CALCIUM ACTION POTENTIALS IN THE SKELETAL MUSCLE FIBRES OF THE STICK INSECT CARAUSIUS MOROSUS

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1 7. exp. BM. (1981), 93, With 8 figures Printed m Great Britain CALCIUM ACTION POTENTIALS IN THE SKELETAL MUSCLE FIBRES OF THE STICK INSECT CARAUSIUS MOROSUS BY FRANCES M. ASHCROFT* Department of Zoology, Cambridge University, England (Received 28 November 1980) SUMMARY The ionic requirements for the generation of action potentials in the ventral longitudinal muscle fibres of the stick insect, Carausius morosus, were investigated. Ca-free Ringer rapidly and reversibly abolished the action potential. In the presence of tetraethylammonium (TEA) ions (to suppress outward currents) the overshoot of the action potential changed 26 mv for a 10-fold change in [Ca] 0. The maximum rate of rise of the action potential (measured in TEA Ringer) showed saturation at high [Ca] 0. Cobaltous ions (20 HIM) and the organic Ca antagonist D 600 (5 x io" 4 g/ml) reversibly inhibited the action potential; the inhibitory effect of 1 mm-la s+ was irreversible. Barium and strontium, but not magnesium, were able to substitute for calcium as charge carriers. These results suggest that an inward movement of Ca 2+ underlies the action potential of Carausius fibres. INTRODUCTION There is good evidence that in a number of insect muscle fibres calcium ions are the principal charge carriers of the inward current underlying electrical excitability. Calcium-dependent action potentials have been recorded from the skeletal muscle fibres of adult Drosophila (Ikeda, Ozawa & Hagiwara, 1976) and Sarcophaga (Patlak, 1976) and from the larval muscle fibres of the moth Ephestia kiihniella (Deitmer & Rathmayer, 1976). In the presence of tetraethylammonium ions action potentials can also be evoked in locust (Washio, 1972) and in beetle (Fukada, Furuyama & Kawa, 1977) skeletal muscle; the overshoot of these action potentials varies with the extracellular calcium concentration as predicted for a purely Ca-selective membrane. Calcium channels are characteristically impermeable to magnesium ions (Hagiwara. 1973) and it is therefore surprising that magnesium has been implicated as a major charge carrier in the muscle fibres of Tenebrio larvae (Kusano & Grundfest, 1967) and of the stick insect (Wood, 1957). In the present study the ionic basis of excitability of the ventral longitudinal muscle fibres of the stick insect, Carausius morosus, was re-examined. The results strongly support the hypothesis that Ca 2 *, but not Rig 2 " 4 ", carries the inward current during the muscle action potential of this insect also. Present address: Department of Physiology, The University, Leicester LEi 7RH, England.

2 258 F. M. ASHCROFT (a) Preparation METHODS Experiments were carried out on dissected ventral longitudinal muscles from the third thoracic segment of the stick insect, Carausius morosus. This muscle consists of about 50 fibres, averaging i-6 mm in length and between 60 and 100 /im in diameter, which insert directly into the cuticle anteriorly and are attached to the posterior cuticle by a common apodeme. The muscle was suspended in the experimental bath, at rest length, by clamping the anterior cuticle with fine forceps and ligaturing the apodeme to a dissecting pin. The bath had a volume of 0-3 ml and was perfused at a constant rate of 2 ml/min, rapid solution changes being produced using a gravity feed system incorporating a multiway non-return valve (Holder & Satelle, 1972). (b) Solutions The normal Ringer had the following composition (ITIM/1); NaCl, 20; KC1, 20; CaCl 2, 20; MgCl 2) 50; HEPES, 5 (ph 7-4); glucose, 10; sucrose, 500; and was continuously aerated with 100% O 