/. exp. Biol. 155, 305-321 (1991) 3 0 5Printed in Great Britain © The Company of Biologists Limited 1991

POTASSIUM AND CALCIUM CURRENTS IN DISSOCIATEDMUSCLE FIBRES OF THE MOLLUSC PHILINE APERTA

BY D. A. DORSETT AND C. G. EVANS

Animal Physiology Group, School of Biological Sciences, University CollegeNorth Wales, Bangor, Gwynedd LL57 2UW, UK

Accepted 1 August 1990

Summary

Dissociated unstriated muscle fibres from the buccal mass retractor muscles ofthe mollusc Philine aperta were studied using a two-electrode voltage-clamp. Themean resting potential of the fibres was —76.3±0.44mV (N=30), and themembrane resistance was 42.2±3MQ. The space constant of the fibres was2.03+0.33mm (N=5).

Three outward potassium currents were resolved in response to a depolarisingstep to zero from resting potential. (1) An early transient current, voltage-activated and blocked by 2 mmol I"1 4-aminopyridine (4-AP). This resembled theA-current described in molluscan neurones and some arthropod muscle fibres.(2) A calcium-dependent late transient current, with slower kinetics, which wassuppressed by 50 mmol 1~* tetraethylammonium chloride (TEA-C1), zero-calciumsaline, 1 mmol \~l Cd2+ and 1 /imol I"1 verapamil. (3) A delayed voltage-activatedcurrent, blocked by 50 mmol I"1 TEA-C1 and with kinetics associated with thedelayed rectifier current IK.

An inwardly directed current, blocked by zero-calcium salines, Cd2+ andverapamil, was considered to be a calcium current whose activation closelymatched that of the Ca2+-dependent potassium current.

A blockade of either the A-current, or exposure to low-calcium artificial seawater, or a combination of both, promoted the development of oscillations andregenerative spikes in the muscle fibre following depolarization.

Introduction

Studies on the membrane properties of molluscan fibres have generally beenrestricted by their small size and inaccessibility, and our knowledge of theirpermeability is derived largely from investigations of the ionic mechanismsdetermining the resting and action potentials (Wilkens, 1972; Brezden andGardner, 1984; Dorsett and Evans, 1989), although recently patch-clamp studieson isolated heart muscle fibres of Lymnaea have identified a potassium-selectivechannel sensitive to membrane stretch (Brezden etal. 1986; Sigurdson etal. 1987).

Key words: mollusc, muscle, potassium currents, calcium current, spiking, Philine aperta.

306 D . A . DORSETT AND C. G. EVANS

In other invertebrate preparations, these difficulties have been overcome by theuse of single fibres obtained by enzymic dissociation of the connective tissuematrix of the muscle (Ishii and Takahashi, 1982; Hernandez Nicaise et al. 1980;Anderson, 1984). In this report the technique was used to isolate fibres from thebuccal mass retractor muscles 4 and 5 of the opisthobranch mollusc Philine aperta(Evans and Dorsett, 1989). These are paired, anatomically distinct muscles withunstriated non-spiking fibres having a simple transverse tubule system (Dorsettand Roberts, 1980), which respond to motoneurone stimulation with summatingexcitatory junction potentials (Dorsett et al. 1989; Evans and Dorsett, 1989).

The present study was undertaken to characterise the ionic currents associatedwith depolarization of a non-spiking muscle fibre that initiates the development oftension in the muscle.

Materials and methods

To isolate single muscle fibres, muscles were dissected free of their attachmentsto the buccal mass and body wall and rinsed in an artificial sea water (ASW)containing OmmolP1 Ca2+ to reduce the possibility of contraction during enzymicdigestion of the sheath. The ASW contained (in mmolP1); Na+, 463; K+, 10;Ca2+, 9; Mg2+, 53; SO4"2, 17; Tris, 5; adjusted to pH8 with HC1. Measurementsof the equilibrium potential involving elevated levels of K+ were made in salineswhere the [K+][C1~] product was kept constant (Dorsett and Evans, 1989).

The muscles were incubated in a 2mgml~1 solution of Type XIV protease(Sigma) in zero-Ca2+ ASW until the outer connective tissue sheath was disrupted.At this point the muscles were carefully transferred to the experimental chambercontaining sea water, and a number of single fibres were dissected free usingelectrolytically sharpened tungsten needles. Fibres separated in this way normallysettled on the bottom of the chamber, retaining their characteristic shape anddimensions (see Fig. 1).

