Archidoris monteryensis and Anisodoris nobilis. Under voltage clamp, inward current

J. Phy8iol. (1979), 286, pp. 41-60 41 With 10 text-ftigure Printed in Great Britain CALCIUM CURRENT IN MOLLUSCAN NEURONES: MEASUREMENT UNDER CONDITIONS WHICH MAXIMIZE ITS VISIBILITY BY JOHN A. CONNOR From the University of Washington, Friday Harbor Laboratories, Friday Harbor, Washington University of Illinois, Department of Physiology and Biophysics, 524 Burill Hall, Urbana, Illinois, U.S.A. (Received 24 October 1977) SUMMARY 1. Membrane currents were studied in isolated somata of molluscan neurones from Archidoris monteryensis and Anisodoris nobilis. Under voltage clamp, inward current displayed a two phase time course, and in some cases a clear reversal potential difference could be shown for the fast and slow phases. The slower phase was carried predominantly by calcium ions. 2. The apparent magnitude of the slower phase was greatly influenced by conditions which altered potassium current flow. Blocking voltage-dependent potassium conductances, either by appropriate conditioning polarizations or by tetraethylammonium (TEA) ion, enhanced the magnitude, while conditions which augmented potassium current made the slow phase disappear. 3. A fraction of the membrane potassium conductance was TEA insensitive. This fraction could be blocked by procedures which prevented internal levels of calcium from increasing during the voltage clamp pulse. Three such procedures were demonstrated; replacement of external calcium by magnesium, internal buffering by EGTA, and replacement of calcium by permeant barium. 4. Internal EGTA buffering or external barium in combination with external TEA produced an extreme change in membrane current as compared with the normal time course. Membrane current, when activated by pulses up to + 50 mv, was net inward and showed only fractional inactivation over time courses running to several seconds. Pulses to voltages greater than + 60 mv resulted in outward current. 5. It is concluded that under normal conditions the calcium conductance has the extended time course clearly evident under the modified conditions of paragraph 4 but that the calcium flux component is easily missed. 6. In agreement with several prior studies it is also concluded that a rise in internal calcium is causally related to a rise in potassium conductance. A transmembrane flux of calcium can be uncoupled from the ge increase by appropriate buffering of internal calcium. 7. The transient potassium current, IA, which bears a resemblance to calciumdependent potassium transients in some muscle cells did not depend upon internal calcium but instead is a voltage-activated mechanism.

42 J. A. CONNOR INTRODUCTION The existence of a transmembrane calcium current during excitation is by now well recognized in a number of nerve and muscle preparations (see reviews by Hagiwara, 1973 and Reuter, 1973). The central neurones of the various gastropod mollusks have been among the more extensively studied of these preparations. Apart from a clear consensus that calcium ions constitute a sizeable fraction of the total inward current, there is a certain amount of disagreement as to the time course of the calcium flux and of the relation of the calcium flux to other components of membrane conductance. For example, several studies have supported the idea that calcium flux under voltage clamp conditions is primarily a rapid transient of similar time course to the sodium transient (cf. Kostyuk, Krishtal & Doroshenko, 1974; Standen, 1975). Other studies have indicated a slower time course (Geduldig & Gruener, 1970; Adams & Gage, 1976; Heyer & Lux, 1976a). Work by Eckert & Lux (1976) on a bursting neurone of Helix has indicated that at small depolarizations the calcium flux does not completely inactivate during a voltage step lasting several seconds. In a previous report (Connor, 1977a) this author has presented evidence to show that there is an inward flux of calcium or barium ions which shows negligible inactivation over a time course of 100 msec even at positive internal voltages. A flux of this nature would be similar to the calcium flux in squid axon and barnacle muscle (cf. Meves & Vogel, 1973; Keynes, Rojas, Taylor & Vergara, 1973; Hagiwara, Fukuda & Eaton, 1974). Whether or not the neurone exhibits bursting as a normal physiological function is immaterial. Meech (1972) showed that micro-injection of calcium ions into the somata of snail neurones was followed by a period of several seconds in which potassium conductance was elevated. Krnjevic & Lisiewicz (1972) performed similar experiments on spinal motorneurones. Transmembrane calcium flux, activated upon depolarization, is also followed by a rise in potassium conductance (see Meech & Standen, 1975; Heyer & Lux, 1976b; Barrett & Barrett, 1976; Thompson, 1977). The relationship between internal calcium and increased potassium conductance is presumably causal because operations which reduce or eliminate inward calcium flow, i.e. application of calcium blockers such as cobalt or manganese ion, calcium free media, or large depolarizations, reduce the potassium flux. Only a fraction of the total potassium current is calcium dependent, however, the remaining being activated by transmembrane voltage changes (cf. Meech & Standen, 1975b; Heyer & Lux, 1976 b; Thompson, 1977). However, Heyer & Lux (1976b) have interpreted their data as indicating that the calcium dependent fraction of potassium conductance is coupled to transmembrane calcium flux and not to internal calcium concentration. This report presents voltage clamp studies on molluscan neurones which were undertaken in an attempt to describe the time course of one phase of calcium entry and the relation of this calcium entry to the generation of a potassium conductance increase. A preliminary report has been given (Connor, 1977b).

