Both IA and ICF in larvae (Wu and Haugland, 1985; Gho and Mallart, 1986) and in adults (Salkoff, 1983; Salkoff and Wyman, 1983) have been shown to inactivate with membrane depolarization. The ICF inactivation in larvae is almost complete at a membrane potential of −20mV (Gho and Mallart, 1986), a potential at which lea or ICF have still not been activated. This implies that the ICF inactivation is voltage-dependent and not Ca2+-dependent. However, there is no study on the recovery of ICF from inactivation in larvae. This was studied by using different experimental protocols and recovery times in recordings from Sh (44 fibers) and Sh;slo (36 fibers). Fig. 2 shows the effect of a 140ms pre-pulse to +20mV on ICF and lea-Their recovery, after different times at the holding potential of −50 mV, was tested by an identical test pulse. The pulse paradigm is shown in Fig. 2A. The first traces in both the Sh and the Sh;slo recordings in Fig. 2B show the membrane current during the pre-pulse, which serves as a control. Since IK and Ics rise slowly with a delay, the early phase (0–40 ms) of this current mainly consists of the inward lea in Sh;slo and the sum of lea and ICF in Sh. The traces on the right show membrane currents in response to the test pulse, after the indicated recovery period at −50 mV. ICF was considerably inactivated by the pre-pulse. Note that the first test pulse (after 0.1s recovery time) shows stronger inward current than the control current seen with the pre-pulse in Sh (Fig. 2B). Since the Sh fibers lack IA, the inactivation of ICF unmasks the inward current to a greater degree. Even if inactivation did occur in lea during the pre-pulse, it recovered during the first 100ms, as indicated by recordings in Sh;slo. In contrast, ICF showed clear inactivation which did not recover completely within 2 s (compare Sh and Sh;slo in Fig. 2B). The recovery from inactivation of IcF was slower than that of IA, which shows substantial recovery within Ics at a comparable temperature (Wu and Haugland, 1985). The main result is that the recovery of 1er from inactivation may not be linked to that of Ica
Relationship between peak ICF and Ica
The possibility of extracting ICF with the use of mutations enabled us to look at the relationship between this current and the inward Ca2+ current that is responsible for its activation. The current-voltage relationship for the extracted ICF is given in Fig. 3A. The values for ICF were obtained by subtracting the averaged current densities recorded in Sh;slo from those recorded in Sh. The current for Sh is shown as the early peak of the outward current at different membrane potentials. For the purpose of subtraction, the inward ICa in Sh;slo was measured at the time when a peak occurred for the outward current in Sh at the corresponding voltage. This time was a few milliseconds after the actual inward peak in Sh;slo (see Fig. 1C and D), which gives values very similar to the ones shown in Fig. 3A.
Fig. 3B shows the relationship between ICF and lca, determined as described for Fig. 3A. It shows a linear dependence of ICF on lea-This is consistent with a one-to-one correspondence between the Ca2+ entering the fiber and the opening of individual ICF channels. A linear relationship between the amount of Ca2+ injected directly into the cell and the value of the Ca2+-activated K+ current has also been reported in the dorsal unpaired median neurons in the cockroach (Thomas, 1984). However, the data cannot be taken as conclusive evidence for a one-to-one correspondence, because the amount of free Ca2+ in the cytosol depends on the influx of Ca2+, on the nature of the Ca2+ sequestering process and on a number of other factors.