2. The purpose of the sucrose was to increase the osmolarity (x 2) and so block contraction (Hodgkin & Horowicz, 1957). In some experiments tetraethylammonium (TEA) ions were used to block outward potassium currents (Stanfield, 1970); 20 mm TEA-Ringer was made by replacing NaCl with TEAC1, and 120 mm TEA-Ringer by replacing NaCl and sucrose with TEAC1. The divalent cation concentration was held constant in all solutions (except low Mg- Ringer), different Ca concentrations being obtained by replacing Ca 2 * with Mg**, and barium and strontium Ringer by substituting Ba*+ or Sr 5^ for Ca^". Co* 4 " was exchanged for Mg 2+. Sodium-free and 5 mm Mg-Ringer were made by replacing NaCl or MgCl 2 with sucrose. Tetrodotoxin (TTX) in citrate buffer was prepared as a 2 x io" 3 M stock in distilled water and diluted to 2 x io~ 6 M with Ringer. D 600 was made up as a stock solution in ethanol and diluted to give a final ethanol concentration of less than 0-2% in Ringer. (Control solutions containing similar concentrations of ethanol or citrate alone were without significant effect.) Potassium solutions for resting potential measurements were made by substitution of KC1 for sucrose or by reciprocal dilution of KC1 for NaCl, keeping KC1 + NaCl constant. These solutions were not oxygenated and the preparations were not superperfused. The preparation was allowed to equilibrate for about 20 min in 20 mm Ca-Ringer (or 20 mm Ca-, TEA-Ringer) before experiments were started. Experiments were carried out at room temperature (18-23 C)- (c) Recording techniques Two microelectrodes, filled with 4 M potassium acetate, were inserted into the middle of the fibre about 100 fim apart. One of these electrodes (resistance MD) was used to record membrane potential and the other (resistance 6-10 MD.) was used to pass current. Current was injected through a 100 MQ resistor and measured with a standard current-to-voltage transducer. The maximum rate of rise of the actioj potential (V max ) was recorded using a differentiating circuit, or measured dire

3 Ca action potentials in insect muscle !_ Fig. 1. Dependence of resting potential on [K] o (KC1 was exchanged for NaCl). The line is drawn to a slope of 58 mv/10-fold change in [K] o and the arrow indicates the potassium concentration at o mv, where [K], should equal [K] o.vertical bars indicate standard errors, where these are larger than the circles representing the experimental points. from records taken on a fast time base, and was used as a measure of the inward current (Hodgkin & Katz, 1949). It is implicit in the use of F max as a measure of the inward current that outward currents are negligible during the maximum rate of rise of the action potential and F max was therefore only recorded in TEA-Ringer where this criterion is valid. RESULTS Electrical properties in normal Ringer (a) Resting potential In normal Ringer the resting potential had a mean value of 54*9 ± 0-8 mv (n = 23). Fig. 1 shows the relationship between resting potential and [K] o when KC1 was exchanged for NaCl. The straight line is drawn to a slope of 58 mv for a 10-fold change in [K]o and extrapolation to o mv suggests that [K]i is around 255 mm in normal Ringer. At potassium concentrations lower then 40 mm the membrane becomes less sensitive to changes in [K] o and the resting potential deviates from a 58 mv slope. A similar insensitivity to [K] o at potassium concentrations less than 50 mm has been reported for moth muscle (Huddart, 1966; Rheuben, 1972). Exchanging KC1 sucrose, instead of NaCl, had no significant effect on the relationship between ^, and resting potential, and the predicted [K]i was the same. There was little

