Nakajima, 1966; Okamoto, Takahashi & Yoshi, 1976; Kostyuk & Krishtal, 1977a;

Transcription

1 J. Physiol. (1984), 356, pp With 7 text-figures Printed in Great Britain TWO CLCIUM CURRENTS IN NENTHES REN CEODENT TUS EGG CELL MEMBRNES BY. P. FOX ND S. KRSNE From the Department of Physiology, hmanson Laboratory of Neurobiology, Jerry Lewis Neuromuscular Research Center, University of California, Los ngeles, C 924, U.S.. (Received 22 March 1983) SUMMRY 1. Two distinct types of Ca currents, Ca(I) and Ca(II), were found in the eggs of the marine polychaete Neanthes arenaceodentatus and studied under voltage-clamp conditions. 2. Ca(I) and Ca(II) channels differ in their selectivity sequences, show different sensitivities to blocking by Cd, and have different activation thresholds. These facts indicate that the channels responsible for Ca(I) and Ca(II) currents are unique and different. 3. Both Ca(I) and Ca(IJ) currents decrease with time under a maintained depolarization. This relaxation exhibits different kinetics, with those for Ca(IJ) being an order of magnitude slower than for Ca(I). The Ca(I) current relaxation has been shown previously to be due to a voltage-dependent inactivation. 4. The magnitude of relaxation of the Ca(JI) current elicited by a test-voltage step immediately following a conditioning-voltage step paralleled the magnitude of the peak of the Ca(II) current flowing during the conditioning pulse. The kinetics of the current relaxation depended upon bath Ca concentration, the kinetics slowing down as the bath Ca was increased. These observations are consistent with an external depletion being the cause of the current relaxation for the Ca(IJ) channel. INTRODUCTION Ca ions have been found in many preparations to be the main inward current carrier, while in others both Na and. Ca carry the inward current (Hagiwara & Nakajima, 1966; Okamoto, Takahashi & Yoshi, 1976; Kostyuk & Krishtal, 1977a; dams & Gage, 1979; see Reuter, 1973 and Hagiwara & Byerly, 1981 for more references). These Ca currents exhibit diverse behaviours in different preparations. One feature displayed by most Ca currents is a decrease in the size of the inward current with time under a maintained depolarization (Standen, 1974; Hencek & Zachar, 1977, kaike, Lee & Brown, 1978; Brehm & Eckert, 1978; Tillotson, 1979; lmers, Fink & Palade, 1981). The unfertilized eggs ofthe marine polychaete Neanthes are no exception. Two types of inward Ca currents, referred to as Ca(J) and Ca(JI), are found in the eggs. Both currents relax with time under a maintained depolarization.

