Voltage-Activated Currents in Somatic Muscle of the Nematode Parasite Ascaris Suum

Ascaris suum is an intestinal nematode parasite of pigs and is capable of maintaining its position in the gut by means of forwardly directed waves of contraction. The somatic muscle cell of Ascaris consist of a contractile spindle region; a balloon-shaped structure, the bag which contains the nucleus; and a thin process known as the arm which passes to a nerve cord where it is electrically coupled to adjacent cells and where it receives excitatory and inhibitory synaptic inputs (Rosenbluth, 1963, 1965). DeBell et al. (1963) and Weisblat et al. (1976) have studied the electrophysiology of Ascaris body muscle cells and described three types of depolarizing potential recorded in the bag region of the muscle which are related to contraction. They described the ‘spike potentials’, the ‘slow waves’ and the ‘modulation waves’. The spike potentials were of varying amplitude up to 30 mV, lasted 5–10 ms and behaved like Ca²⁺ spikes; the slow waves were up to 20 mV in amplitude and lasted 100–1000 ms; the modulation waves lasted several seconds, were up to 5 mV in amplitude but were directly associated with muscle contraction. The currents responsible for depolarization and repolarization of the cells have yet to be isolated and identified.

Muscle cells of Ascaris are large enough to allow a two-microelectrode voltage-clamp to be used to examine membrane currents (Martin, 1982). This technique has revealed the presence of a voltage-sensitive Ca²⁺ current and two K⁺ currents that underlie the spike potential and are the subject of this paper.

Ascaris suum, approximately 20 cm in length, were obtained from the local slaughterhouse, maintained in Locke’s solution at 32 °C and used within 4 days. An anterior section of the worm, 2 cm long, was cut along a lateral line and then pinned cuticle side down onto a layer of Sylgard in the experimental chamber. The gut was then removed, exposing the muscle cell bodies. The experimental chamber consisted of a central bath with a liquid capacity of 1.8 ml. The bath was surrounded by a water jacket through which water was circulated to maintain the preparation at 37 °C. The worm was viewed from above through a Bausch and Lomb zoom microscope and cross illuminated using a fibre-optic light source. The preparation was continuously perfused with a Shuco pump and, by means of a broken pipette, positioned near (500 gm) the cell recorded from, in order to accelerate solution changes. The flow rate for the total perfusion system was set at 8 ml min⁻¹ and cleared the bath in 4.5 min when filled with blue dye. An interval of 7–15 min was therefore used after a change of solution and before recording.

Two micropipettes were placed in the bag region of the muscle cells in order to carry out the two-microelectrode voltage-clamp experiments. The micropipettes were pulled on a David Kopf puller from filamented glass tubing (Clarke Electromedical Instruments GC 150F-15). The current micropipettes had a resistance of 4 MΩ and the voltage micropipette had a resistance of 11 MΩ. Both were filled with 3 mol l⁻¹ potassium acetate. 3 mol l⁻¹ magnesium sulphate was used to fill the micropipettes when reversal potentials were being measured.

Initially artificial perienteric fluid (APF) was used which contained (mmol l⁻¹): NaCl, 23; sodium acetate, 110; KC1, 24; CaCl₂, 6; MgCl₂, 5; glucose, 11; Hepes, 5; pH7.6 with NaOH. For recording Ca²⁺ currents, a solution with TEA⁺ to block K⁺ currents and a high Ca²⁺ concentration was used containing (mmol l⁻¹): TEACI, 69; calcium acetate, 69; potassium acetate, 24; magnesium acetate, 5; glucose, 11; pH 7.6 with maleic acid. In most of the experiments described here, a Ca²⁺-free lanthanum solution based on Ascaris Ringer was used which contained (mmol 1⁻¹): NaCl, 135; KC1, 3; MgCh, 15.7; glucose, 3; Tris, 5; EGTA, 0.5; LaCl, 1 ; pH 7.6 with maleic acid. The use of this type of low-Ca²⁺ solution is likely to abolish secondary channel currents produced as a result of entry of Ca²⁺ into the cell: one example of this type of channel current in Ascaris is the Ca²⁺-activated Cl⁻channel (Thom and Martin, 1987). In order to modify the reversal potential of tail currents, Ca²⁺-free lanthanum solutions were used in which sodium was substituted for potassium; they contained (mmol 1⁻¹): NaCl, 0.1–138; KC1,0.1–138 (total K⁺+Na⁺=138); MgCh, 15.7; glucose, 3; Tris, 5; EGTA, 0.5; LaCl, 1; pH7.6 with maleic acid.