4 26o F. M. ASHCROFT 1 I 40 mv 20 mv 10" 7 A -200 ms (a) 10" 7 AJ _" 100m$ Fig. 2. Effect of Ca-free Ringer on the action potential, (a) Normal Ringer; (i) io min after exposure to Ca-free Ringer. The upper, middle and lower traces represent the zero potential, the membrane potential and the injected current respectively. Records taken from different fibres. (b) difference between resting potentials recorded in unperfused preparations and those recorded in the oxygenated, perfused preparations used in the following experiments. (b) Electrical excitability In normal Ringer about 30 % of the fibres responded to stimulation with a graded regenerative response which was characterized by a variation of amplitude with stimulus intensity. All-or-none action potentials, distinguished by a constant amplitude independent of stimulus intensity, were elicited in the remaining fibres and only these fibres were used in the experiments described in this paper. The overshoot of the action potential had a mean value of ±1-2 mv (n = 7) in normal Ringer. Effect of calcium concentration. I Calcium-free Ringer caused membrane depolarization (average reduction in resting potential, 19 mv) and a fall in membrane input resistance, and completely abolished the action potential within 10 min (Fig. 2). These effects were reversible on return to normal Ringer. Increasing the stimulus intensity to compensate for the fall in input resistance did not elicit excitable activity in Ca^-free Ringer. When [Ca] 0 was reduced, the number of fibres producing graded responses increased from around 30% in 20 mm Ca-Ringer to almost all fibres in 5 mm Ca- Ringer. This increased frequency of graded responses suggests that outward currents also affect the peak of the action potential and that when Ca-permeability is low, as at low [Ca] 0, spike amplitude is influenced more strongly by outward currents. The relationship between [Ca] 0 and action potential overshoot was therefore studied in the presence of 20 mm tetraethylammonium (TEA) ions, to suppress outward currents (Stanfield, 1970). Effect of calcium concentration. II All fibres produced all-or-none action potentials in 20 mm TEA-Ringer, and boik the amplitude and duration of the action potential were increased, indicating t

5 Ca action potentials in insect muscle 261 (a) 7 (b) V m (mv) mv 20 10" 7 A,100 ms 50 Ca 1 * 10 1 I 5 10 [Ca] 0 (mm) Fig. 3 (a). Action potentials recorded from the same fibre after 10 min in 5 mm Ca-Ringer (above) and after 10 min in 50 mm Ca-Ringer (below) containing 20 mm TEA. In each case the upper, middle and lower traces represent the zero potential, the membrane potential and the injected current respectively, (b) Relationship between [Ca] o and action potential overshoot for 8 fibres (4 muscles). The slope of the line is 26 mv/10-fold change in [Ca] 0. Vertical bars represent the standard error. (b) (a) i [Ca] 0 (mu) l/[ca] 0 (mm-') Fig. 4. (a) Effect of [Ca] 0 on the maximum rate of rise of the action potential (n = 7). Vm* shows saturation at high [Ca] 0. Vertical bars give standard errors, (b) Relationship between i/[ca] 0 and 1/Km.,. The arrow indicates the dissociation constant of the Ca binding site which was 121 mm. 0-2

6 262 F. M. ASHCROFT outward currents were significantly reduced by TEA. In 20 mm Ca-, TEA-Ringefl the action potential had a peak value of ± 2-3 (n = 15) and a maximum rate of rise of io-o ± 0-3 V/sec. Fig. 3 a shows the action potential of a typical muscle fibre recorded at different Ca concentrations in the presence of 20 mm TEA. The overshoot and maximum rate of rise of the action potential decreased with a decrease in [Ca] 0 and no action potentials could be elicited in Ca-free Ringer. The relationship between [Ca] 0 and action potential overshoot, for 8 