2 492. P. FOX ND S. KRSNE The relaxation of the Ca(I) current has been described previously and shown to be due to a voltage-dependent inactivation (Fox, 1981). In this paper we will investigate the mechanism of Ca(II) current relaxation. Several possibilities can account for the current decrease with time; the following four will be entertained. First, there might be no decrease in the Ca current at all, and the relaxation process could be due to a turn-on of outward-going current, probably carried by K, which masks the inward Ca current and makes it appear to decrease. Since delayed rectifiers and Ca-activated K channels have both been well described in other preparations (Hodgkin & Huxley, 1952a, b, d; Meech and Standen, 1975; Hermann & Gorman, 1979), their presence must be considered. Secondly, there could be a Ca-induced inactivation of the Ca conductance. Ca itself has been shown to have an inhibitory effect on Ca currents (Hagiwara & Nakajima, 1966; Kostyuk & Krishtal, 1977b; Takahashi & Yoshii, 1978). Recently, a Ca-induced Ca inactivation has been discovered in Paramecia and plysia whereby Ca entering the cell decreases the size of the Ca currents (Brehm & Eckert, 1978; Tillotson, 1979). third possibility to account for the current decrease with time could be a voltage-dependent inactivation much like that seen for Na channels in squid giant axon and described by Hodgkin & Huxley (1952c). Finally, the current relaxation could be due to an external depletion or internal accumulation of Ca ions. n external depletion of Ca has been reported for frog skeletal muscle, although these findings have been challenged (lmers et al. 1981; Cota, Nicola Siri & Stefani, 1982). In this paper we will attempt to show that, unlike the voltage-dependent inactivation of the Ca(I) channel, the Ca(II) current relaxation is due to an external depletion of Ca ions. We will also explore, to a limited extent, some of the other properties of the two Ca channels found in Neanthes. It will be shown that there are differences in the Ca(I) and Ca(II) currents with respect to the kinetics of the current decrease, threshold of activation, pharmacological sensitivity to blockers, as well as selectivity ratios (as determined from peak inward currents). METHODS Gravid Neanthes arenaceodentata were purchased from Donald J. Reish (California State University, Long Beach). The eggs were removed from the female worm by severing its anterior and posterior portions and then flushing them out of the body by using a slow jet of sea water from a syringe through a needle inserted into the coelom. The eggs recorded from had diameters between 4 and 6 gsm. The eggs were surrounded by a tough vitelline membrane which was 1-2 1um thick and which was sufficiently tightly apposed to the plasma membrane that no space could be resolved between the two by electron microscopy up to magnifications of 4 times. The solutions used are given in Table 1. Experiments designed to test the effects of Co were done using unbuffered solutions to prevent any precipitation between the buffers and Co-containing solutions. In order to test whether there were any effects of ph on the experiments, controls were done where two identical Co-free solutions were made and then 1 mm-tris, ph 7-6, was added to one while the other had 1 mm-nacl added to maintain osmotic strength. No difference in results was observed between the solution containing the ph buffer and the one that did not. two-micro-electrode voltage clamp was used to control the potential of the unfertilized Neanthes eggs. The settling time for the voltage clamp ranged from 1 to 3 ms for a 1 mv jump depending on the resistance ofthe micro-electrode used. The micro-electrodes were filled with 3 M-KCl and had resistances of 4-1 MO. reference electrode was positioned near the egg to minimize bath series resistance. The bath was held at virtual ground by a current-to-voltage converter (joined to the bath by a 3 M-KCI-agar bridge) which also measured total bath current. The voltage-clamp holding potential was either set equal to the egg resting potential (between -7 and -85 mv) or

3 TWO Ca CURRENTS IN EGG CELL MEMBRNES was set to more positive potentials to inactivate the Ca(I) channel and to remove any Ca(I) current contamination from the current trace. Some of the data presented in this paper have been corrected for passive membrane leak. In Neanthes eggs there are only two types of voltage- and time-dependent currents in the range of potentials from -12 to -2 mv; these are the anomalous rectifier and the Ca(I) current. In order to measure the leak, both of these currents were suppressed. The anomalous rectifier was totally TBLE 1. Solutions used Permeant NaCl KCl divalent ion* MgCl2 Tris Name (mm) (mm) (mm% (mm) (mm) ph loca 51 1 Ca (1) loba 51 1 Ba (1) Sr 51 1 Sr (1) Ca 47 1 Ca (6) 7 6Ba 47 1 Ba (6) 7 6Sr 47 1 Sr(6) 7 1 Ca, 5 Mg 47 1 Ca (1) Ca, 45 Mg 47 1 Ca (15) Ca, lomg 47 1 Ca (5) 1 7 5Ca, 6Co, 4Mg 47 1 Ca (5) 4t 7 * s chloride. t Solution also contains 6 mm-co. 493 blocked by adding Ba at a concentration of 5,UM or larger while the Ca(I) current was removed by long, depolarizing pre-pulses. (For experiments in Ca and Sr solutions, the Ba was added to the bath and the leakage determined after the experimental data-gathering run was completed.) The leak measured by these procedures was ohmic and showed no time dependence. To get values for the leak at more positive potentials a linear extrapolation of the leak current was made from the -12 to -2 mv region. To verify the correctness of our leak subtraction procedure, control records taken with Cd in the bath, which suppresses any Ca(II) currents (see Fig. 1 B), were sometimes subtracted from Ca(II) current records. (For these experiments inactivating pre-pulses were used to remove the Ca(I) currents.) The same results were obtained as from our linear leak subtraction protocol over most of the voltage range (see Fig. 4). The experimental data were filtered by a six-pole Bessel analog filter having a corner frequency at 86 Hz, after which they were digitized at sampling rates of either 2 or 1lpoints per second by an eight-bit /D converter. The Bessel filter corner frequency was kept as large as possible to decrease the settling time of the current trace while maintaining acceptable filtering. No aliasing was detected at the 1 Hz sampling rate. The data were then stored on a Northstar Horizon II microcomputer and subsequently plotted on a Houston DMP-4 digital plotter or an Epson MX-8 graphics printer. RESULTS Comparisons between two Ca currents Examples of the two types of Ca currents found in Neanthes eggs are illustrated in Fig. 1. two-step protocol was used to control the membrane potential. The first step was a depolarization to -25 mv lasting for 2 ms. This step elicited Ca(I) current and some leak. (The threshold of activation for Ca(IJ) currents was between -5 and -1 mv; therefore no Ca(II) current was seen in the step to -25 mv.) Following this initial step, the membrane was repolarized to the holding potential, -75 mv, for 1 ms, after which the second pulse, a depolarization to + 25 mv lasting 3 s, was applied. (Experiments on the Ca(I) current show that this current totally