A Dagan 8500 voltage-clamp was used and all output was displayed on a Gould OS400 digital storage oscilloscope with an IEEE 488 interface. The gain on the amplifier was adjusted to 2500-5000 V/V and the phase lag was adjusted for each cell to give optimal recording. Data were recorded on a Racal Store Four DS tape recorder with a bandwidth of up to 2.5 kHz. The signal was played back into a Lectromed chart recorder or a Gould Series 6000 XY plotter to provide hard copy. All pulse regimes were controlled by a Digitimer DI00. Stimulus input was provided by Digitimer DS2 isolated stimulators. Leak subtraction, where used, was performed by summation of two current responses to equal but opposite-polarity voltage steps. The current response to hyperpolarizing steps was linear in the range used and therefore represented the leak current.

It was possible to check the quality of the space-clamp in the bag region of the muscle cell by placing a third micropipette in the base of the bag, at a site near the junction with the spindle, while the other two micropipettes for the voltage-clamp were placed at the top of the bag. Under these conditions it was found that the third micropipette could record a voltage response that was 95 % of the command potential within 500 μs. In view of the particular anatomy of the muscle cells of Ascaris it is, however, likely that there may be poorly controlled regions of membrane: these were recognised as ‘notches’ of inward cunent which occurred at a relatively long but irregular time after the start of the depolarizing pulse, producing a very abrupt rise in the inward current–voltage relationship. Such events led to the rejection of the muscle cell for recording: only currents from cells which showed adequate voltage control were analysed.

The peak amplitude and time to peak of current responses were measured from graphs of currents plotted against time. The limiting equivalent voltage sensitivity for the Ca²⁺ current was determined from the maximum slope of the plot of log_e(peak Ca²⁺ current/maximum peak Ca²⁺ cunent) against voltage (Hille, 1984). A BBC microcomputer with a Unilab interface was used to digitize the currents. Sampling rates were adjusted between 1 kHz and 0.05 kHz, depending on the duration the sample. The rate of decay was then analysed using the BMDP P3R non-linear regression program. The best fits to the Boltzmann equation were obtained using the NAG subroutine E04CCF.

This paper describes results from 49 successful voltage-clamp experiments. The resting membrane potentials of recorded muscle cells were around −35 mV with a mean input conductance of 2.4±0.18 μS (mean ± S.E., N=9) measured using 150 ms hyperpolarizing voltage pulses.

Depolarizing voltage steps from a holding potential of −35 mV elicited an outward current in cells bathed in APF solution (Fig. 1A). These currents rose to a peak within 20 ms and had an inwardly directed inflection in the rising phase: these inflections were not because of ‘breakthrough’ as a result of poor space-clamping because they occurred in most cells at regular intervals after the onset of the depolarizing pulse. After the peak current there was a slow decay. As the depolarizing steps were made more positive, the outward-current peak became more pronounced. Hyperpolarizing steps did not induce active currents (Fig. 1B). A plot of current amplitude 20 ms after the voltage step against the potential shows the current increasing non-linearly on depolarization but linearly on hyperpolarization (Fig. 1C). The linearity observed following hyperpolarization was subsequently used to allow for ‘leakage’ correction.