fibres (4 muscles), had a mean slope of 26 mv for a 10-fold change in [Ca] 0 (Fig. 36), close to the 29 mv slope predicted for a calcium electrode. Individual Nernst slopes ranged between 22 and 29 mv. These results indicate that the action potential of Carausius muscle results from a transient increase in membrane permeability to Ca 2 " 1 ". Fig. \a is a plot of the maximum rate of rise of the action potential against [Ca] 0 and demonstrates that the Ca permeability shows saturation at high [Ca] 0 similar to that described for the maximum rate of rise of the Ca action potential of barnacle muscle (Hagiwara & Takahashi, 1967) and for the Ca current of snail neurones (Akaike, Lee & Brown, 1978). This suggests that Ca 2 " 1 " binds to a saturable site either at the membrane surface (Hagiwara & Takahashi, 1967) or within the channel itself (Akaike et al. 1978) during its passage through the membrane. A plot of the reciprocal of f^max against the reciprocal of [Ca] 0 (Fig. 46), can be fitted by a straight line, indicating that the Ca permeability obeys Michaelis-Menten kinetics, and the intercept with the abscissa gives the dissociation constant of the Ca-binding site, 12-1 mm. Low [Ca] 0 decreased both the resting potential and input resistance of the fibre, but had little effect on the threshold for initiation of action potentials. Effects of inhibitors Tetrodotoxin (TTX) blocks the Na channel of a variety of tissues at very low concentrations ( nm, Narahashi et al. 1964), but had no effect on the action potential of Carausius muscle even at a concentration as high as 2 x io~ 5 M in normal Ringer. Lanthanum and cobaltous ions block the Ca action potential of barnacle muscle fibres (Hagiwara & Takahashi, 1967; Hagiwara, 1973) and of smooth muscle (Tomita, 1975), the Ca current of snail neurones (Standen, 1974; Akaike et al. 1978) and the TTX-insensitive component of Ca entry in squid axons (Baker, Meves & Ridgway, 1973). The effect of these inhibitors was therefore tested. The action potential of Carausius muscle was irreversibly inhibited within 2 min after exposure to 1 mm-lacl 3 in normal Ringer. The effect of cobaltous ions is shown in Fig. 5 (three other preparations gave similar results): 20 mm-co** caused a reversible reduction in action potential overshoot of about 6 mv and an increase in the threshold and duration of the action potential. On return to normal Ringer there was a consistent increase in action potential overshoot of about 8 mv relative to its amplitude before exposure to Co-Ringer, similar to that found for the Ca action potential of Ephestia larvae (Deitmer, 1977). As the membrane is permeable to both Ca 2 " 1 " and K + at the peak of the spike this effect could be caused by an increase in Ca permeability, a decrease in K permeability, or a reduction in the rate or extent of inactivfl tion of the Ca permeability.

7 Ca action potentials in insect muscle m 10" 7 A 100 ms (a) (b) 20 mm-co J * (c) Fig. 5. Effect of ao mm cobaltous iona on the action potential, (a) Normal Ringer; (6) 20 mm Co-Ringer after 10 min; (c) 10 min after return to normal Ringer. The upper, middle and lower traces give the zero potential, the membrane potential and the injected current. Records were taken from the same fibre. \ 40mV 5X 100 ms ( fl ) (ft)d600 (c) Fig. 6. Effect of D 600. (a) Normal Ringer; (b) normal Ringer +5 x io~* g/ml D 600 after 10 min; (c) 10 min after return to normal Ringer. The upper, middle and lower traces give the zero potential, the membrane potential and the injected current. Records were taken from the same fibre. The organic Ca antagonist, D 600, at a concentration of 5 x io~* g/ml in normal Ringer reversibly blocked the action potential (Fig. 6). On