The experimental chambers were lined with sea water agar or with Sylgard(Dow Corning), the hydrophilic nature of the former offering an advantage inallowing the volume of fluid in the chamber to be reduced, thus minimisingcapacitance artefacts associated with the command pulse. The fibres were voltage-clamped with a Dagan 8500 two-electrode amplifier. Both current and voltageelectrodes had resistances in the range of 6-10 MQ and were shielded to within ashort distance of the tip with silver conducting paint. The driven shield of thevoltage electrode was insulated and that of the current electrode grounded. Theindifferent electrode consisted of an agar bridge to a KCl/AgCl junctionconnected to the virtual ground current monitor of the amplifier. Rectangularcommand pulses 70ms in duration, derived from a pulse generator (Farnell),resulted in membrane potential changes that were completed in 600 /JS. No attemptwas made to compensate for the series resistance as the cells were isolated andsurrounded by a medium of high ionic strength. Membrane voltage and current

Ionic currents of molluscan muscle fibres 307

Fig. 1. (A) A fibre within the retractor muscle injected with carboxyfluorescein. Notethat the fibre does not extend over the entire length of the muscle, and has filamentousprocesses at the ends. (B) A dissociated fibre preserves its shape and has similarprocesses at each end. Scale bar, 100 fim.

traces were displayed and photographed on a Tetronix 5103N storage oscilloscope,digitised on a GTCO Digipad and stored for subsequent manipulation on aDEC VAX 8650. Leakage currents were measured for a series of hyperpolarisingpulses and a straight line projection was used to estimate leakage currents in thedepolarizing range. Temperatures ranged between 16 and 18°C.

308 D. A. DORSETT AND C. G. EVANS

ResultsProperties of the muscle fibres

The morphology of single fibres in the living muscle was studied by injectingthem with 5-carboxyfluorescein (Rao et al. 1986). (Fig. 1A). They have acylindrical form with tapering ends, and are between 1 and 2 mm long and about20 pan in diameter. One end of each fibre is often forked, and the tapered end iscommonly serrated, as if to provide a firm insertion into the connective tissuesheath. Dissociated fibres extracted from the muscle retained a similar mor-phology when relaxed (Fig. IB), but became sigmoid or almost spherical whenfully contracted, with the contractile apparatus consolidated as a central core.

In the present series of experiments 30 muscle fibres sampled in haemolymphwere found to have a mean membrane potential of — 74.9±1.35mV ( ± S . E . ) . Themean membrane potential of a further sample of 30 dissociated fibres in sea waterwas — 76.3±0.44mV and the mean membrane resistance was 42.2±3mQ. It wasconcluded that the fibres were not unduly stressed by the isolation procedures.

When subjected to small depolarisations through the voltage-recording elec-trode, single fibres were observed to twist as they contracted and relaxedsmoothly, the degree of contraction depending on the membrane potential. Thisobservation is of particular interest as it establishes that contraction is initiated byand depends on depolarization, and supports a previous observation of smalltwitch-like contractions of the whole muscle in response to single excitatoryjunction potentials (EJPs) (Dorsett et al. 1989).

Estimations of the space constant (A) of the fibres were made following themethod outlined by Hernandez-Nicaise etal. (1980) and Dubas etal. (1988), usingdata obtained from the spatial decay of injected current at two voltage-recordingelectrodes inserted simultaneously at different points along the fibre. Oneelectrode was inserted close to the current electrode (E) while the other wasplaced closed to the end of the fibre. The distances between E and the ends of thefibre {L\ and L2) and the recording electrodes {dx and d2) were measured andsubstituted in the equation for a short cable (Hodgkin and Nakajima, 1972):

V1/V2=cosh[(L2-d1)/k]/cmh[L2-d2)/X] , (1)

where Vi and V2 are the potentials recorded at dx and d2. The value of A was foundto be 2.0310.33 mm (N=5) for hyperpolarising pulses. Substituting the value of Ain Weidmann's equation (1952):

VjV0=l/cosh(L/X) ,

where Vo and VL are the potentials at the current electrode and at distance L,respectively, the fibre will be isopotential (within 2 %) for 400/im on either side ofthe current electrode. With depolarizing voltages potassium currents will beactivated and the isopotential length will be reduced, but this reduction will bepartly offset by the contraction of the fibre. Although it is possible that the spaceclamp may not be complete towards the ends of the fibre, we do not consider thatthis prejudices the evidence provided for the profile of the potassium currents.

Ionic currents of molluscan muscle fibres 309

0mV<

40 n A

-75 mV 20 msI

Fig. 2. The total outward current recorded from an isolated muscle fibre held at—75 mV, when the membrane potential is stepped to OmV. Note the inflection in theleading edge of the current (arrow) leading to a transient peak which inactivates to asteady-state condition. The upper trace is the voltage record.