In five experiments, substitution of sodium in APF with 69mmol l⁻¹ tetraethylammonium (TEA⁺) and 69mmol l⁻¹ calcium revealed an inward current activated by depolarizing voltage-clamp steps from a holding potential of −35 mV. This inward current was identified as a Ca²⁺ current on the grounds that the permeant cation was calcium in the high-Ca²⁺ TEA⁺ solution. As further confirmation of the identity of the inward current, it was found that bath-application of lanthanum (using 1 mmol l⁻¹ LaCl) as a calcium channel blocker rapidly abolished the inward current (not shown). The Na⁺ channel blocker tetrodotoxin was not used because of its lack of effect in Ascaris (Jarman and Ellory, 1969). A family of Ca²⁺currents evoked by increasing depolarizing steps and corrected for leakage is shown in Fig. 2A. The threshold for activation of the inward current was around −15 mV. More depolarized voltage-clamp steps increased the amplitude of the inward-current peak up to a value of about +5 mV and thereafter decreased it with further depolarization; the time to peak decreased monotonically with increasing depolarization. At the most depolarized potentials, the peak of the inward current was followed by a small outward cunent which may be because of residual outward current not blocked by TEA⁺. However, there was no evidence of any maintained current; at the end of the 150 ms pulses all the currents had inactivated and there was tail current on the return to the holding potential of −35 mV. The time constant for inactivation of the Ca²⁺ cunent was, therefore, less than 150 ms, the length of the voltage pulse. All the data from this cell relating peak inward cunent to voltage are shown in Fig. 2B. The maximum inward cunent was 411 nA, the extrapolated reversal potential was +45 mV and the limiting equivalent voltage-sensitivity (Hille, 1984) was 7.5 mV per e-fold increase in Ca²⁺ cunent.

The predicted Nemst Ca²⁺ potential with 69mmol 1⁻¹ extracellular calcium and 1 μmol l⁻¹ intracellular calcium is +140mV, some 95 mV more positive than estimated here. Despite the theoretical prediction of + 140 mV, many other authors report values as low as +40 mV to +70 mV, which they explain by the Ca²⁺ channels also being permeable to other monovalent ions (K⁺) moving outwards through the Ca²⁺ channels (Reuter and Scholtz, 1977; Fenwick et al. 1982; Lee and Tsien, 1982).

The aim of this series of experiments was to examine the permeability of the outward current activated by depolarization. The experimental approach was to activate the currents and then to determine the reversal potentials of the tail currents at different potentials, in solutions of different potassium concentrations (Fig. 3A–D). Cells were bathed in Ca²⁺-free lanthanum solutions to block Ca²⁺ currents (Meech and Standen, 1975; Thomas, 1984) and potassium was replaced by sodium. The experiments were carried out using a two-pulse voltage-clamp protocol: i.e. a holding potential of−35 mV, a prepulse potential of +55 mV for 10–20 ms to activate the outward current and a step potential to between -125 mV and +55 mV. The amplitude and duration of the tail current elicited during the step potential then allowed the reversal potentials of the current activated by depolarization to be determined.

These experiments are consistent with depolarization activating cation channel currents which are about twenty times more permeable to potassium than to sodium. The extracellular potassium concentration is around 20 mmol l⁻¹ in Ascaris (Brading and Caldwell, 1971) and the reversal potential of the outward current is expected to be in the linear region of the plot (Fig. 3E) under physiological conditions. Subsequently in this paper the voltage-activated outward current is referred to as the K⁺ cunent.

Prolonged depolarizing voltage steps produced a K⁺ current which rapidly rose to a peak and then slowly declined. This decline was taken to indicate slow inactivation of the K⁺ current during depolarization; at hyperpolarized potentials the inactivation would have to be removed to allow subsequent activation by depolarizing steps. In order to study the time course of recovery from inactivation, a two-step depolarizing protocol was used: the depolarizing steps were separated by intervals lasting up to 12 s at a holding potential of −35 mV (Fig. 4A). A delay of 1 min between each series of two depolarizing pulses was adequate to allow complete recovery of the K⁺ current.