return to control Ringer action potentials could again be elicited, but they were of greater amplitude and longer duration than those recorded initially (Fig. 6). These effects were still apparent 1 h after return to control Ringer, and as in the case of the effect of Co 2 " 1 ", may be attributed to a reduction in K permeability, an increase in Ca permeability or a decrease in Ca inactivation. Permeability to other ions Sodium-free Ringer had no effect on either the resting potential or the overshoot of the action potential (average reduction 1 1 mv, n = 3). Ca channels are characteristically permeable to Sr 24 " and Ba*+ but impermeable to Mg*+ (Hagiwara, 1973, 1975). Since Ba*+ and to a lesser extent Sr 2 " 1 " block potassium currents (Hagiwara, Fukada & Eaton, 1974; Hagiwara et al. 1978; Standen & Stanfield, 1978; Eaton & Brodwick, 1980) the ability of these ions to carry current was tested in the presence of 120 mm TEA, which blocks about 85% of the outward j^tassium current in Carausius muscle (Ashcroft & Stanfield, unpublished). The ^brshoot and maximum rate of rise of the action potential were not significantly affected by increasing TEA from 20 to 120 mm. In 20 mm Ca-Ringer the action

8 264 F. M. ASHCROFT 40 mv 2X1O' 7 A - _ d ±d (a) 20 nm-tca],, (fr) 20 mm-[sr] 0 ( c ) 20 nw- [Ba] 0 Fig. 7. Action potentials recorded from different fibres in (a) 20 mm Ca-Ringer, (b) 20 nun Sr-Ringer and (c) 20 mm-ba-ringer in the presence of 120 mm TEA. Records were taken about 20 min after exposure to the experimental solution. The traces from top to bottom give the rero potential, the membrane potential, the differentiated membrane potential and the injected current. Time calibration bar represents 100 ms in (a), 1 s in (6) and 200 mi in (c). potential overshoot had a mean value of ± 2-5 (n = 14) compared with a mean value of ± 17 (n = 10) in 20 mm Sr-Ringer and of ± 3-4 mv (n = 9) in 20 mm-ba-ringer (Fig. 7). Corresponding values for F max were IO-I io-6 V/sec (n = 14) in Ca, F/sec (n = 8) in Sr and F/sec (n = 8) in Ba. These values are not significantly different, suggesting that Ba 2 " 1 ", Sr 2 " 1 " and Ca 2 " 1 " are about equally permeable through the Ca channel of Carausius muscle. Action potentials recorded in Ba- and Sr-Ringer were significantly longer than those in Ca- Ringer ( s (n = 9) in Ba, a (n = 11) in Ca), probably due to the reduced rate and extent of inactivation of the inward current found in Ba 2 "*" and Sr*+ solutions (Ashcroft & Stanfield, 1980). The Ca channel appears to be impermeable to Mg^ as no action potentials were elicited in Ca-free Ringer containing 50 mm Mg 2 *. A 10-fold reduction in [Mg] 0 from 50 to 5 mm (in the presence of 20 mm Ca 8 " 1 " and 120 mm TEA) was without significant effect on either V max or the overshoot of the action potential indicating that Mg 24 " does not significantly block the Ca channel either. DISCUSSION The experiments described here constitute good evidence that the action potential of Carausius muscle fibres results from a transient increase in membrane permeability to calcium ions: (a) Ca-free Ringer rapidly and reversibly abolished the action potential. (b) A 26 mv change in action potential overshoot for a 10-fold change in [Ca] 0 was found in TEA-Ringer. This is close to the 29 mv slope predicted for a calcium electrode. (c) Lanthanum ions, cobaltous ions and D 600, which are known to block Ca action potentials in other preparations (Hagiwara, 1973; Reuter, 1973), inhibited the action potential. (d) Strontium and barium, but not magnesium, could substitute for calcium as charge carriers. A similar selectivity is found for Ca channels in other preparatia>na (Hagiwara et al. 1974; Deitmer & Rathmayer, 1976).