The Ca2+-activated slow Ics
In addition to the fast Ca2+-activated ICF, the larval muscle fibers in Drosophila also show a slow Ca2+-activated outward Ics (Gho and Mallart, 1986; Singh and Wu, 1989). We performed experiments with 0 mmoll−1 Ca2+ (20 fibers for Sh and 10 fibers for Sh;slo) or 20 mmol 1−1 Ca2+ (19 fibers for Sh and 13 fibers for Sh;slo) in the saline. Ics was observed in recordings made in saline with 20 mmol 1−1 Ca2+. Sh and Sh;slo gave similar results because ICF is almost completely inactivated during the 500 ms pulse used in these experiments (Fig. 2 shows inactivation produced by a 140 ms pulse) and the contamination from residual ICF is expected to be minor. Fig. 4A shows the voltage-activated IK in Ca2+-free saline, which reaches a steady state before the end of the 500 ms pulse. In Fig. 4B,C, the delayed outward current recorded in saline with Ca2+ consisted of IK and Ics-Ics can be seen as changes in the overall amplitude of the slow outward current and the tail currents after the voltage pulse has ended. Unlike ICF, ICS did not inactivate, and in fact continued to rise for at least 500 ms. This contrast between the fast and the slow Ca2+-activated outward currents, ICF and Ics, parallels the difference between the two voltage-activated outward currents, the fast transient IA and the delayed sustained IK
The tail currents were recorded by stepping the membrane potential to +20 mV and then repolarizing it to different voltages (Fig. 4). The tail currents immediately after the end of the voltage pulse are either inward or outward, depending on the membrane potential reached. Inward tail currents are expected if the membrane potential is repolarized below the reversal potential for the current, and outward tails are expected if it is above the reversal potential. Only outward tail currents were seen in recordings made in Ca2+-free saline (Fig. 4A). This is because IK does not show inward tail currents as there is strong rectification (Wu and Haugland, 1985). Inward tails were seen when recordings were made in the presence of Ca2+. Fig. 4B shows inward tail currents for Ics and combined outward tail currents for IK and ICs-Inward tails are seen for potentials up to −40 mV. At −30 mV, the tails were completely outward. At this potential, Ics makes only a marginal contribution to the outward tail in Fig. 4B, as can be seen by comparing with the tail at −30 mV in Fig. 4A. The reversal potential for ICs in Fig. 4B thus lies between −40 and −30mV, near to −30mV. In different fibers, the reversal potential ranged between −30 and −50 mV.
In some fibers, depolarization by repeated test pulses showed a cumulative effect on the outward current and gave rise to more complex tail currents. Fig. 4C shows an example where inward tail current is seen up to −20 mV. The outward tail for −10 mV (Fig. 4C) is also not as strong as the IK tail at the same voltage (Fig. 4A). The reversal potential for the current in addition to IK in this case seems to be more positive than −10 mV. A possible interpretation of this is suggested in the Discussion.
The relative role of 1A and ICF in membrane excitability
The ability to eliminate individual K+ currents by means of mutations and pharmacological agents allows an analysis of the role of different currents in membrane excitability. Mutational elimination of IA and ICF has been used in adult dorsal longitudinal flight muscles (DLM) of Drosophila for this purpose (Elkins and Ganetzky, 1988). We used the lack of IA in Sh and the lack of ICF in slo to study the role of these currents in the excitability of larval muscle fibers. This was done by looking at the membrane potential in response to constant current injection.
Regenerative potentials have recently been demonstrated in larval preparations in which air was supplied through tracheoles to maintain the resting potentials up to −80mV (Yamaoka and Ikeda, 1988). Most previous reports, which used unaerated preparations, described lower resting potentials and only graded potentials in response to current injection in normal physiological saline, which contains 1.8mmoll−1 Ca2+ (Suzuki and Kano, 1977; Wu and Ganetzky, 1988). We found that regenerative responses were easier to elicit when the resting potential was sufficiently negative, either in a freshly dissected preparation or because of injection of a hyperpolarizing current even in the absence of aeration. Recordings were made from the normal (4 fibers), Sh (9 fibers) and slo (13 fibers). Fig. 5 shows recordings made in fibers with the resting potential kept near −60 to −70 mV by injecting hyperpolarizing current. The regenerative peak in these recordings is more pronounced in slo and in Sh. A smaller amount of current was required to elicit regenerative potentials in slo as compared to Sh. This indicates that ICT is more effective than IA in repolarizing the regenerative potential supported by ICa.