Total outward current (I,)

Isolated muscle fibres in sea water were subjected to a two-electrode voltage-clamp. The voltage-recording electrode was inserted into the central region of thefibres and normally recorded potentials in excess of -70 mV, close to the normalresting potential of undissociated fibres in haemolymph or sea water. The currentelectrode was then inserted and the fibres clamped at — 75 mV. The membranepotential was stepped to 0 mV by a 70 ms command pulse and the fibres respondedwith an outward current which rose to a delayed transient peak after approxi-mately 25 ms before declining to a steady-state current that persisted until the endof the command pulse (Fig. 2). The complex profile of the outward currentsuggested the presence of more than one component.

A feature of the outward current is an inflection in the rising edge some 9-10 msafter the onset of the pulse when the current has attained a value of 25-30 nA(Figs 2, 4, inset i), marking a change in the rate at which the late transient currentapproaches its maximum. Stepping the membrane potential to zero from holdingvoltages of —60 and — 80 mV did not substantially alter the profile of the totaloutward current other than to vary the value of the transient peak and the steady-state level.

Several lines of evidence support the conclusion that the outward current ismainly carried by potassium. The reversal potential of the tail currents wasmeasured in three sets of muscle fibres following exposure to constant product[K+][CP] ASW solutions containing 10, 20 or 40mmoir 1 potassium. The fibreswere stepped to 0 mV for 70 ms and returned to a series of membrane potentialsbetween -30 and -70mV to give the instantaneous current-voltage relationshipfor the steady-state current (Fig. 3). The regression of the equilibrium potentialplotted against [K+]o had a slope of 51 mV for a 10-fold change in [K+]o, whichcompares well with the figure of 51.2 mV obtained for isolated radial fibres ofBeroe ovata (Bilbaut et al. 1988). The mean equilibrium potential in 40mmoll~1

[K+]o was -46.7 mV compared with a predicted value of 45.8 mV from the Nernstequation assuming a [K+]i of 247mmoll~l (Dorsett and Evans, 1989). Finally,

310 D . A . DORSETT AND C. G . EVANS

>:n

tial

(

oo.

rsal

Rev

e

—fu-

-49-

-58-

-66-

-75-

>-=50.94JT-127.8

/• /

^=0.97

• /

/

0.9 0.99 1.09 1.18 1.27

log [K+]

1.37 1.46 1.56 1.65

Fig. 3. Plot of the reversal potential of the instantaneous current-voltage relationshipof three sets of fibres in 10 ( • ) , 20 (•) and 40 (A) mmoll"1 K+ artificial sea water inwhich the [K+][CF] product is kept constant to avoid chloride fluxes. The regressionslope is 51 mV per decade.

application of saline containing the potassium channel blockers TEA-C1 and 4-APabolished all outward currents.

The current-voltage relationship

The current-voltage relationship of 10 muscle fibres was measured in sea waterwith an extended range of command pulses (+30 to +210 mV) from a membraneholding potential of — 75 mV. At each step the current was measured after25-35 ras to coincide with the late transient peak, and again at 70 ms to record themaintained current (Fig. 4, inset i). As the membrane was stepped to positivevoltages the increase in the peak transient current became progressively smallerand eventually began to decline (Fig. 4, inset ii), whereas the steady-state currentincrease remained approximately linear until levelling out at about 90 mV.

The N-shaped current-voltage relationship shown by the transient current hasbeen considered to indicate the presence of a calcium-dependent component ofthe total potassium current (Meech and Standen, 1975). The experiment was thenrepeated, first measuring the current at the time of the late transient peak innormal, and then in Ca2+-free, ASW (Fig. 5). Removal of calcium eliminated therapid growth of the current associated with the transient peak, converting the Nshape into a shallow curve that meets the former at the lowest point of its decline.Comparable results were obtained by adding the calcium current blockersImmoll"1 Cd2+ or 1 jumoll"1 verapamil to theseawater bath. These treatments allabolished the transient current peak at 25-30 ms, giving an approximately linearrelationship over the range, and supporting the interpretation that the late

Ionic currents of molluscan muscle fibres 311

500

400

300

200

100

- 2 5 25 75 125Membrane potential (mV)

Fig. 4. A representative current-voltage relationship of an isolated fibre over anextended range of command pulses from a holding potential of -75 mV. The currentwas measured to coincide with the late transient peak after 25-30 ms (Ic) and at thesteady-state after 70 ms (IK). The N-shaped relationship indicates a Ca+-dependentcomponent which fails to activate as the membrane potential exceeds the reversalpotential for Ca2+ and calcium fails to enter the fibre. Inset i shows the growth in thelate transient peak (C) and steady-state current (K) in the physiological range as thefibre is stepped between —35 and OmV. An inflection marking the beginning of the A-current inactivation can be seen on the leading edge. In inset ii, depolarizations in thepositive range show the typical reduction in the late transient peak (C) as the calciumequilibrium potential is approached. The amplitude of the steady-state currentincreases with increasing depolarization and exceeds C.

transient peak represents a calcium-dependent component of the potassiumcurrent.