MOLLUSCAN NEURONE Ca CURRENT 43 METHODS Studies were performed on specimens of Archidori8 monteryen8i8 and Ansidori8 nobilis obtained both from waters around Friday Harbor, Washington and from the southern Californian coast (R. Fay, Pacific Biomarine Supply, Venice, California). Experiments were conducted at the University of Washington Friday Harbor Laboratories using the local animals and at the University of Illinois using California and Washington specimens maintained in artificial seawater (Instant Ocean Systems, Wycliff, Ohio). There were not major differences in the properties reported herein between the two populations except that the time course of the various conductance changes at a given temperature appeared to be somewhat more rapid in the northern animals than in the southern animals, possibly reflecting an acclimation to different ambient temperature. A systematic study of this has not been made. Left plural Cerebral Right pleural ganglion \ A,' ganglion - L~~~l2~ ~ / ALPO2-RPI 2 L~e1--- ~" LPI 3 / --RPe 1 Left pedakilp Right pedal ganglion ganglion LPe 2 R Circumesophageal commisure Fig. 1. Diagram of Archidris brain from dorsal aspect showing usual locations of identified neurones. For illustration purposes the ganglia have been given a slight lateral spread. The numerous connexions arising from the ganglia are not shown. Connective tissue was removed from the brain with and without the aid of enzymic softening. Both protease at 2 mg/ml. and trypsin (Sigma Chemical Co. St Louis, Mo.) at 4 mg/ml. were used on different occasions. Trypsin was used in about 90 % of the experiments and was rinsed away as best possible before the sheath was dissected away. Possible artifacts arising from enzyme treatment are recognized and a number of dissections were done without the aid of enzyme treatment. The most satisfactory medium found for this dissection was a calcium-free saline made approximately 1-5 normal osmolarity by the addition of sucrose. The brain was allowed to equilibrate approximately 20 min in the dissection medium before the initial rend in the connective tissue sheath was made. With this pretreatment the cells did not 'balloon' out through the initial cut as they do when dissection is attempted in normal saline. Presumably the calcium deficit prevents contraction of muscular elements in the sheath and the hypertonicity reduces the volume of the packed neurones. There were no detectable changes due to enzyme treatment in the membrane parameters measured in this study. In five control experiments, trypsin treated, desheathed brains were maintained for 3 days in a nutrient media (10% fetal calf serum and 5% minimum Eagle's media added to artificial sea-water). Parameters such as action potential overshoot and resting potential changed by less than 20 % over that period. Enzyme treatment was preferred because it facilitated sheath removal with minimum disruption of cells and cell ordering. Most experiments were performed on three clearly identifiable cell bodies (diameters 300-600 #sm) from the left pleural ganglion of Archisori8, labelled LP1 1, 2,

44 J. A. CONNOR and 3 in Fig. 1. These neurones were generally quiescent. These cell bodies are located in the extreme upper left of the ganglion and are readily cut off from the brain. A good removal leaves only 200-300 #m of axon attached to the cell body, apparently sealed at the end. This truncation procedure greatly reduces electrical artifacts arising from the axon. Records showing the effects of truncation on current records have been published (Connor, 1977 a). Fifteen other identifiable neurones in Archidori8 and Anisodoris brains have also been studied in the course of these experiments. Standard micro-electrode voltage clamp procedures were used (cf. Connor & Stevens, 1971 a). Resistance of micro-electrodes was typically 2 MQ. Membrane voltage was measured differentially with one micro-electrode inside the cell and one outside. Internal voltage homogeneity was tested in several experiments by inserting two potential measuring micro-electrodes into the cell. Currents recorded externally from the soma surface (see Kado, 1973; Connor, 1977a) were also compared with total clamp currents. Both of these tests indicated good spatial control of membrane voltage. Experimental temperatures were controlled by a Peltier effect cooler and ranged from 5 to 12 0C, but for a given experiment the temperature was controlled to within 0 5 'C. Composition of saline solutions is given in Table 1. Records were photographed from the oscilloscope face (Tektronix 5103) using a Grass C4 camera. Film images were reversed and printed for Text figures except, as noted, where tracings were made. TABLE 1. Composition of saline solutions (mm) Normal low-ca saline Ca-TEA TEA Ba-TEA NaCl 490 390 390 390 KCl 8 8 8 8 CaCl2 15 15 0 0 MgCl2 18 50 65 50 MgSO4 32 BaCl2-15 TEA-Cl 100 100 100 Tris or MOPS 5 5 5 5 ph 7.4 Preparation of EGTA solutions: A stock solution was prepared by bringing 9.5 g EGTA (Sigma, St Louis, Mo.) into solution by adding concentrated KOH. Final ph was set at 7-4 and water was added to give an EGTA concentration of 250 mm. A ph buffer (MOPS, Sima) was added by combining the EGTA stock and MOPS stock (1 m at ph 7.4) to give molar ratios (MOPS: EGTA) of 1 and greater. Injections of buffered and unbuffered EGTA were made. RESULTS The basic current pattern of interest is shown in Fig. 2. For small positive steps the membrane current displayed the usual wave form of well clamped excitable membrane, an inward transient followed by a maintained outward current (Hodgkin, Huxley & Katz, 1952; Dodge & Frankenhaeuser, 1959). For larger steps the inward transient became larger and faster but in addition a slower inward phase became appreciable. With increasing step size the magnitude of the slower phase increased while the faster transient decreased. At the voltage where the faster transient reversed direction from inward to outward, the slow phase was still inward-flowing. For this illustration a cell was chosen with a rather low reversal potential for the fast transient. Generally the fast transient reversed around + 40 to + 50 mv. Outward current increased steadily with step size. Note that the step duration has been decreased in successive steps to keep outward current within bounds at the current

MOLLUSCAN NEURONE Ca CURRENT 45 sensitivity used. Outward currents developed by molluscan neurones are very large compared to inward currents (Connor & Stevens, 1971 a; Meech & Standen, 1975). This two phase pattern of the inward current is very similar to records obtained from a very diverse group of preparations: eggs of tunicate (Harumasa, Takahashi & Yoshii, 1976); starfish (Hagiwara, Ozawa & Sand, 1975); cardiac muscle (Rougier et al. 1969; Beeler & Reuter, 1970). It has been concluded that calcium ions carry much of the slower inward current in these other preparations and evidence has -24 +20-18 + 26 Xf -- -- -13 / +31 ~ _lo%-- - -. _7 +60 _"-_! oft +10 A/ -,- AV =-50 Fig. 2. Voltage clamp series illustrating the activation time course of two-phase inward current. Test voltages appear at the right of corresponding currents. Zero current is indicated by dotted line. Outward current runs off scale for the traces at 20, 31, and 60 mv. Scales: -24,-18,-13 mv; 100 msec.-7 mv; 40 msec. + 10, + 20 mv; 20 msec. 26, 31, 60 mv; 10 msec. Current scale 200 na for all panels except + 60 where scale is 400 na. Cell: Arch. LPl 1. T = 8 0C.