4 .- I 494. P. FOX ND S. KRSNE 6 mm-ca 1 n B 5 ms 6 mm-ca 1 mm-cd Test pulse I nactivation pre-pulse Fig. 1. Ca(IL) current and relaxation process., the current elicited from a two-pulse procedure; the first pulse activates the Ca(I) conductance, and the second pulse (to+ 25 mv) activates the Ca(IL) conductance, producing a net inward current which decays under the maintained depolarization. B, same cell and the same pulse protocol as in but with 1 mm-cd added to the bath. Note that the Ca(I) current is not affected by Cd, while the Ca(II) current is virtually abolished by it. No leak subtraction was used in either or B. Zero current is indicated by a dashed line in all Figures with current traces. C, voltage pulse protocol for measuring currents through the Ca(IL) channel. The first pulse, to inactivate Ca(I) channels, is always to -25 mv and lasts 2 ms. The second pulse elicits the Ca(II) current and is 3 s long. The first and second pulses are separated by a 1 ms return to the holding potential. This same pulse protocol was used to obtain the data in all subsequent Figures except Fig. 5. inactivates at voltages more positive than -4 mv and shows no recovery within 1 ms; thus there is no contamination of the Ca(II) current records by the Ca(I) current.) The most striking difference seen in Fig. 1 is in the time scales of the relaxations for the two currents. Ca(II) currents decrease an order of magnitude slower than Ca(I). (The times for Ca(J) currents to decrease to half their maximal values are in the 1-4 ms range while for Ca(II) they are in the 1-5 ms range.) number of observations suggest that these two currents are mediated by different

5 TWO Ca CURRENTS IN EGG CELL MEMBRNES channels. First, both Ca(J) and Ca(II) currents show different sensitivities to blockers. Fig. 1 B shows a current record from the same cell as Fig. 1, using the same pulse protocol and the same conditions as was described for Fig. 1 except that 1 mm-cd was added to the bathing medium. The Cd virtually abolished the Ca(II) current while leaving the Ca(I) current almost totally unaffected. (The only noticeable effect on the Ca(I) current was a shift of the current-voltage relationship along the voltage axis by about +11 mv; this shift is likely to be due to an effect of the Cd on the surface-charge density near the Ca(I) channel.) Co has also been found to be more effective in blocking Ca(II) than Ca(I) channels (by about times) but is less effective than Cd in blocking the Ca(II) currents. second difference between the Ca(I) and Ca(II) currents is in the selectivity sequences determined from the peak inward currents carried by the permeant divalent cations Ca, Ba, and Sr. For Ca(I), the sequence is Ba < Sr = Ca (. P. Fox & S. Krasne, unpublished observations), while for Ca(II) the sequence is Ba > Sr > Ca (see Fig. 2C). third observation suggesting that Ca(I) and Ca(II) currents are mediated by two distinct channels is that eggs have been found with little or no Ca(I) current but quite large Ca(II) currents, and conversely, eggs have been found with little or no Ca(II) current but quite large Ca(I) currents. The weight of the above evidence argues strongly that Ca(I) and Ca(II) currents are produced by different channels, referred to here as the Ca(I) and Ca(II) channels, respectively. The following evidence indicates that the mechanism underlying the time-dependent relaxation of Ca(IJ) currents is also different from that of Ca(I) currents, further supporting this conclusion. Comparisons of the Ca(lI) currents produced by Ca, Ba or Sr Fig. 2 illustrates the currents elicited, with no leak subtraction performed by depolarizing the same egg to + 15 mv in the presence of either 6 Ca, 6 Sr or 6 Ba solution. Note that the kinetics of the current traces in Fig. 2 are quite similar to each other. This observation is in contrast to what has been reported (see, for example, Brehm & Eckert, 1978; Tillotson, 1979) for Ca channels whose mode of inactivation is due to Ca-induced Ca channel inactivation; for these channels, the kinetics of the currents obtained in Ba are grossly different from those obtained in Ca. Fig. 2B plots the time for the currents to relax to halfway between the peak and steady-state values as a function of potential, again comparing data taken in 6 Ca, 6 Sr and 6 Ba solutions. This Figure shows that the time constants are always within a factor of two of each other, at any given potential, and that the fastest current decays are those recorded in Ba, which is again quite different from what has been reported for Ca-induced inactivation (Tillotson, 1979). Indeed, in Fig. 2B much of the difference in relaxation times between Ca, Ba and Sr at any given potential is caused by shifts along the voltage axis due to differential binding, and thus different surface potentials, which we have observed for these divalent cations (. P. Fox & S. Krasne, unpublished observations). (These effects, which have not been corrected for in the present paper, will exist even if the current relaxation is current- rather than voltage-dependent, since the magnitude of the peak inward current will depend upon the number of channels activated, which in turn depends upon the membrane's electric field.) Fig. 2C shows the peak current-voltage 495