The calcium-dependent current (Ic)

The time course of the Ca2+- and Cd2+-sensitive potassium current wasexamined by comparing the total current in response to a 70 ms depolarising stepfrom a holding voltage of — 75 mV before and after the addition of lmmoll"1

Cd2+ to the sea water bathing the fibre (Fig. 6A). This treatment abolished thetransient current peak at 25-30 ms and reduced the steady-state component of thecurrent by about 30 %. The profile of the calcium-dependent potassium currentwas then obtained from the total outward current by digital subtraction of the

312 D . A . DORSETT AND C. G . EVANS

300 r

<c

200

100

-25 25 75Membrane potential (mV)

125

Fig. 5. Current-voltage plot of the late transient current 25-30ms after a series ofcommand pulses in normal (A) and zero-Ca2+ (A) sea water. Removal of Ca+

eliminates the current inflection.

B20 nA

I,Cd2+

Cd2++TEA4

7Fig. 6. (A) The outward currents before and after addition of 1 mmol 1 ' Cd2+ to theseawater medium. The late transient peak of the Ca2+-dependent current is suppressedand an earlier peak is revealed at the level of the inflection on the leading edge of theoutward current. The steady-state current is reduced but not eliminated. (B) Currentsin the same fibre with the further addition of 50mmoll~l TEA+. This eliminates thesteady-state current but does not affect the early transient current which resembles IA.

Cd2+-resistant component (see Fig. 11), after correction for an inward currentthought to be carried by calcium (see below).

Although the most prominent feature of the Ca2+-dependent current is thetransient peak, the reduction in the steady-state current following treatment withcadmium or l^imolP1 verapamil could have one of several explanations. Theremay be a residual component of the Ca2+-dependent current that inactivates veryslowly or shows a voltage-dependency or, possibly, the delayed rectifier current IK

is partially affected by cadmium. Alternatively, it may be due to an entirelyseparate current that has yet to be isolated from the other three. At present wehave not resolved this question.

Ionic currents of molluscan muscle fibres 313

12

3

- 5 0 - 4 0 - 3 0 -20Membrane potential (mV)

- 1 0

Fig. 7. The activation of the Ca2+-dependent potassium current (Ic), the delayedcurrent (IK) and the early transient current (IA)- Each point represents the averagecurrent from four different fibres as the membrane was stepped to the indicated levelsfrom a holding potential of —75 mV. IA was measured after blocking Ic with 1 mmol I"1

Cd2+

Activation of the outward current

The activation thresholds of the Ca2+-dependent transient peak and steady-state components of the outward current were determined from I/V plots of thecurrent measured at 25-30 ms and at 70 ms during a series of step depolarizationsof a fibre from a holding potential of -75 mV (Fig. 7). As the membrane wasstepped to potentials more depolarised than -45 mV a small outward current wasactivated which rose slowly to a maintained plateau. With larger depolarizationsthe current increased and developed a peak which reached its maximum amplitudebetween 25 and 30 ms, before inactivating to the steady-state level (Fig. 4, inset i).

Both the Ca2+-dependent transient and steady-state components of the outwardcurrent have activation thresholds at around — 40 mV, although the transientcurrent rapidly exceeds the maintained current with greater depolarization.

If the calcium-dependent K+ current is blocked with l m m o l P 1 Cd2+, thetransient peak at 25-30 ms is replaced by a smaller early transient outward currentwhich peaks 10-12ms after the onset of the pulse (Fig. 6B). The activationkinetics of this early current were faster than those of the late calcium-dependenttransient current and its inactivation coincided with the inflection in the leadingedge of the total current profile previously noted (Figs 2, 4, inset i).

The early transient current (IA)

The addition of 50 mmol I"1 TEA+ to the bath containing sea water andl m m o i r 1 Cd2+ blocked both the residual Ca2+-dependent and steady-statecomponents of the potassium current, leaving an early transient current which

314 D . A . DORSETT AND C. G . EVANS

0.8-

0.6-

0.4-

0.2-

-70 -60 -50 -40Membrane potential (mV)

-30 -20

Fig. 8. Steady-state inactivation of IA measured as the ratio l/lmax- The membranewas held at —75 mV before stepping to the test potential for 1 s and then stepping toOmV. The fibres were in sea water containing 1 mmolF1 Cd2+ and50mmoll~1TEA+.