46 J. A. CONNOR been presented that this is also the case in molluscan somata (Geduldig & Greuner, 1970; Eckert & Lux, 1976; Heyer & Lux, 1976a). The results of the present study are in general agreement with this conclusion. The fast transient is carried by both sodium and calcium in varying proportions in different molluscan neurones (Connor & Stevens, 1971 a; Standen, 1975; Chamberlain & Kerkut, 1969; Krishtal & Magura, 1970) but was not studied in detail in the present series of experiments. It should be noted however that the fast transient was insensitive to tetrodotoxin (10-5 M) in enzyme treated and untreated preparations. This contrasts to the A +5 B +10 1sec 200 na 40 mv 1 0 msec 200 na +20 ------ ~--- -- ---- -- --- --1-- -- -- -- ------- -- - ---e Fig. 3. A, effect of conditioning voltage on inward current time course. Paired test pulses at + 5, + 10, + 20 mv from conditioning levels of -35 mv (left column) and -50 mv (right column) were applied. B, low sweep speed records showing membrane current during two conditioning pulses and the fully developed outward current at the test level. Initial voltage, -50 mv. Cell: Arch, LPe 1, dissected without enzyme treatment. T = 12 'C. observation of Lee, Akaiki & Brown (1977) who found that tetrodotoxin sensitivity was present in certain neurones of Helix and was abolished by trypsin treatment. The conditioning voltage, the level at which membrane voltage was held between test pulses, of Fig. 2 was -40 mv. Resting potential for Dorid neurones usually lies between -40 and -60 mv. For conditioning voltages between -30 and -50 mv the net slow inward current upon depolarization underwent marked changes as shown in Fig. 3A. In the left hand column of the Figure the conditioning voltage was -35 mv and the slow inward current was quite prominent. For a conditioning voltage of -50 mv (Fig. 3A, right column) there was little evidence for it at all. The interpretation of this effect, much of it based upon data to follow, is that more outward-

MOLLUSCAN NEURONE Ca CURRENT flowing current is activated from the more negative conditioning voltage and masks the inward current in the total records. The magnitude of outward-flowing current is a function of Vcofld, decreasing as Vcond becomes more positive (Connor & Stevens, 1971 a). In addition to delayed potassium current, the transient outward current (IA) also activates upon depolarization when 'Kond is more negative than -40 mv(see Connor & Stevens, 1971b; Neher, 1971; Gola & Romey, 1971). Activation of this component can produce a large decrease in the total inward current both for the rapid transient and slower phase (see discussion in Neher, 1971). In Fig. 3A, current flow from both of these components probably contributes to the masking of inward current. To illustrate this point in more detail, the records of Fig. 3B show the effects of conditioning voltage on a much slower time scale. All three runs were started from the same voltage, -50 mv. In the second and third run a conditioning voltage was interposed before the test step. These conditioning steps, as amplitude increased, progressively decreased the magnitude of the outward current during the test pulse. With prepulse 47 200 na No prepulse ' l0msec Fig. 4. Current wave form during a step to + 20 my with and without an inactivating prepulse (+ 35 my). Same neurone as Fig. 3. In the third run there was a sizeable outward transient during the conditioning step. In the second run there was no appreciable current flow during the conditioning step but the outward current still underwent inactivation during the step, as evidenced by the magnitude of current during the test step. The situation is much the same as with axonal sodium conductance upon a voltage step from -100 mv to -60 mv; the conductance inactivates by approximately one half but there is little sodium current flow. In most neurones tested the slow inward current could be almost completely masked by making Kond more negative than -50 mv. There was some species difference noted, with Anisodoris neurones showing more outward current relative to inward than Archidoris neurones. In many of the Anisodoris neurones there was little or no direct evidence of a slow inward current phase even for conditioning voltage of -40 mv (cf. Fig. 3, Connor & Stevens, 1971 a). More detailed examination, however, readily showed that the slow flux was present. The rapid and slow inward current components could be separated by one of two routine conditioning techniques; either making the conditioning voltage more

48 J. A. CONNOR positive than - 25 mv, or preceding the test step with a short positive pulse. The second procedure is illustrated in Fig. 4. The long test pulse was, in one sweep, preceded by a short pulse (a 4 msec) during which the fast transient activated and partially inactivated. In the second, superimposed, sweep there was no short pulse. In the record shown the pause between pulses was too short for the fast transient to reactivate appreciably, and following the make of the longer pulse, inward current activated slowly. Where the pause was more extended, the fast transient underwent partial recovery and the time course of inward current showed two phases. For tests where the short pulse was too brief to inactivate the fast transient, the two phases remained during the test pulse, with the fast one reduced in amplitude. In Fig. 4 the total inward current during the test pulse is less where a prepulse was applied than where there was no prepulse. Two processes, occurring individually or in combination, could give rise to this difference: (1) either the slow inward current undergoes a partial inactivation during the short pulse and does not recover during the pause or (2) potassium conductance is activated during the prepulse and does not inactivate completely during the pause. These possibilities are explored below. Since the time course of the inward current can be masked by simultaneous outward current, either delayed rectifier or transient potassium fluxes, it seemed advantageous to attempt procedures which block the potassium currents. Blockage of the potassium transient, IA, is easily done by employing conditioning voltages more positive than -40 mv. For the delayed potassium conductance tetraethylammonium (TEA) ion was used. In squid axon this agent acts only from the internal surface of the membrane (Armstrong & Binstock, 1965) while in vertebrate node it acts from either surface (Hille, 1967; Armstrong & Hille, 1972; Koppenhofer, 1967). In molluscan neurones it also is effective when applied externally or internally but the effective concentration and the action of the drug are different. Internally applied, a block of both I' and IA is achieved at concentrations in the range of 10 mm (Neher & Lux, 1972; Walter & Connor, 1975) while from the outside, concentrations of 50-100 mm must be used in marine saline (Hagiwara & Saito, 1959). Even these large amounts of external TEA reduce IA only by a factor of 2 or so (see Connor & Stevens, 1971 b). Internal TEA strongly affects the kinetics of IA at low concentrations; external TEA only reduces amplitude leaving the kinetics unaffected (Walter & Connor, 1975). This finding has been used to demonstrate that very little, if any, TEA crosses the membrane and enters these particular neurones over the time course in which its action occurs. Voltage clamp records before and after the external application of 100 mm-tea are shown in Fig. 5. Records were taken starting 5 min after TEA exposure. The reduction in outward current is dramatic, as is the corresponding increase in the magnitude and duration of net inward current. Outward current in control saline was in general not allowed to activate fully at large depolarizations in order to avoid dealing with the problems introduced by large currents, series resistance and external ion accumulation most notably. Also, the recovery times necessary to restore initial conditions following long positive pulses were often a minute or more, probably reflecting the time course of recovery from large calcium influx. Large, fully activating steps were thus avoided in the interest of compressing the time span of an