6 496. P. FOX ND S. KRSNE Ca 5 B E4 E~~~~ Ba E W 3-4- ~ * I 2 1 I 5 m 1 n C a 1 Ba 5 ms a1os moo o1ca * Sr -1 O ~~ c -2 _3 * ~~ -4 a Potential (mv) Fig. 2. Comparison of inward currents in 1 Sr, 1 Ba and 1 Ca., current traces obtained after depolarizations to + 15 mv in 1 Sr, 1 Ba and 1 Ca solutions. Dashed line represents the zero current level. Note the similarity of the currents in all three solutions. None of the currents has been leak-gubtracted. The current trace in Ba shows that a holding current was necessary to keep the membrane at -8 mv since the inward rectifier, the channel which dominates the conductance at rest, was blocked by the Ba. B, plots of the relaxation half-times (i.e. the time for the first half of the current decay process to take place) vs. voltage in 1 Sr, 1 Ba and 1 Ca. This plot illustrates that the kinetics of the current relaxations are quite similar in all three divalent cations. However, note that at any given potential the relaxation times are fastest in Ba and slowest in Ca. C, plot of the peak inward currents v8. transmembrane voltage in 1 Sr, 1 Ba and 1 Ca solutions. These currents have been corrected by subtracting the currents recorded in the presence of 2 mm-cd. Data in, B and C have been obtained on the same egg. relationships obtained in the same cell for 6 Ca, 6 Sr or 6 Ba solution. The largest currents, recorded in Ba, are almost twice the size of the largest currents obtained in Ca at the same concentration. Ca(II) current relaxation is not due to development of an outward current Tail current measurements indicate that the time-dependent relaxations of Ca(II) current represent a conductance decrease rather than being due to a conductance increase for a net outward current which would mask the inward current. The magnitudes of tail currents observed on repolarizing the membrane to more negative potentials following a step depolarization decrease the longer the time of the depolarization. In Fig. 3, the egg was depolarized from a holding potential of -75 mv to + 25 mv for 1 ms, 2 ms or 1t5 s. fter each of these depolarizations the