peaked at 12-15 ms and declined over 40-50 ms (Figs 6B, 9A). This current couldalso be isolated by blocking the late transient and steady-state currents with TEA+

in the absence of Cd2+, in which case its magnitude was reduced and itsinactivation sometimes revealed a small inward current (see Fig. 9A). The earlytransient current was voltage dependent, resistant to SOmmoll"1 TEA+ andunaffected by either low-calcium saline or the presence of cadmium in the saline. Ithas many features associated with the potassium A-current described for mol-luscan neurones (Connor and Stevens, 1971a,b). At holding voltages of —75 mV,and in the presence of Cd2+ and TEA4", the activation threshold of the earlytransient current was — 40 mV, a value close to that of the other two potassiumcurrents (Fig. 7). The activation kinetics are faster than those of the calcium-dependent current, maximum current amplitude occurring 12-15 ms after theonsets of depolarization (Figs 6B, 9A,B). Many neuronal A-currents are known toinactivate around the resting potential (Rudy, 1988), but measurement of thesteady-state inactivation of IA in these muscle fibres indicates that, althoughinactivation begins as the membrane is held depolarised to the resting potential, itis not complete until it reaches — 30mV (Fig. 8).

Inward current (Ica)

If isolated muscle fibres are treated with both 50 mmol l~l TEA+ and 2 mmol 1~1

4-AP, the three potassium conductances are blocked, revealing a small inwardcurrent (Fig. 9A-D). The activation threshold of the inward current measuredfrom a holding potential of — 75 mV was around — 35 mV (Fig. 10), the current

Ionic currents of molluscan muscle fibres

B c

\ l . 25 nA

315

20 ms

Fig. 9. Isolation of the inward current. (A) The total outward current (It) following avoltage step to 0 mV from -75 mV. The fibre is then treated with 50 mmol I"1 TEA"1" toisolate IA- Note that IA inactivates to leave a small inward current. (B) Twosuperimposed sweeps of IA at an interval of 2 min prior to the addition of 2 mmol I"1

4-AP. 4-AP suppresses IA to reveal the inward current (Ica)- (C) Two sweeps at a 2 mininterval show that prolonged exposure to 4-AP removes the last vestige of IA,increasing the development of Ica- The inward current is then blocked by the additionof 1 mmol I"1 Cd2+ to the saline (Cd2+). Upper traces monitor the membrane voltageduring the step. (D) A fibre depolarised in the presence of 2 mmol I"1 4-AP, whichremoves IA. Note the inward current preceding the late outward transient peak (Ic)and a second dip in the trace as I c inactivates, denoting the continuing inward current.After addition of lmmolF1 Cd2+, Ic and Ica are blocked and the delayed rectifiercurrent (IK) remains.

12 nA

increasing with greater depolarization to peak between 20 and 30 ms.'Inwardcurrent amplitudes were generally in the range of 12-15 nA, but currents up to25 nA were recorded from large (2 mm) fibres.

The inward current was blocked by the addition of calcium channel blockerssuch as l m m o i r 1 Cd2+ or ljumoll"1 verapamil to the bath containing TEA"1",with no sign of any residual inward current carried by other cations (Fig. 9C).

Separation of individual currents

Profiles of the individual currents cannot be obtained directly from experimentson a single fibre, as the procedures necessary to isolate the Ca2+-dependentpotassium current also block the inward calcium current. The total outwardcurrent recorded in response to the standard depolarising command pulse to 0 mVis opposed by the inwardly directed calcium current. The three potassium currentswere derived by simple manipulation of digitized current records of the totaloutward current (It), the cadmium-resistant current (Icd)> t n e early transientcurrent (IA) and the inward current (Ica) obtained from experiments on separatefibres (Fig. 11). In this instance the figure is derived from data on five separate

316 D . A . DORSETT AND C . G . EVANS

fibres, in which the ratios of the component currents were similar, and were scaledto match on the initial total outward current profile. Subtraction of IA from ICd

leaves a TEA+-sensitive current which rises slowly to a maintained plateau anddoes not inactivate, having the characteristic profile of the well-known delayedrectifier current (IK). IK can also be demonstrated directly by depolarization of

28

24

20

1 16

12

-40 -30 -20 -10 0Membrane potential (mV)

Fig. 10. Activation of the inward calcium current. Potassium currents were blockedwith SOmmoll"1 TEA+ and 2mmoll~1 4-AP. The maximum inward current wasmeasured as the membrane was stepped through a series of depolarizations from aholding voltage of —75 mV.

145-

0 10 20 30 40 50 60 70Time (ms)

Fig. 11. Computer-generated plot of the total (It) and individual potassium currents(IA, IK, IC) ar)d the inward calcium current (Ica) derived from three individualexperiments. Note the general correspondence in the amplitude of Ic and Ica- Thedotted line is zero current.