MOLLUSCAN NEURONE Ca CURRENT 49 experiment. At low depolarizations in the TEA saline, net current is inward for the duration of the pulses shown. Peak magnitudes ofthe fast inward current and the current measured at 80 msec are plotted against membrane voltage in part B of the Figure. The magnitude of the fast transient was reduced in TEA saline over the lower part of the voltage range. This observation is consistent with the decreased sodium Control +15 TEA A -5 i >1J00 na 50 msec - J o100na 5 msec +30 +27 +20 +15 m-e -5, I~~~+5 At 100 na 50 msec J100nA 20 msec +32 +25 +20 li +52 / /+40 /,_ 1J0o0nA 5 msec +60 +56 +50 /7 ~~44 I+ +38 J100nA 5 msec 800r Im(lnA) 600 - A /(t =80msec) A Control V TEA B 400 1-200 -10 w A V v Leakage current I~~ I I A I I I i I ] I -400 _- v10v 2i0 30 40 50 ' 60 0 0 g 0 dog V,,(fmV) /(Peak) * Control O TEA Fig. 5. A, the effects of TEA (100 mm) on membrane current. Test voltages paired to approximately the same value. Note change in sweep speeds necessary to keep outward current within bounds. Data traced from film. B, plots of membrane current vs. voltage in control and TEA saline. Cell: Arch. LP1 1. T = 9 0C.

50 J. A. CONNOR concentration; TEA-Cl was substituted for sodium (see Table 1). For large positive voltages the difference was negligible, possibly reflecting a compensating reduction in the potassium current. As can be seen from the data tracings it also became difficult to discriminate between fast and slow currents at large voltages. Exposure times to TEA were generally limited to less than 30 mi. Although TEA treatment reduced net outward current it did not eliminate it in any of the neurones studied. The TEA insensitive outward current and the slow A B C Vcond =-30 Vcond =-30 Vcond =-40-6 -7-5 I 100 na 1 00 msec +10 +10 +10 - --- A -----t +20 +20 +17 -A~~f------ -----, { r~---- +28 +28 +28 Fig. 6. The effects of removing external calcium on membrane current in the presence of TEA. A: 15 mm-calcium. B and C: calcium replaced by magnesium. Cell: Arch. RPe 1. T = 9 C. inward current were both dependent upon the presence of calcium in the external saline. This is illustrated in Fig. 6 where normal calcium-tea records are compared with records taken in low calcium-tea saline. Calcium was replaced by magnesium (see Table 1). The primary effects noted were a reduction in amplitude of the outward current and a virtual elimination of the slow inward current. As the calcium was being flushed from the bath, cells tested in this manner fired spontaneously for 3-5 min and then settled to a resting potential which generally lay between zero and -20 mv as magnesium exchange for calcium became complete. The concomitant reduction of both inward and outward current by calcium free saline is in agreement with findings reported by Meech & Standen (1975) and Heyer & Lux (1976a) under