Steady-state inactivation was studied by holding cells at potentials between − 70 mV and − 10 mV and stepping to a potential of +50 mV. The amplitude of the peak K⁺ current was reduced in all cells as the holding potential was made more depolarized. The effect of holding potential on K⁺ current is plotted in Fig. 5 for a representative cell: the amplitude of peak K⁺ currents at +50 mV obtained from different holding potentials was expressed as a percentage of the peak K⁺ current obtained from a holding potential of − 70 mV. It can be seen that the reduction in peak K⁺ current, because of inactivation, was partial, since the current only declined by 34% when very depolarized holding potentials (− 20 mV) were used. Similar results were obtained from three other cells.

The selective effect of inactivation on the early component (the peak) of the K⁺ current and the fact that only part of the K⁺ currents may be inactivated by very depolarized holding potentials (Figs 4 and 5) suggest that the K⁺ currents may be divided into two currents corresponding to an inactivating K⁺ current and a non-inactivating K⁺ current.

A rapidly inactivating, transient K⁺ cunent, known as I_a, and a slowly inactivating current, known as Ik, are prominent features of many other cells (e.g. Connor and Stevens, 1971; Neher, 1971; Kostyuk et al. 1981; Thompson, 1977; Belluzzi et al. 1985). The I_a currents of these cells are selectively blocked by the potassium channel antagonist 4-AP.

The effect of 5 mmol l⁻¹ 4-AP on the K⁺ currents of Ascaris was therefore examined using cells bathed in a lanthanum solution and given depolarizing steps to between − 25 mV and +55 mV from a holding potential of − 35 mV (Fig. 6A). The currents obtained in the presence of 4-AP showed an increase in the time to peak and a decrease in the peak amplitude (Fig. 6B). At a potential of +55 mV the amplitude of the current was reduced to around 70% of its original value; washing the preparation for 30 min did not reverse the effect of 4-AP.

The currents obtained after application of 5 mmol l⁻¹ 4-AP were then subtracted from the control records to determine the 4-AP-sensitive current. Fig. 6C shows the currents at +55 mV. It can be seen that the 4-AP-sensitive current was a transient current which rose to a peak and decayed rapidly.

The peak amplitudes of the 4-AP-sensitive and the 4-AP-resistant currents were plotted against the step potential: all the data from the cell shown in Fig. 6, relating the peaks of the 4-AP-sensitive and 4-AP-resistant cunents to voltage, are shown in Fig. 7A. The threshold for both currents was near − 15 mV but between − 15 mV and +25 mV the 4-AP-sensitive cunent peak was greater.

Our main conclusion is that a voltage-sensitive Ca²⁺ current and two K⁺ currents can be isolated and observed in the soma of muscle cells of Ascaris suum. The large amplitude and relatively fast kinetics are consistent with these currents in combination generating the spike potentials described by Weisblat et al. (1976). Slower and smaller-amplitude currents which may underlie ‘slow waves’ and ‘modulation waves’ were not revealed in this study. It was pointed out in Materials and methods that low-Ca²⁺ solutions, as used in this study, were likely to abolish currents activated as a result of calcium entry. Thus, Ca²⁺-activated channel currents could be present and be involved in the production of the slow waves and modulation waves.

Exogenous neural control and initiation of each spike potential at an initiation zone in the syncytium have been suggested for Ascaris (DeBell et al. 1963); the spikes were suggested to start at the syncytium and then to propagate actively or electrotonically via the arm to the rest of the muscle cell. The observations reported here show that the bag region (soma) possesses currents that could initiate spike potentials. It is therefore suggested that each muscle cell of Ascaris is capable of self-generated depolarizing activity and that the role of the electrical coupling at the syncytial region is to produce coordination of the depolarization of adjacent muscle cells for contraction of the body wall. The excitatory and inhibitory chemical synaptic inputs at the syncytium could speed or slow the depolarizations.