7 TWO Ca CURRENTS IN EGG CELL MEMBRNES 497 membrane was repolarized to -2 mv and the tail current observed. The inward tail currents seen upon repolarizing the cells to -2 mv were largest at 1 ms and became progressively smaller the longer the depolarization was maintained. The tail current at 1-5 s was quite small. (Note that the traces in Fig. 3 have not been leak-subtracted.) The fact that the tail currents never disappeared totally indicates 1 n 'b ;a a 1 ms 1 n C _ - Fig. 3. Time dependence oftail currents. The conductance of Ca(II) channels was activated with a depolarization and then monitored by stepping to a new, more negative potential and measuring the 'tail current'. The top trace is a current record obtained in 6 Ca solution after depolarization to + 25 mv. (Ca(I) currents were inactivated with a pre-pulse.) No leak subtraction has been employed. rrows on the trace represent different times at which the membrane was repolarized to -2 mv: a, 2 ms; b, 1 ms; c, 1-5 s. The traces labelled a, b and c are the tail currents occurring following depolarizations at the times indicated as a, b and c, respectively, on the top trace. Zero current is represented by the dashed line. The first five points of each tail current have been blanked out as they represented only the saturation value of the input stage of the computer. that there is a small component of steady-state inward current (also seen in some records of Fig. 4, as mentioned below). The records for Fig. 3 were made with Ca in the bath while those of Fig. 4 were made with Ba in the bath, showing that the maintained conductance for an inward current is not dependent on the ion used, rather it is a general feature of the Ca(JI) currents. Because the early tail-current size is directly related to the conductance at the time the depolarization is interrupted, the decrease in tail-current magnitude with time is a clear indication of a conductance decrease with time. This decrease may represent either a true decrease in the slope conductance or a decrease in the driving force on the ion. Repolarization to -2 mv was chosen because the tails were greatly slowed at this potential. Tails could clearly be seen down to -4 mv and qualitatively the same results could be obtained with repolarizations to several different potentials between -2 and -4 mv; however, below -4 mv the clamp could no longer resolve the tail currents in time. The peak

8 498. P. FOX ND S. KRSNE amplitudes of the tails appear smaller than the peak Ca current in the upper trace because of saturation of the input stage to the computer. We have removed the first five points of each tail current from the Figure as these points show the saturation value of the /D converter. In fact the tails at 1 and 2 ms produced significantly more Ca current than the peak current shown in the upper trace. Because of the limited time resolution of our voltage clamp, we also do not have confidence in points recorded earlier than 5-6,s after the start of the voltage step; thus we were unable to determine whether these tail currents might have a second, faster component whose time constant is buried in this range. The decrease in the magnitude of the tail currents with increasing time at which repolarization occurred, suggests that the current relaxation for the Ca(II) channel is due to a decrease in conductance and not to the turning on of an outward current. In addition, note that if these tails were due to a K current turning off, they would be in the opposite direction, representing an outward-going current, as the equilibrium potential for K is about -8 mv. Further specific arguments can be made against the net inward current relaxation being due to turning on of a Ca-activated K channel or a delayed rectifier. The kinetics and magnitudes of the current relaxations are very similar in Ca, Ba and Sr solutions (see Fig. 2). Such a lack of selectivity has never been found before for a Ca-activated K channel; previously, Ba has always been unable to turn on this K conductance. If an outward, non-ca-dependent current were turning on, the magnitude of the relaxation should be the same for 1 and 6 Ca; however, it was several times larger in the higher Ca solution (not shown). If the current relaxation were due to a delayed rectifier similar to the one in the squid giant axon, the magnitude of relaxation should be smaller in Ba than in Ca solutions, since very low concentrations of internal Ba (such as could get through the Ca(II) channel) block this channel; however, somewhat larger magnitudes of relaxation were observed in Ba than in Ca solutions, as discussed above for the data in Fig. 2. n argument against turning on of an outward rectifier not blocked by Ba (at least not for the levels of Ba which result from its entry through the Ca(II) channel) can also be made based upon the data in Fig. 4. Fig. 4 shows the currents obtained in the 6 Ba solution for depolarizations to the potentials shown to the right of each trace. The records have been leakcorrected by the linear extrapolation technique described in the Methods, and the Ca(I) current has been inactivated with a depolarizing pre-pulse. ddition of Cd to the bath to block the Ca(II) channel removes any time-dependent component of this current as seen in the traces in Fig. 4B (which have not been leak-corrected) for the same egg. (Note that, except for the largest depolarizations, the currents remaining in Fig. 4B are comparable to those subtracted as leak from the traces in Fig. 4, as discussed in the Methods.) The lack of time dependence after Cd is added indicates that the process of current decay can be entirely attributed to the Ca(II) current relaxation. The magnitudes of the currents in Fig. 4B correspond almost identically to the values of leakage estimated, for the corresponding voltages, in correcting the traces in Fig. 4. Note that large depolarizations result in outward, steady-state currents in the uncorrected records (as inferred, for example, by summing the traces at +56 mv in Fig. 4 and B).