9 Ca action potentials in insect muscle i SO 100 Fig. 8. Relationship between action potential overshoot and [Ca] o predicted by constant field theory using a permeability ratio PC«^K of 10:0015. The filled circles give the experimental points. The main conclusion of this paper, that the inward current in Carausius muscle is primarily carried by Ca 2 *, conflicts with an earlier report (Wood, 1957). One explanation for this discrepancy may be that in the previous paper action potentiate were neurally evoked, making it difficult to distinguish between effects of ionic substitution on neuromuscular transmission and on the muscle membrane itself. In common with Ca action potentials in other preparations the overshoot in Carausius muscle does not reach the calculated calcium equilibrium potential. Ashley & Ridgway (1970) found that the resting intracellular Ca concentration in barnacle muscle was around io~ 7 M and if [Ca]! is assumed to be io~ 7 M in Carausius muscle, in 20 mm [Ca] 0 the calculated equilibrium potential would be mv. The membrane is therefore not completely Ca-selective at the peak of the action potential. Constantfield theory (Goldman, 1943) has been used by Fatt & Ginsberg (1958) to calculate the relative permeabilities to Ca 24 " and K+ at the peak of the Ca action potential of the barnacle muscle fibre. At the peak of the action potential the relative permeability to Ca*+ and K + is given by: ([K] t exp (VF/RT)-[K] 0 ) (exp (VF/RT)+i) 4 ([Ca]! exp (2 VF/RT)- [Ca] 0 ) (0 where V is the membrane potential at the peak of the spike and R, T and F have their usual thermodynamic meanings. Taking [Ca] 0 and [K] o as 20 mm, [K]! as 255 mm (Fig. 1) and [Ca]i as io~ 7 M, equation (1) gives a permeability ratio Pa'-P^ = i-o: 0015 in 20 mm TEA-Ringer. Fig. 8 shows the relationship between action potential overshoot and [Ca] 0 predicted by constant-field theory using this permeability ratio. The slope of line was 31-5 mv for a 10-fold change in [Ca] 0 ; a 26 mv slope/io-fold Bange in [Ca] 0 was found experimentally. In normal Ringer (TEA-free) where the

10 266 F. M. ASHCROFT K permeability is larger the predicted permeability ratio POL'-PK was i'o:o-o66 arni the slope 34mV/io-fold change in [Ca]o. Constant-field theory can therefore be used to predict action potential overshoots similar to those observed experimentally by assuming a small permeability to K + at the peak of the spike. In these calculations I have assumed that [Ca] t did not change when external calcium was altered, as the fibres were only exposed to the test solutions for about 15 min. However, if changes in fca] 0 also affected [Ca],, and therefore E&, the slope of the relationship between [Ca] 0 and the peak of the action potential would become less steep. This may account for the deviation from the predicted slope in Fig. 8. The maximum rate of rise of the action potential in TEA-Ringer showed saturating Michaelis-Menten type kinetics with increasing [Ca] 0, similar to that described for the Ca spike of barnacle muscle (Hagiwara & Takahashi, 1967) and for the Ca current of snail neurones (Akaike et al. 1978), which suggests that permeating Ca 2 " 1 " bind to a saturable site either within the channel or at its entrance. Akaike et al. (1978) found that in snail neurones the Ca-binding site was voltage-dependent, the dissociation constant becoming larger with increasing membrane potential, implying that the site must lie within the membrane voltage field. Voltage clamp experiments are required to determine if K& is also voltage-dependent in Carausius muscle. This work was carried out during the course of an S.R.C. studentship. I should like to thank Dr J. E. Treherne for his constant encouragement and invaluable advice. REFERENCES AKAIKB, A., LEE, K. S. & BROWN, A. M. (1978). The calcium current of Helix neuron. J. gen. Physiol. 71. S 9-53i- ASHCROFT, F. M. & STANFIELD, P. R. (1980). Inactivation of Ca currents in skeletal muscle fibres of an insect depends on Ca entry. J. Phytiol. 