Ionic currents of molluscan muscle fibres 317

fibres in sea water containing 2mmoll~1 4-AP, which blocks IA, and l m m o l P 1

Cd2+, which blocks I c (Fig. 9D).When I c is blocked with cadmium it has been noted that there is also some

reduction in the steady-state current at the end of the 70 ms depolarising commandpulse. This suggests that some part of I c persists until the inward calcium currenthas inactivated. This did not always occur with the pulse duration used in theseexperiments. However, the results of experiments such as those illustrated inFig. 9D indicate that, at times, IK can account for almost all the steady-statecurrent.

Physiological role of the transient outward currents

In spite of their normal non-spiking mode of operation, spike-like transients areoccasionally observed in the fibres of these and other molluscan buccal muscles(Tattershall and Brace, 1987; Dorsett and Evans, 1989). As the two transientpotassium currents IA and Ic dominate the membrane conductance in the first40 ms following a depolarization, the question of their possible role in preventingspike activity arises.

When isolated fibres in sea water are depolarised in a series of steps, themembrane voltage remains steady at each level. If the medium is exchanged forsea water containing 2mmoll~1 4-AP to block IA and the sequence repeated, asthe membrane is stepped to potentials more depolarised than — 40 mV, itcontinues to depolarise, eventually producing a series of regenerative overshoot-ing spikes which may attain amplitudes of 40-50mV (see Fig. 12C). In thismedium a spike train frequently occurs on penetration of a fibre. Individual spikesare preceded by a relatively slow depolarization and followed by a rapidrepolarization, having a half-peak amplitude duration of around 40 ms. Each spikeis accompanied by a twitch-like contraction of the fibre.

To investigate the possible role of the calcium-dependent Ic, a series ofexperiments was carried out in low-calcium ASW. In zero-calcium solutions nospiking was obtained, but in 2mmoll~1 Ca2+ ASW depolarising the membrane to-40 mV initiated slow oscillations that eventually developed 20 mV spike-liketransients (Fig. 12A). Addition of l m m o l P 1 Cd2+ to the bath caused theoscillations to become slower and rather erratic, and it became impossible toinduce spiking (not shown).

Isolated fibres were then depolarised in 2mmoll~1 Ca2+ saline containing2mmoll~1 4-AP (Fig. 12B). Under these conditions even small depolarizationsbecame difficult to control, the membrane continuing to depolarise when steppedto around -60 mV, to produce large overshooting 50 mV spikes which attained afrequency of around 4 Hz. Further investigations into the mechanisms underlyingthe transition to a spiking state will be reported elsewhere.

Discussion

Isolated fibres of a molluscan unstriated muscle have an array of ionic currents

318 D . A . DORSETT AND C. G. EVANS

2mmoir'Ca2+ ASW

Fig. 12. (A) Oscillations and spiking induced in a muscle fibre by depolarization to—40 mV in 2 mmol P 1 Ca2+ artificial sea water (ASW) which presumably reduces theCa2+-dependent potassium current. (B) Regenerative 50mV spikes induced in a fibreimmersed in 2mmoll~L Ca2+ and 2 mmol I"1 4-AP ASW which will suppress both IA

and Ic. Note that the membrane depolarizes spontaneously to spike after stepping toaround -60mV. (C) Large spikes follow depolarization of a fibre to — 40 mV in ASWcontaining 2 mmol \~l 4-AP to block IA. Note the spontaneous depolarization after themembrane was returned to resting potential. Resting potential of fibres; A, — 76 mV;B, -70mV; C, -70mV.

comparable to those found in molluscan neurones (Thompson, 1977). Depolariz-ation of the fibre activates a voltage-dependent transient outward current which issimilar in its pharmacology and kinetics to the A-current first described by Connorand Stevens (1971b), but, unlike many neuronal A-currents, its activationthreshold is shifted to levels more depolarised than the resting potential (Rudy,1988). Also, the steady-state inactivation, which in many neurones occurs close tothe resting potential, begins as the membrane potential falls below — 70 mV, but isonly complete at membrane potentials around -35 mV. Comparable departuresfrom what might be considered 'typical' A-current properties are also seen inDrosophila muscles (Salkoff and Wyman, 1983), and may be correlated with themore positive activation threshold of systems where the inward current is carriedmostly by calcium.

There seems to be no generalised role for either IA or I c in excitable cells(Rogawski, 1985; Rudy, 1988). One function of the two transient currents in themuscle fibres of Philine seems to be to prevent spikes, which are readily induced ifthe early transient A-current is blocked with 4-AP or the fibres are immersed in a2 mmol P 1 Ca2+ saline, which may reduce the late calcium-dependent current I c .