MOLLUSCAN NEURONE Ca CURRENT 51 different experimental conditions. Cobalt ion applied externally or internally reduced both currents as did manganous ion applied externally. Increasing the external concentration of calcium also increased the magnitude of the slow inward current. A systematic study whereby changes in inward current v8. calcium concentrations are determined has not been done at this time, because the TEA-insensitive potassium current activates over a similar time course and obscures true changes in magnitude. The magnitude of the fast transient was changed and its reversal potential lowered somewhat in the calcium-free saline. Two sets of calcium-free records are shown (Fig. 6B, C) to illustrate the effects of conditioning voltage on the fast transient. A decrease in the reversal potential would be expected if the fast transient were partially carried by calcium ions. Changes in peak current (or conductance) are more difficult to interpret because of the well known ability of external calcium to control the voltage dependence of membrane parameters in excitable cells and hence the magnitude of currents carried by other ions (Frankenhaeuser & Hodgkin, 1957; Hille, 1968; Schauf, 1975). The data presented thus far show that there is a dependence of two oppositelydirected membrane currents upon external calcium concentration and indicate that the currents are coupled in some manner. Change in internal calcium concentration brought about by calcium influx has been suggested by Meech (1972) as a likely mechanism of coupling; however, this mechanism has been questioned recently by Heyer & Lux (1976b) who have suggested that the transmembrane flux of calcium is the activator of the potassium conductance. A series of experiments was conducted in which the calcium chelating agent EGTA was injected into 30 neurones in an attempt to hold the internal level of calcium ions at a low value in spite of transmembrane influx. This group included samples of all the identified neurones. The EGTA was generally injected under pressure from a micro-electrode filled with 250 mm EGTA at ph 7*4. Results from one neurone are shown in Fig. 7. which compare current records in calcium-tea saline before and after injection of EGTA. The primary effect of the treatment was to convert the inward-outward current pattern into one which was entirely inward. In the last panel of Fig. 7A the sweep speed was reduced tenfold to illustrate that the inward current was maintained for extended periods. Where the tip of the injecting electrode was large, about 2,sm, giving a large efflux of EGTA, the effect on membrane current was noticeable within a few seconds. Smaller tips required longer injection periods and for intermediate injection amounts (between no effect and maximum) the outward current showed steady decrease with increased dose. The quantity of EGTA injected under pressure could not be measured in the experimental set up, but estimates of effective EGTA concentration were made from electrophoretic injections. Assuming a charge carried by EGTA of + 2 and a transport number of 0 5 (the latter value probably high), suppression of the outward current as shown in Fig. 7A was achieved at EGTA concentrations of around 1-2 mm. In the neurones analysed the fast inward transient was reduced by as much as 40-60 % following EGTA injection. However, the initial magnitude and activation time course of the slow inward current were little affected by the EGTA. The differences in the records of Fig. 7 were then not brought about by a large increase

~~~~~~500 52 J. A. CONNOR in the slow inward current but seem best interpreted as showing that the EGTA treatment greatly reduced outward-flowing membrane current leaving the inward current to predominate in the total records. Within the framework of this interpretation, the calcium inward current inactivates very slowly, if indeed at all, during a voltage clamp pulse, making its behaviour in this respect identical to the calcium current in barnacle muscle (Keynes et al. 1973; Hagiwara et al. 1974) and squid axon (Meves & Vogel, 1973). A B Control Internal EGTA -6-8 1250 1000 / +6 +5 < 750 +24 +21 Leakage current (~ io0 +10 ±20 +30 +40 +60 / ~~~~~~~~400nA VM(MV +40 / +40-250 200msec Fig. 7. A, membrane currents recorded in calcium-tea saline before and after the injection of EGTA. Conditioning voltage was -30 mv in both cases. Sweep speed has been reduced in the last panel. B, I-V relations from the cell of part A. Total membrane current at the termination of 650 msec pulses before (0) and after (A) EGTA injection are given as well as the minimum current (ignoring the fast transient) during the pulse (FL) Cell: LPI 1. T = 9 OC. Various levels of ph buffering were used for pressure injection of EGTA. Where no ph buffer was injected with the EGTA, the calcium influx during a voltage clamp pulse resulted in a sizeable decrease in internal ph as measured by indicator dye absorbance changes (Z. Ahmed & J. Connor, in preparation). An injection mixture of at least 10: 1 molar ratio of MOPS buffer to EGTA was necessary to stabilize the internal ph during and after a voltage clamp pulse. Membrane current was unchanged by ph buffering; i.e. the effect of EGTA injection on the outward current is probably not mediated by ph. Fig. 7B shows plots of the peak slow current (LI) and the current measured at 650 msec before (0) and after (A) EGTA injection. For the relatively long pulses from which the data were taken the inward current generally showed significant decay, relaxing from inward to outward at approximately 50 mv. Where the test voltage was greater than + 50 mv, current was outward for the duration of the pulse. This behaviour was observed in all the EGTA injected neurones, with the range for current reversal between + 40 and + 60 mv. For small voltage steps (V < +IO mv) there was little difference in the

MOLLUSCAN NEURONE Ca CURRENT 53 membrane current records before and after EGTA injection. In this voltage range total current remained inward for the duration of the pulses shown and indeed for several seconds. Fig. 8A shows small step records in more detail. External saline was calcium-tea and there was no EGTA injection. For the smallest step net current remained inward for the duration of the step. Upon repolarization, the current fell quickly to zero. An inward deactivation tail of rapid time course is obscured by the A B -15-10 -8 0V a_ - _dm foi_ I_ +5 +10 U- ft- 200 msec loona 0 LLI200 na 40 msec Fig. 8. A, time course of membrane currents for small voltage steps in TEA saline. The decay of slow inward current to zero is accompanied by an outward tail of increasing size. Fast inward transient is off scale in the two lower panels. B, superimposed records of current before and after injection of EGTA in normal saline. Injection reduced the magnitude of outward current at the larger voltage steps. The preinjection records were taken after insertion of the EGTA electrode but before pressure was applied. Conditioning potential, -30 mv. Records were photographically superimposed. Cell: Arch, identity not noted. T = 6 0C. slow sweep (see Connor, 1977a). For steps of large amplitude, the total current rose toward zero, but upon repolarization there was significant outward current which decayed with a slow time course. These data can be interpreted within the framework outlined in connexion with Figs. 6 and 7, if it is supposed that the inward current activated by the small step did not give rise to a potassium conductance increase but that the two larger steps did. There are at least two mechanisms which make this a reasonable possibility. First, the steady inward current could be carried predominantly by sodium ions for small steps. Smith, Barker & Gainer (1975) have

~~~~~~~~~~~I 54 J. A. CONNOR proposed that the fast inward conductance does not inactivate completely at small depolarizations in some burster neurones, thereby giving rise to a steady sodium ion current. Secondly, if the steady current is carried partially or entirely by calcium ions it is possible that for small influx the internal uptake mechanisms can buffer the internal concentration effectively enough to prevent a conductance increase. Regardless of the underlying mechanism, the observation that small inward currents are not followed by appreciable potassium conductance activation provides a useful control experiment for establishing the actions of TEA and EGTA. Fig. 8B A 8 C D -12-6 -6 +6 +6 +6 v Ba-TEA Saline 0-10 10 20 30 40 50 60 70 80 v -0.5 v Vm(mV) +13 +14 +14 V - SAC ------a ---a ~~~-1.0 0-1.5 V A - f - ~~~~ ~~ - ~~ I ~(pa) 0.5 o Ca-TEA Saline +18 +20 +25 0 v0 g 1 pa -2.0 v0 V 0 +36 +36 - +30 200msec -2.5 V V 0 +42 +43 *JA 40msec Fig. 9. The effects of replacing external calcium by barium on membrane current. A, calcium-tea saline. B and C, barium-tea saline. More complete washout going from B to C. D, plots of peak slow inward current in calcium and barium for the runs shown in A and B. Cell: Arch. RPl G. T = 8 "C. shows superimposed records made before and after EGTA injection without TEA in the external saline. The records are consistent with what would be expected from the foregoing analysis. For the clamp to -10 mv, there is little difference in the records taken before and after injection. Since TEA was absent, a sizeable outward current developed. This was presumably due to a purely voltage-dependent conductance increase. With TEA present (Fig. 8A) there was little if any outward current measurable at a comparable voltage. For larger voltage steps the currents of Fig. 8B clearly diverge over the latter portion of the time course but are quite similar during the initial phase. With TEA present, as in Fig. 8A, the records diverged in the same way except that there was no voltage activated potassium conductance. These observations are inconsistent with the idea that TEA or EGTA, either singly or acting in conjunction, has a major effect on the time course or magnitude of the slow inward current. The records of Figs. 7 and 8 illustrate that calcium influx can be decoupled from