Two major types of Ca²⁺ current have been described in other cell types. There is a transient Ca²⁺ current which has been variously named low-threshold inactivating, LTI (see Kostyuk et al. 1988), transient (Bossu et al. 1985), low-voltage activated, LVA (Carbone and Lux, 1984), T (Fox et al. 1987) and type 1 (Narahashi et al. 1987). This current is transient and inactivates with a time constant in the region of 20 ms, may be activated at relatively negative potentials (−40 to −20mV) but also requires relatively negative potentials (more negative than −40 mV) to remove inactivation. The second type of Ca²⁺ current seen in many cells decays slowly with a time constant of at least 1 s. It could be activated from less negative holding potentials than the transient current and has been referred to as high-threshold inactivating, HTI (Kostyuk et al. 1988), high-voltage activated HVA (Carbone and Lux, 1984) and L (Fox et al. 1987).

The Ca²⁺ current of Ascaris seen in our experiments had some but not all of the properties of both of these two major types of Ca²⁺ current: it inactivated but required a relatively depolarized potential for activation and was not inactivated by a holding potential of −35 mV. The current allows the production of a spike in Ascaris muscle cells with a resting membrane potential around −35 mV.

Little pharmacology has been done on the Ca²⁺ spikes of Ascaris but the report of Wann (1987) shows that the vertebrate Ca²⁺ antagonists verapamil and cinnarazine have little effect in Ascaris-, the channel may therefore have a distinctive pharmacology.

It has already been pointed out in this paper that two major types of K⁺ cunent have been recognised in a wide variety of cells and that these types are referred to as I_a and R. The I_a is a transient current which significantly inactivates with time constants of 10-50 ms at holding potentials more depolarized than −40 mV (Connor and Stevens, 1971; Kostyuk et al. 1975; Thompson, 1977) and is usually blocked specifically by 4-AP (Connor and Stevens, 1971; Cook, 1988; but see Yeh et al. 1976; Ginsborg et al. 1991). The Ik current is a maintained current which is not significantly inactivated at holding potentials more depolarized than −40 mV (Connor and Stevens, 1971; Kostyuk et al. 1975; Thompson, 1977).

In this paper the K⁺ cunent of Ascaris was separated into two components on the grounds that maintained depolarization and 4-AP selectively blocked a lower-threshold (V_h=+14mV) transient component (inactivation rate about 10 ms) but left a maintained current (inactivation rate constant about 1 s) which required greater depolarization (V_h =+29 mV) to produce activation. The 4-AP-sensitive current of Ascaris is therefore I_a-like and the 4-AP-resistant current is I_k-like.

The function of I_a currents as suggested by Connor and Stevens (1971) for neurones of Anisodoris is to serve as a damper on the interspike interval to space successive action potentials. This is also possible in Ascaris because inactivation of the I_a-like cunent appears to be complete at − 20 mV (Fig. 5B). Activation occurs at potentials more positive than − 30 mV (Fig. 7B) so that, at the steady state, only a small fraction of the channels may conduct in the narrow range of − 30mV to − 20mV. At the typical membrane potential of − 35 mV, most of the I_a-like current would be inactivated. At the end of a Ca²⁺ spike the subsequent hyperpolarization removes inactivation of the I_a-like channel as well as closing the Ik-like channel and allowing the membrane to depolarize slowly again; the I_a-1 ike cunent at this stage activates and slows the rate of depolarization until it becomes inactivated; the cell can then again reach threshold. Rates of recovery from inactivation are significant and have to be sufficiently rapid if the I_a-like current is going to behave in this manner. In this study most of the recovery of the transient current took place in less than 1 s but full recovery did not occur until after 12 s. Comparable recovery rates have been seen in Archidoris and Anisodoris neurones (Aldrich et al. 1979) and in the neural somata of Helix pomatia (Kostyuk et al. 1975; Heyer and Lux, 1976). This slow recovery from inactivation may permit the K⁺ currents to contribute to the production of the ‘slow waves’ and ‘modulation waves’ of Weisblat et al. (1976). This effect and the effect of possible Ca²⁺-activated channel currents are subjects for further investigation.

We are pleased to acknowledge the financial support of the SERC.