The relative role of different outward currents can be seen more clearly by using saline containing 20mmoll−1 Ca2+. With increased Ca2+ concentration, nounced regenerative potentials could be elicited when one or more outward currents were removed by mutations or drugs. This could be achieved without aeration in preparations with resting potentials as low as −20 mV. Fig. 6 shows recordings made in saline containing 20 mmol 1−1 Ca2+ without injection of hyperpolarizing current. The normal fibers showed graded potentials in response to increasing amounts of current injection (Fig. 6A) and did not show action potentials. Elimination of IA by Sh led to increased depolarization, which was still graded in nature (Fig. 6B). However, when ICF was eliminated by the slo mutation, the fibers underwent all-or-none action potentials (Fig. 6C). This is consistent with the observation shown in Fig. 5 that ICF plays a stronger role than IA in repolarizing the membrane. As expected, slo fibers treated with 4-AP (100μmoll−1), which preferentially blocks IA at this concentration, also exhibit all-or-none action potentials, but requiring even less injection of current (Fig. 6D) than slo (Fig. 6C). The number of fibers tested was 11 for the normal, 10 for Sh, 16 for slo and 22 for slo with 4-AP.
Blockade of IK by quinidine
Quinidine, which blocks different K+ currents in several systems (Fishman and Spector, 1981; Hermann and Gorman, 1984; Imaizumi and Giles, 1987), blocks IK in Drosophila (Singh and Wu, 1989). The inset in Fig. 7 shows membrane currents in response to voltage steps from the holding potential of −80 mV to −10, +10 and +30mV in Ca2+-free saline with and without quinidine. IA was removed by the use of Sh, and 0.1 mmol 1−1 quinidine substantially blocked the only remaining current IK-This concentration of quinidine has earlier been shown not to block IA, ICF or Ics (Singh and Wu, 1989). The double-reciprocal plot in Fig. 7 shows the effect of different concentrations of quinidine on the amplitude of IK. Quinidine at 10 μmol 1−1 reduced IK by more than half. In this plot, the reciprocal of the fraction of inhibition is plotted against the reciprocal of quinidine concentration in micromoles per liter. A linear relationship would signify a simple saturation with one-to-one binding of quinidine molecules to the IK channels. Fig. 7 shows a deviation from a linear relationship. Although IK is blocked more than 50% by 10μmoll−1 quinidine (as indicated by the upper-most point in the plot), even 1.0 mmol 1−1 quinidine leaves some current intact (the lower-most point in the plot). It is not clear at this stage if this is a manifestation of sub-populations of IK with different sensitivity to quinidine.
Some K+ channel blockers are known to show voltage-dependent action. The extent of inhibition by quinidine was examined at +10 and +30 mV in Drosophila. The plot shows small differences at the two potentials (Fig. 7). These differences could not be substantiated statistically, owing to the small sample sizes, but were far less pronounced than the voltage-dependence reported for 4-AP (Yeh et al. 1976) and tetraethylammonium (Armstrong, 1971).
The role of IK in membrane excitability
Selective blockade of IK by quinidine provided an opportunity to examine the role of this current in determining the membrane potential. Fig. 8 shows current clamp recordings in saline containing 20mmoll−1 Ca2+ and 100μmoll−1 quinidine. In the absence of quinidine, normal and Sh fibers did not show action potentials, as discussed above. Removal of IK by quinidine did not show a strong effect except for a slight regenerative potential, as indicated by a shoulder in Sh after the end of the current injection (Fig. 8B). In the case of slo and Sh;slo, regenerative potentials were initiated even during weak current pulses. After the end of the pulse, the regenerative potential was sustained for hundreds of milliseconds (Fig. 8C,D). The duration of the prolonged depolarization was highly variable even between fibers of the same genotype and no consistent differences were found between slo and Sh;slo. Elimination of IK thus prevents repolarization of Ca2+ regenerative potentials initiated either by the lack of ICF alone or by the elimination of both ICF and IA (compare Fig. 8 with Fig. 6). The number of fibers tested was 9 for the normal, 8 for Sh, 10 for slo and 7 for Sh;slo.
Properties of K+ currents
The availability of the two mutations, Sh and slo, allows a comparison between IA and ICF underlying the early phase of the membrane current. Fig. 1B,C shows that the generation of the Ca2+-activated ICF is preceded by the voltage-activated IA. Presumably, ICF requires Ca2+ influx following ICa activation, whereas IA is activated by membrane depolarization without a lag following lea activation. As previously reported (Gho and Mallart, 1986), ICF, like IA, was inactivated by membrane depolarization. ICF recovery from inactivation followed a time course much slower than that of Ica recovery (Fig. 2), suggesting a process independent of the availability of cytosolic free Ca2+.