MOLLUSCAN NEURONE Ca CURRENT 55 potassium efflux, the presumed decoupling mechanism providing adequate buffering of internal free calcium. Fig. 9 illustrates a second condition under which the two fluxes are decoupled; where barium ion substitutes for calcium as the inward charge carrier. Equimolar substitution of barium for all the calcium in the bathing saline was made. The series of records in the left hand column (9A) was taken in calcium- TEA saline while those of the right hand columns (9B, C) were taken in barium- TEA saline. The records of Fig. 9B and 9C were made following the exchange of approximately 8 and 12 chamber volumes of barium-tea saline. With barium-tea saline as the external medium the current records were very similar to those taken under combined EGTA injection and calcium-tea superfusion. In both cases the outward current fails to develop and the inward current inactivated only fractionally. As with EGTA injection the effect was prominent within seconds following the introduction of the barium-tea saline and reached a steady condition as the exchange of barium and calcium became complete. Exposures of more than 20 min to the barium-tea saline were avoided because the magnitude of the inward current decreased irreversibly. Peak inward current is plotted as a function of test voltage in Fig. 9D. For voltages below + 30 mv the maximum inward current in barium was greater than in calcium saline in all of the cells tested but the reversal voltage for total current was smaller in barium than in calcium. As the replacement of calcium by barium progressed, action potentials increased from the control values of several hundred milliseconds to periods of well over 30 sec. The lengthening reflected the decrease in outward current under voltage clamp. None of the effects of barium was as prominent in the absence of TEA in the external saline because the voltageactivated potassium conductance contributed a large outward current even in the presence of barium. With regard to calcium-linked potassium current, barium over the short term is not so much a blocker but an agent which fails to activate the conductance mechanism as well as calcium. Experiments were run in which current from the same cell was measured in the following sequence of superfusing solutions: (1) [Ca] = 15 mm, [Ba] = 0mM; (2) [Ca] l 0, [Ba] = 15 mm; 3) [Ca] 5 mm, [Ba] 10 - mm.teawas present throughout. For solution (1) there was the usual outward current; in solution (2) there was little if any as in Fig. 9B, C. For solution (3), where there should be a mixed influx of calcium and barium during a voltage step, outward current activated to an intermediate level. The maximum value attained by the slow inward current was not greatly altered in any of the mixtures. This type of result implies that the potassium conductance activation depends on how much Ca enters the cell regardless ofwhether barium is simultaneously present or not. Barium, then, if it did actually block the potassium channel from the inside, would have to have a weak affinity for the channel compared to calcium. Following short exposures to barium-tea saline, less than 10 min, the complete restoration of calcium led to a recovery of outward current to at least 90 % of its control value within seconds. Strontium was a slightly better current carrier than calcium judging from records where equimolar substitution for calcium was made. The effect on activation of potassium conductance by this ion was intermediate between that of barium and calcium. That is, there was much less outward current in strontium than in calcium saline, but the reduction was never so dramatic as in barium.

56 J. A. CONNOR Fig. 10 demonstrates activation of IA under two conditions where the delayed outward rectifier and calcium dependent potassium conductances were blocked. The delayed rectifier was blocked in both cases by external TEA. The calcium dependent flux was eliminated by internal EGTA buffering in IOA and by barium substitution in lob. In either case, with an appropriate conditioning voltage (< -40mV), a large outward transient developed upon depolarization. The inward flux, which was there all the time, was expressed in the total current records only toward the end of the transient. The superimposed inward records resulted from steps from conditioning voltages which inactivated the IA mechanism leaving nothing but inward current. A I100 na 400 msec B I l. A 1 00 msec Fig. 10. Records showing that the transient outward current (IA) can be activated under conditions which suppress other outward current mechanisms. A, cell LPe 1 of Archidoris bathed in calcium-tea saline and injected with EGTA. Two sets of clamp currents are shown, -70 to + 10 to -70 mv, which activates 'A' and -40 to + 7 to -40 mv, which does not. B, cell of Fig. 9 in barium-tea saline. Clamp sets: -50 to 0 to -50 mv activates 'A' -40 to + 2 to -40 my, does not activate. DISCUSSION This report has presented evidence showing that the time course of calcium flux into Dorid molluscan neurones is maintained for periods of several seconds under voltage clamp conditions, more than two orders of magnitude longer than net inward

MOLLUSCAN NEURONE Ca CURRENT 57 current flows in normal saline. This flux was present in all the neurones examined. Identification of the charge carrier as calcium was made by indirect methods in this study, ion replacement and cobalt block, but in the following report (Ahmed & Connor, 1978) a more direct determination using the indicator dye arsenazo III is presented. The greatest hinderance to determining the true time course of the calcium flux is the simultaneous presence of potassium current arising in any of three known, operationally distinct conductance channels. Of the three conductances, the transient, IAN activates with a time course most similar to the calcium conductance, and, if the conditioning voltage used in experiments is more negative than -45 mv, this current will activate and obliterate any trace of the slow calcium flux development (cf. Fig. 3). The remaining two potassium conductances, voltage and calcium dependent, activate more slowly and permit a glimpse of the calcium flux but make it appear as a transient (see Figs. 2-5). The techniques employed in the present study for displaying the inward flux as a quasi-steady state phenomenon instead of a millisecond-order transient were direct buffering of internal calcium by injected EGTA and replacement of calcium as the transmembrane current carrier by barium. Both of these techniques should prevent an increase in internal calcium level during a period of influx but should do so by very different means. The current patterns which developed were very similar. That is, for voltages between 0 and + 40 mv, the normally large outward current simply failed to develop and the net current, while showing an apparent inactivation over a very slow time course, always remained below the extrapolated leakage current. For voltages below zero the inward current was nearly flat. Akaike, Lee & Brown (1977) in experiments on Helix neurones showed that when outward current was blocked by the replacement of cesium for potassium, a non-inactivating calcium current remained similar to the one described in the present study. It seems safe to conclude, in agreement with earlier work, that an elevated internal calcium concentration is causally related to increased potassium conductance (see Meech, 1972; Meech & Standen, 1975). Whether the change in calcium concentration is but one of several intermediate steps has not been determined. Two other potassium conductance mechanisms are clearly demonstrable in these molluscan neurones, neither of them dependent on internal calcium levels in the way that the first is. There are the delayed rectifier (IK) and the transient (IA) both of which seem to be primarly sensitive to voltage. Although it has not been shown that these three phases are the result of distinct membrane mechanisms it is certainly useful to regard them as such in discussing the ensemble of events which create the membrane I-V relationship. At voltages more positive than + 60 mv, voltage clamp records became difficult to interpret. Total current, both initial and at long durations, was more positive than leakage current extrapolated from negative-going steps. Leakage corrected current, measured at its most negative point, reversed from inward-going to outward around + 60 mv (range 50-70 mv) in cells bathed in TEA. It is very unlikely that this represents the reversal potential for a calcium flux since the Nernst equation with 15 mm external calcium would predict an internal concentration of approximately 90 AM. Estimates of intracellular free calcium concentration generally are less than