308, 36P. ASHLEY, C. C. & RIDGWAY, E. G. (1970). On the relationship between calcium transient and tension in single barnacle muscle fibres. J. Phytiol. 009, BAKER, P. F., MEVES, H. & RJDGWAY, E. B. (1973). Effects of manganese and other agents on the Cauptake that follows depolarization in squid axons. J. Pkytiol. 331, DETTMER, J. W. (1977). Effects of cobalt and manganese on the calcium action potentials in larval insect muscle fibres (Ephestia kohniella). Comp. Biochem. Phytiol. 58, 1-5. DEITMER, J. W. & RATHMAYER, W. (1976). Calcium action potentials in larval muscle fibres of the moth Ephettia kuhniella Z. (Lepidoptera). J. comp. Phytiol. na, EATON, D. C. & BRODWICK, M. S. (1980). Effects of barium on the potassium conductance of squid axon. J. gen. Physiol. 75, FATT, P, & GINSBORG, B. L. (1958). The ionic requirements for the production of action potentials in crustacean muscle fibres. J. Phytiol. 14a, FUKADA, J., FURUYAMA, S. & KAWA, K. (1977). Calcium dependent action potentials in skeletal muscle fibres of a beetle larva. Xylotrupet dictotomut. J. Intect Phytiol. 33, GOLDMAN, D. E. (1943). Potential, impedance and rectification in membranes. J. gen. Phytiol. 37, 37-6o. HACrwARA, S. (1973). Calcium spike. Adv. Biopkyt. 4, HAGIWARA, S. (1975). Ca-dependent action potentials. In Membranes, a Series of Advances, vol. m (ed. G. Eisenmann), pp New York: Marcel Dekker. HACIWARA, S. & TAKAHASHI, K. (1967). Surface density of calcium ions and calcium spikes in the barnacle muscle fibre membrane. J. gen. Physiol. 50, HAGIWARA, S., FUKADA, S. & EATON, D. C. (1974). Membrane currents carried by Ca, Sr, and Ba barnacle muscle fibre during voltage clamp. J. gen. Physiol. 63, I

11 Ca action potentials in insect muscle 267.OIWARA, S.,MIVAZAKI, S., MOODY, W. & PATLAK, J. (1978). Blocking effects of barium and hydrogen ions on the potassium current during anomalous rectification in the starfish egg. J. Phytiol. 279, HODGKIN, A. L. & HOROWICZ, P. (1957). The differential action of hypertonic solutions on the twitch and action potential of a muscle fibre. J. Phytiol. 136, 17-18P. HODGKIN, A. L. & KATZ, B. (1949). The effect of sodium ions on the electrical activity of the giant axon of the squid. J. Phytiol. 108, HOLDER, R. E. D. & SATELLE, D. B. (1972). A multi-way non-return valve for use in physiological experiments. J. Phytiol. 226, 2-3P. HUDDART, H. (1966). The effect of potassium ions on resting and action potentials in Lepidopteran muscle. Comp. Biochem. Phytiol. 18, IKEDA, K., OZAWA, S. & HAGIWARA, S. (1976). Synaptic transmission reversibly conditioned by single gene mutation in Drosophila melanogatter. Nature, Lond. 359, KUSANO, K. & GRUNDFEST, H. (1967). Ionic requirements of synaptic electrogenesis in neuromutcular transmission of mealworm larvae Tenebrio molitor. J. gen. Phytiol. 50, NARAHASHI, T., MOORE, J. W. & SCOTT, W. (1064). Tetrodotoxin block of sodium conductance increase in lobster giant axons. J. gen. Phytiol. 47, 965. PATLAK, J. B. (1976). The ionic basis of the action potential in the flight muscle of thefly.sarcophaga bullata.j. comp. Phytiol. 107, REUTER, H. (1973). Divalent cations as charge carriers in excitable membranes. Prog. Biophyt. Molec. Biol. 26, RHEUBEN, M. B. (1972). The resting potential of moth muscle fibre. J. Phytiol. 325, STANDEN, N. B. (1974). The effect of calcium on the electrical properties of an identified snail neurone. Ph.D. Thesis, University of Cambridge. STANFIELD, P. R. (1970). The effect of tetraethylammonium ion on the delayed current of frog skeletal muscle. J. Phytiol. 209, STANDEN, N. B. & STANFIELD, P. R. (1978). A potential and time-dependent blockade of inward rectification in frog skeletal muscle by barium and strontium ions. J. Phytiol. 280, TOMITA, T. (1975). Electrophysiology of mammalian smooth muscle. Prog. Biophyt. Molec. Biol. 30, WASHIO, H. (1972). The ionic requirements of the initiation of action potentials in insect muscle fibres. J. gen. Phytiol. 59, WOOD, D. W. (1957). Effect of ions upon neuromuscular transmission in phytophagous insects. J. Phytiol. 138,

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