The late outward currents consist of IK and Ics-IK can be isolated by using Sh in Ca2+-free saline. This allows an analysis of the effect of quinidine on IK. A double reciprocal plot between the fractional inhibition and the quinidine concentration did not indicate a linear relationship (Fig. 7). This observation suggests that there are different sub-populations of IK channels with different sensitivities to quinidine, a possibility that requires further investigation.
As mentioned above, IK does not show inward tail currents because of rectification (Fig. 4A). Outward currents are seen at potentials more positive than −60 mV but not at a potential of −70 mV. The reversal potential for this current is therefore between −60 and −70 mV, consistent with the earlier reported reversal potential for IK (Wu and Haugland, 1985). Fig. 4B shows both outward and inward tail currents, recorded in saline containing Ca2+. In different fibers, the component contributed by Ics reversed at a potential between −50 and −30 mV (see Results). This is consistent with Ics being a K+ current in larvae (Gho and Mallart, 1986) as well as in adults (Wei and Salkoff, 1986). The currents observed in the presence of Ca2+ show a greater degree of variation in some fibers. Fig. 4C shows such a fiber with a reversal potential that is more positive than −10 mV. Such a reversal potential was generally associated with stronger tail currents as well as a cumulative effect on the variable outward currents during repeated voltage pulses (Fig. 4C). This type of behavior was generally elicited in fibers that were relatively leaky, which may be an indication of deterioration or damage. A more positive reversal potential may be accounted for by the presence of yet another set of channels or a non-selective conductance in addition to the Ics channels.
The roles of K+ currents in membrane excitability
Yamaoka and Ikeda (1988) have recently shown that the tracheole aeration of the larval preparation is essential in maintaining the resting potential of the muscle fibers and in initiating a regenerative response. Lack of aeration results in a drop in the resting potential from about −80 mV to about −30 mV within l h at room temperature. It is known that the resting potentials in many insect muscles are maintained below the K+ equilibrium potential by electrogenic pumps (Huddart and Wood, 1966; Rheuben, 1972; Henon and Ikeda, 1981). The metabolic state, which would depend upon effective aeration, is thus expected to influence the resting potential. However, it is not clear if the effect of aeration on the regenerative response is due to the membrane potential, which would determine the state of channel inactivation, or to a direct deteriorative effect on channel machinery or to some other cellular factors. Recordings in this study and in other reports (Jan and Jan, 1976; Suzuki and Kano, 1977; Wu and Ganetzky, 1988) have been made in the larval preparation without tracheole aeration. Our recordings from muscle fibers with resting potentials at different levels indicated that the metabolic condition was probably not a prerequisite for regenerative responses. Regenerative responses were easier to elicit in fibers with resting potentials between −60 and −70 mV, maintained by injection of hyperpolarizing currents or by low temperature (4°C) in freshly dissected preparations. For example, regenerative potentials could be seen in Sh fibers only if the resting potential was more negative than −60 mV (compare Figs 5B and 6B; note the inflexion points in Fig. 5B). An effect of resting potential on the regenerative events is expected since the Ca2+ channels mediating inward current inactivate to different degrees depending upon the extent of membrane depolarization. The main aim of the present study was to compare the relative role of different K+ currents in membrane excitability. We found clear differences between the consequences of eliminating different currents on excitability, even without tracheole aeration. However, it is important to investigate these differences under normal physiological conditions with effective aeration.
An important consideration is the free Ca2+ concentration around the fibers as well as inside the fibers. This can be a source of variation in the Ca2+-activated K+ currents as it can affect the amount of Ca2+ influx and the Ca2+ available to trigger these currents. From electron microscopic observations (Jan and Jan, 1976; C.-F. Wu, unpublished data), the larval muscle fibers are enveloped by relatively little connective tissue. In addition, ion replacement and drug additions to the recording saline show immediate effects, indicating a lack of effective diffusion barriers (Wu and Haugland, 1985; Haugland, 1987; Singh and Wu, 1989; Wu et al. 1989). The level of intracellular free Ca2+ can conceivably vary in relation to metabolic state, e.g. a weakened sequestering mechanism, and affect the Ca2+ concentration gradient across the plasma membrane as well as the available free Ca2+ for activating ICF and Ics. It will be important to determine the free Ca2+ level directly, employing other available techniques in future investigations.