58 J. A. CONNOR 1 /SM (see Tuley, Brown & Baur, 1975; Meech, 1974). Also, the low reversal was noted with internal EGTA. There are three likely factors which could give rise to the low reversal potential; (1) the TEA block of potassium current becomes ineffective at large positive potentials, (2) the calcium conductance mechanism is permeant to some extent to sodium and/or potassium ions and these ions leave the cell at large positive voltages, (3) the sodium conductance fails to inactivate completely at large positive voltages as in squid axon (see Bezanilla & Armstrong, 1977). It has proved very difficult to sort out the various ion fluxes at large voltages using purely electrical techniques. An extended approach using arsenazo III is reported in the following article. The records of Fig. 10 were included in this report to illustrate two points, one which bears upon the conductances of different excitable membranes, the other of direct relevance to the calcium conductance. Vassort (1975) and Mounier & Vassort (1975) have described a transient potassium current in uterine smooth muscle and certain crustacean muscle. The time course and general appearance of this current is similar to IA of molluscan neurones. In the muscle preparations, however, the current is abolished by treatments which block transmembrane calcium flux; by inference it is activated by internal calcium. There is no such dependence of the molluscan IA; this transient current is activated even though internal calcium level is held at a low value either by EGTA injection or by external substitution of another divalent ion for calcium. Secondly, the records of Fig. 10 show the enormous error in estimating the value of inward flux which can result from employing conditioning voltages in the range which permits activation of IA. In the period immediately following the fast inward transient, total current is outward; only as IA decays does total current again become inward. By this point under normal conditions (no EGTA, barium or TEA), voltage and calcium dependent potassium current would have developed and obscured the inward component. I wish to thank Dr A. 0. D. Willows for making available the facilities of the Friday Harbor Laboratories. Appreciation is also expressed to Dr R. Fay and Mrs C. Eaton for supplying animals. The work was supported by National Science Foundation grants GB-39946 and BMS-19938. REFERENCES ADAMS, D. J. & GAGEE, P. W. (1976). Gating currents associated with sodium and calcium currents in an Aplysia neuron. Science, N.Y. 192, 783-784. AHMED, Z. & CoNNoR, J. A. (1978). Measurement of calcium influx under voltage clamp in molluscan neurones using the metallochromic dye Arsenazo III. J. Physiol. 286, 61-82. AxAE, N., LEE, K. S. & BROWN, A. M. (1977). Calcium currents in molluscan neurons. Biophys. J. 17, 192a. ARMSTRONG, C. M. & BINSTOCK, L. (1965). Anomalous rectification in the squid giant axon injected with tetraethylammonium chloride. J. gen. Phy8iol. 48, 859-872. AR MONG, C. M. & HiiT, B. (1972). The inner quarternary ammonium ion receptor in potassium channels of the node of Ranvier. J. gen. Physiol. 59, 388-400. BA=T-T, E. F. & BAmRETI,J. N. (1976). Separation of two voltage-sensitive potassium currents, and demonstration of a tetrodotoxin-resistant calcium current in frog motoneurones. J. PhyAiol. 255, 737-774. BEELER, G. W. & REUTER, H. (1970). Voltage clamp experiments on ventricular myocardial fibres. J. Phy8iol. 207, 165-190. BEZAN 9, F. & ARMSTRONG, C. M. (1977). Inactivation of the sodium channel. I. Sodium current experiments. J. gen. Phy8iol. 70, 549-566.