Ionic currents of molluscan muscle fibres 319

A combination of these treatments both lowers spike threshold and inducesregenerative large-amplitude spikes, which can be blocked by the addition of Cd2+

to the saline. In normal fibres, the faster activation kinetics and lower threshold ofIA, followed by the development of I c , would be expected to shunt the inwardcalcium current and prevent its full expression. Indications that the muscle spikesare mediated by calcium are supported by preliminary experiments (not reportedhere) where spiking was observed in 2 mmol I"1 Ca2+ ASW when choline chloridewas substituted for sodium.

Normally non-spiking muscle fibres of crustaceans retain their capacity todevelop calcium spikes following the application of TEA+ or when the internalcalcium activity is reduced (Fatt and Ginsborg, 1958; Hagiwara and Naka, 1964).This implies a suppression of some component of the potassium current, but thedetails are unknown. In Drosophila melanogaster, the A-current is responsible forthe rapid repolarization of the spike in flight muscle and in cervical giant axons(Salkoff and Wyman, 1983; Tanouye et al. 1981), while a similar function isperformed by a transient calcium-dependent current in short Purkinje fibres of thecalf (Siegelbaum and Tsien, 1980). In a preparation of Drosophila muscle fibres, apartial blockade of IA with dendrotoxin can lead to explosive discharges ofprolonged or giant EJPs when IK is also blocked or reduced (Wu et al. 1989).

Long-term changes in excitability of some neurones have also been associatedwith reduction in the voltage- and calcium-dependent potassium currents. Duringconditioning trials of the mollusc Hermissenda crassicornis, a persistent increase inthe excitability of the B photoreceptors results from a reduction in IA and I c

brought on by the learning paradigm (Alkon, 1984).The soma membranes of some apparently inexcitable neurones also retain the

capacity for regenerative activity and are converted to spiking by perfusion withTEA+ or by reducing the calcium gradient (Pitman, 1979; Goodman and Heitler,1979) but, again, details of the potassium currents involved in these preparationswere not studied. However, in Procambarus clarkii, conversion of a non-spikingneurone to a calcium-dependent spiking one results from the voltage-dependentinactivation of the A-current (Czternasty et al. 1989). From the functionalviewpoint, if the twitches which accompany the spikes in isolated fibres in low-Ca2+ and 4-AP saline were to occur during the normal feeding activity of Philinethey would result in tetanic contractions of the entire muscle. In molluscs, whereone motoneurone and all the fibres of a muscle may constitute a single motor unit,fine control of tension depends upon a graded depolarization of the fibre bysummation of individual junction potentials, and could not be achieved withspiking fibres. In this instance, the two transient outward currents may constitutean important control mechanism in the development of tension by regulating thedepolarization leading to calcium influx and preventing regenerative spiking.

ReferencesALKON, D. L. (1984). Changing of membrane currents during learning. /. exp. Biol. 112,

95-112.

320 D. A. DORSETT AND C. G. EVANS

ANDERSON, P. A. V. (1984). The electrophysiology of single smooth muscle cells isolated fromthe ctenophore Mnemiopsis. J. comp. Physiol. B 154, 257-268.

BILBAUT, A., MEECH, R. W. AND HERNANDEZ-NICAISE, M. (1988). Isolated giant smooth musclefibres in Beroe ovata: ionic dependence of action potentials reveals two types of muscle fibre./. exp. Biol. 135, 343-362.

BREZDEN, B. L. AND GARDNER, D. R. (1984). The ionic basis of the resting potential in a cross-striated muscle of the aquatic snail Lymnaea stagnalis. J. exp. Biol. 108, 305-314.

BREZDEN, B. L., GARDNER, D. R. AND MORRIS, C. E. (1986). A potassium selective channel inisolated Lymnaea stagnalis heart muscle cells. J. exp. Biol. 123, 175-190.

CONNOR, J. A. AND STEVENS, C. F. (1971a). Inward and delayed outward membrane currents inisolated neural somata under voltage clamp. J. Physiol., Lond. 213, 1-20.

CONNOR, J. A. AND STEVENS, C. F. (1971ft). Voltage clamp studies of a transient outward currentin gastropod neural somata. J. Physiol., Lond. 213, 21-30.

CZTERNASTY, G. C , KADO, R. T. AND BRUNER, J. (1989). Analysis of mechanisms of spiking innormally non-spiking motoneuron somata in crayfish. /. exp. Biol. 147, 91-110.

DORSETT, D. A. AND EVANS, C. G. (1989). The ionic basis of the resting potential of molluscanunstriated muscle. J. comp. Physiol. 159, 305-312.

DORSETT, D. A. AND ROBERTS, J. B. (1980). A transverse tubular system and neuromuscularjunctions in a molluscan unstriated muscle. Cell Tissue Res. 206, 251-260.