Interactions between the outward and the inward currents determine the membrane potential in response to a current stimulus. The inward current depolarizes the membrane and the outward current counteracts this by repolarizing the membrane. As in other arthropod species, the inward current in Drosophila muscles is carried by Ca2+ (Suzuki and Kano, 1977; Salkoff and Wyman, 1983; Elkins et al. 1986; Gho and Mallart, 1986). A tetrodotoxin-sensitive inward current was recently suggested by current-clamp experiments (Yamaoka and Ikeda, 1988). It remains to be determined by voltage-clamp experiments whether it is a Na+ inward current. The outward currents in Drosophila muscles consist of two voltage-activated (IA and IK) and two Ca2+-activated (ICF and Ics) K+ currents. The effectiveness of different K+ currents in repolarizing the membrane depends on their channel density (current amplitude) and their kinetic characteristics. Because of these differences, they give rise to a variety of excitability patterns.
An ability to manipulate different currents, by the use of mutations and drugs, provides an opportunity to examine the role of individual currents in excitability. The two fast outward currents (IA and ICF) in the normal fibers limit the membrane depolarization immediately after the initiation of the current injection. As compared to the normal muscles, therefore, a lack of IA in the Sh muscles allowed a larger initial depolarization with the same amount of current injection (compare Fig. 6A and 6B). However, removal of ICF alone, in slo, had a greater effect, resulting in action potentials, even at a low resting membrane potential (see Fig. 6C). This implies that ICF plays a more effective role in membrane repolarization than does IA during prolonged current injections. It is known that IA inactivates rapidly (Salkoff and Wyman, 1983; Wu and Haugland, 1985) and is effective in delaying membrane excitation (Salkoff and Wyman, 1983; Elkins and Ganetzky, 1988) but would not have a prolonged repolarizing effect. Although ICF is similar in magnitude to IA, its activation is coupled to the activation of Ica-In addition, it inactivates at more depolarized potentials (Gho and Mallart, 1986) and with a slower time course (S. Singh and C.-F. Wu, unpublished observations) than IA. The effect of IA removal became more apparent only when the resting potential was more negative (Fig. 5B) or when ICF was also removed (action potentials in Fig. 6D require less current injection compared to Fig. 6C). These observations on the relative role of IA and ICF in the excitability of larval muscles are consistent with the earlier conclusions on their role in the adult flight muscles (Elkins and Ganetzky, 1988).
In contrast to the early currents, the slow outward currents activate with delay but show little inactivation. The role of IK in muscle repolarization is revealed by the use of quinidine. In normal and Sh fibers, blockade of IK by quinidine led to only marginal differences. However, when the membrane undergoes an action potential, as when ICF is removed in slo or Sh;slo, IK assumes an important role in repolarization. The action potentials lasted only a few tens of milliseconds when IK was present (Fig. 6C,D) but lasted for several hundred milliseconds when IK was blocked by quinidine (Fig. 8C,D). In the experiments shown in Figs 6 and 8, IA and ICF are expected to have inactivated to a great extent by the end of the current pulse. The repolarization of the action potential, once initiated, thus depends mainly on IK and Ics-With the further removal of IK, the termination of the prolonged action potential after the end of the current pulse will depend primarily on the amplitudes of Ica and Ics, and how they change with time. The membrane during this prolonged depolarization is presumably in an unstable equilibrium and the time taken by the membrane to repolarize varied considerably, regardless of the genotype (data not shown). The sustained action potential is similar to, but less prolonged than that seen in the larval muscle fibers treated with tetraethyl ammonium (Suzuki and Kano, 1977; S. Singh and C.-F. Wu, unpublished observations), which blocks all four K+ currents in this system to different degrees (Wu and Haugland, 1985; Gho and Mallart, 1986). So far no mutations or drugs remove Ics specifically. Such a possibility would be very useful for determining the role of Ics in membrane excitability.
This work was supported by USPHS grants NS-15350, NS-18500 and NS-26528.