MOLLUSCAN NEURONE Ca CURRENT CHAMBERLAiN, S. G. & KERKuT, G. A. (1969). Voltage clamp analysis of the sodium and calcium inward currents in snail neurons. Comp. Biochem. Physiol. 28, 787-801. CONNOR, J. A. (1977a). Time course separation of two inward currents in molluscan neurons. Brain. Re8. 119, 487-492. CONNOR, J. A. (1977b). Calcium and calcium activated ion flux in isolated somata of molluscan neurons. Biophys. J. 17, 43a. CONNOR, J. A. & STEVENS, C. F. (1971 a). Inward and delayed outward membrane currents in isolated neural somata under voltage clamp. J. Phy8iol. 213, 1-20. CONNOR, J. A. & STEVENS, C. F. (1971 b). Voltage clamp studies of a transient outward membrane current in gastropod neural somata. J. Phy8iol. 213, 21-30. DODGE, F. A. & FRANKENHAEUSER, B. (1959). Sodium currents in the myelinated fibre of Xenopu8 laevi8 investigated with voltage clamp technique. J. Phyaiol. 148, 188-200. ECKERT, R. & Lux, H. D. (1976). A voltage-sensitive persistent calcium conductance in neuronal somata of Helix. J. Phqyjiol. 254, 129-151. FRANKENHAEUSER, B. & HODGKIN, A. L. (1957). The action of calcium on the electrical properties of squid axons. J. Phy8iol. 137, 218-244. GEDULDIG, D. & GRUENER, R. (1970). Voltage clamp of the Aplysia giant neurone: early sodium and calcium currents. J. Physiol. 211, 217-244. GoLA, M. & RoMEY, G. (1971). R6ponses anomales a des courants sous liminaires de caertaines membranes somatiques (neurones giants d' Helix pomatia). Analyse par la m6thode du voltage impose. Pfliiger8 Arch. 237, 105-131. HAGIWARA, S. (1973). Ca spike Adv. Biophys. 4, 71-102. HAGIWARA, S., FUKUDA, H. & EATON, D. C. (1974). Membrane currents carried by Ca, Sr and Ba in barnacle muscle fiber during voltage clamp. J. gen. Phy8iol. 63, 564-578. HAGIWARA, S., OZAWA, S. & SAND, 0. (1975). Voltage clamp analysis of two inward current mechanisms in the egg cell membrane of a starfish. J. gen. Physiol. 65, 617-644. HAGIWARA, S. & SAITO, N. (1959). Voltage-current relations in nerve cell membrane of Onchidium verruculatum. J. Physiol. 148, 161-179. HARUMASA, O., TAKAHASHI, K. & YosHii, M. (1976). Two components of the calcium current in the egg cell membrane of the tunicate. J. Phyeiol. 255, 527-561. HEYER, C. B. & Lux, H. D. (1976a). Properties of a facilitating calcium current in pace-maker neurones of the snail, Helix pomatia, J. Phyeiol. 262, 319-348. HEYER, C. B. & Lux, H. D. (1976 b). Control of delayed outward potassium currents in bursting pace-maker neurones of the snail, Helix pornatia, J. Phyeiol. 262, 349-382. HILLT, B. (1967). The selective inhibition of delayed potassium currents in nerve by tetraethylammonium ion. J. gen. Phyaiol. 50, 1287-1302. HIUE, B. (1968). Charges and potentials at the nerve surface. Divalents ions and ph. J. gen. Phyeiol. 51, 221-236. HODGKIN, A. L., HuxLEY, A. F. & KATZ, B. (1952). Measurement of current-voltage relations in the membranes of the giant axon of loligo. J. Phyeiol. 116, 424-448. KADO, R. (1973). Aply8ia giant cell: Soma-axon voltage clamp current differences. Science, N. Y. 182, 843-845. KEYNES, R. D., RoJAS, E., TAYLOR, R. E. & VERGARA, J. (1973). Calcium andpotassium systems of a giant barnacle muscle fibre under membrane potential control. J. Physiol. 229, 409-455. KOPPENHOFER, E. (1967). Die wirkung von tetraauthylammoniumchlorid auf die membranestroume Ranvierscher Schnurringe von Xenopu8 laevi8. Pfluiger8 Arch. ge8. Phy8iol. 313, 361-380. KosTYuT, P. G., IsnE:TAL, 0. A. & DOROsHNEO, P. A. (1974). Properties of inward current channels in the somatic membrane of a nerve cell. Bioelectrochem. & Bioenerg. 1, 350-354. KRIsirEA, 0. A. & MAGURA, I. S. (1970). Calcium ions as inward current carriers in mollusk neurones. Comp. Biochem. Phy8iol. 35, 857-866. KRNJEVI6, K. & Lism:wicz, A. (1972). Injections of calcium ions into spinal motoneurones. J. Phyeiol. 225, 363-390. LEE, K., AxAiI, N. & BROWN, A. M. (1977). A specific action of trypsin on neuronal membranes. Biophy8. J. 17, 192a. MEECH, R. W. (1972). Intracellular calcium injection causes increased potassium conductance in Aplyeia nerve cells. Comp. Biochem. Phy8iol. 42, 493-499A. 59

60 J. A. CONNOR MEECH, R. W. (1974). The sensitivity of Helix aspersa neurones to injected calcium ions. J. Phy8iol. 237, 259-277. MEECH, R. W. & STANDEN, N. B. (1975). Potassium activation in Helix aspersa under voltage clamp: a component mediated by calcium influx. J. Physiol. 249, 211-239. MEvES, H. & VOGEL, W. (1973). Calcium inward currents in internally perfused giant axons. J. Physiol. 235, 225-265. MOuNIER, Y. & VAssORT, G. (1975). Evidence for a transient potassium membrane current dependent on calcium influx in crab muscle fibre. J. Phyaiol. 251, 609-625. NEHER, E. (1971). Two fast transient current components during voltage clamp on snail neurones. J. gen. Physiol. 58, 36-53. NEHER, E. & Lux, H. D. (1972). Differential action of TEA on two K-current components of a molluscan neurone. PJlugers Arch. 336, 87-100. REuTER, H. (1973). Divalent cations as charge carriers in excitable membranes. Prog. Biophys. molec. Biol. 26, 1-43. ROUGIER, O., VAssoiw, G., GARNIER, D. GARGOUIL, Y. & CARABOEUF, E. (1969). Existence and role of a slow inward current during the frog atrial action potential Pfluger8. Arch. 308, 91-110. SCHAUF, C. (1975). The interactions of calcium with Myxicola giant axons and a description in terms of a simple surface charge model. J. Physiol. 248, 613-624. SMITH, T. G., BARKER, J. L. & GAINER, H. (1975). Requirements for bursting pacemaker potential activity in molluscan neurons. Nature, New Biol. 253, 450-453. STANDER, N. B. (1975). Voltage clamp studies of the calcium inward current in an identified snail neurone: comparison with the sodium inward current. J. Physiol. 249, 253-268. THOMPSON, S. H. (1977). Three pharmacologically distinct potassium hannels in molluscan neurones. J. Physiol. 265, 465-488. TuLEY, F. H., BROWN, A. M. & BAUR, P. S. (1975). Calcium and light evoked membrane hyperpolarization in Aplysia giant neurones. Biophy8. J. 15, 170a. VASSORT, G. (1975). Voltage-clamp analysis of transmembrane ionic currents in guinea-pig myometrium: evidence for an initial potassium activation triggered by calcium influx. J. Physiol. 252, 713-734. WALTER, D. & CONNOR, J. A. (1975). An investigation of the effects of tetraethylammonium ion on a transient potassium current in molluscan neurones. Biophys. J. 15, 262a.