DORSETT, D. A., SKELTON, M. E. AND EVANS, C. G. (1989). The innervation and physiology ofthe extrinsic buccal retractor muscles of Philine aperta. J. Moll. Stud. 55, 193-208.

DUBAS, F., STEIN, P. G. AND ANDERSON, P. A. V. (1988). Ionic currents of smooth muscle cellsisolated from the ctenophore Mnemiopsis. Proc. R. Soc. 233, 99-121.

EVANS, C. G. AND DORSETT, D. A. (1989). Pre- and post-synaptic actions of the cerebral ventralgiant cell on buccal muscles of Philine. J. comp. Physiol. B 159, 509-515.

FATT, P. AND GINSBORG, B. (1958). The ionic requirements for the production of actionpotentials in crustacean muscle fibres. /. Physiol., Lond. 142, 516-543.

GOODMAN, C. S. AND HEITLER, W. J. (1979). Electrical properties of insect neurones withspiking and non-spiking somata: normal, axotomised and colchicine-treated neurones. J. exp.Biol. 83, 95-121.

HAGIWARA, S. ANDNAKA, K. (1964). Initiation of spike potential in barnacle muscle fibres underlow intracellular calcium. /. gen. Physiol. 48, 141-162.

HERNANDEZ-NICAISE, M. I., MACKIE, G. O. AND MEECH, R. W. (1980). Giant smooth muscles ofBeroe. J. gen. Physiol. 75, 79-105.

HODGKIN, A. L. AND NAKAJIMA, S. (1972). The effect of diameter on the electrical constants offrog skeletal muscle fibres. J. Physiol., Lond. 221, 105-120.

ISHII, N. AND TAKAHASHI, K. (1982). Length-tension relation of single smooth muscle cellsisolated from the pedal retractor muscle of Mytilus edulis. J. Muse. Res. Cell Motil. 3, 25-38.

MEECH, R. W. AND STANDEN, N. B. (1975). Potassium activation in Helix aspersa neuronesunder voltage clamp: A component mediated by calcium influx. J. Physiol., Lond. 249,211-239.

PITMAN, R. M. (1979). Intracellular citrate or externally applied tetraethylammonium ionsproduce calcium-dependent action potentials in an insect motoneurone cell body. /. Physiol.,Lond. 291, 327-333.

RAO, G., BARNES, C. A. AND MCNAUGHTON, B. L. (1986). Intracellular fluorescent staining withcarboxyfluorescein: a rapid and reliable method for quantifying dye-coupling in mammaliancentral nervous system. J. Neurosci. Meth. 16, 251-263.

ROGAWSKI, M. A. (1985). The A-current: how ubiquitous a feature of excitable cells is it? TrendsNeurosci. 83, 214-219.

RUDY, P. (1988). The diversity and ubiquity of K channels. Neuroscience 25, 729-749.SALKOFF, L. B. AND WYMAN, R. J. (1983). Ion currents in Drosophila flight muscles. J. Physiol.,

Lond. 337, 687-709.SIEGELBAUM, S. A. AND TSIEN, R. W. (1980). Calcium-activated transient outward current in calf

cardiac Purkinje fibres. /. Physiol., Lond. 299, 485-506.SIGURDSON, W. J., MORRIS, C. E., BREZDEN, B. L. AND GARDNER, D. R. (1987). Stretch

activation of a K+ channel in molluscan heart cells. J. exp. Biol. 127, 191-210.

Ionic currents of molluscan muscle fibres 3il\

TANOUYE, M. A., FERRUS, A. AND FUJITA, S. C. (1981). Abnormal action potentials associatedwith shaker complex locus of Drosophila. Proc. natn. Acad. Sci. U.S.A. 78, 6548-6552.

TATTERSHALL, J. E. H. AND BRACE, R. C. (1987). Physiology and motor innervation of thesupralateral retractor muscles of the snail Planorbarius corneus. J. comp. Physiol. 160A,115-125.

THOMPSON, S. (1977). Three pharmacologically distanct potassium channels in molluscanneurones. /. Physiol., Lond. 265, 465-488.

WEIDMANN, S. (1952). The electrical constants of Purkinje fibres. /. Physiol., Lond. 118,348-360.

WILKENS, L. A. (1972). Electrophysiological studies on the heart of the bivalve molluscModiolus demissus. The ionic basis of the membrane potential. J. exp. Biol. 56, 273-292.

Wu, C.-F., TSAI, M.-C, CHEN, M.-L., ZHONG, Y., SINGH, S. AND LEE, C. Y. (1989). Actions ofdendrotoxin on K+ channels and neuromuscular transmission in Drosophila melanogaster,and its effects in synergy with K+ channel-specific drugs and mutations. J. exp. Biol. 147,21-41.