9 TWO Ca CURRENTS IN EGG CELL MEMBRNES 499 Ca(II) current decrease is not due to a voltage-dependent inactivation The next point to resolve is whether the current decrease observed is a currentor voltage-dependent phenomenon. To distinguish between these mechanisms we investigated the dependences of the magnitudes and kinetics of current relaxations on the transmembrane voltage. Experiments were done using a two-pulse procedure.-- B -- r -4 mv mv am6mv - t r6mv - 21 mv 21mV -.' _,p~'srnrnwun 56 mv I 56 mv I 1 n 5 ms Fig. 4. Currents observed in the presence and absence of Cd., the current traces were all obtained in 6 Ba solution. The eggs were depolarized to the potentials indicated to the right of the current traces. ll the traces have been leak-subtracted by the linear extrapolation technique described in the Methods. B, same as but with 2 mm-cd added to the bath to suppress the Ca(II) currents and no leak subtraction performed. Note that there is no time-dependent current in the presence ofcd. In addition, there is a maintained, inward, steady-state current after the Ca(II) current relaxation, at least up to 2 s. Zero current is indicated by the dashed line. The Ca(I) current has been removed by an inactivating pre-pulse, as in Fig. 1. in which the first, 'conditioning' pulse was a 1 s step to a variable voltage and produced the current relaxation, while the second, 'test' pulse was always to a fixed potential and assayed the amount of current remaining at that voltage following the conditioning pulse. The two pulses were separated by a return to the holding potential of 1 ms. (This repolarization did not appear to produce any significant recovery from the relaxation process as such recovery follows a much slower time course.) The results illustrated in Fig. 5B, where the amplitude of the peak inward current recorded during the test pulse is divided by the peak current obtained with no conditioning pre-pulse and is then plotted as a function of potential, show that the magnitude of current relaxation increased monotonically as the voltage was increased to about + 29 mv, but then decreased as the membrane potential became more

10 5. P. FOX ND S. KRSNE 6 Ba -8 _-16 - d Conditioning Test pulse pulse -32 la oca B9 - & Ba -8 DSr ~~~ ^ ot V (mv) Fig. 5. Steady-state 'inactivation' of Ca(II) channels in Sr, Ca and Ba. Experiments were performed using a two-pulse procedure. In this series of experiments, the conditioning pulses were to different potentials and lasted 1 s, and the test pulses were depolarizations to + 2 mv and lasted for 1 s. The voltage was returned to the holding potential for 1 ms between the conditioning and test pulses. The amount of current elicited during the test pulse depended on the amplitude of the conditioning pulse. The inset illustrates the pulse protocol. plots the currents for the voltages applied during the conditioning pre-pulses in 6 Ba solution only. B shows a plot of the peak current amplitudes obtained during the test pulse divided by the peak current obtained by a depolarization to + 2 mv with no conditioning pre-pulse. Note that there is recovery from inactivation at positive potentials. ll solutions were 6 mm-divalent cations, with only one divalent cation in the bath at any time. ll data points for and B have been leakage-corrected by the linear extrapolation method described in the Methods. positive, i.e. as it approached the estimated Ca reversal potential. Thus the magnitude of the current relaxation is not governed simply by voltage. By contrast, comparison of Fig. 5 and B, where 5 is simply a current-voltage plot for the Ba currents obtained during the conditioning pre-pulses, demonstrates that the magnitude of the current relaxation parallels the magnitude of the inward current. (The data of Fig. 5 and B were leak-corrected using the linear extrapolation technique described in the Methods.) The absolute level of I/Imax in Fig. 5B is slightly affected by the fact that the current relaxation has not quite reached a steady-state value for some of the potentials by the end of the 1 s pre-pulse. Unfortunately, for this experiment, in which we sampled very large, positive potentials and several solutions for the same egg, we had to restrict our pre-pulses to 1 s in order to maintain the membrane in

11 TWO Ca CURRENTS IN EGG CELL MEMBRNES 51 r *1-15 -n- -&V i -25 B 33 r 29 F V (mv) a 35 5 E._ 4- I F 17 in,u V (mv) Fig. 6. Ca(II) currents and their half-times as a function of transmembrane voltage. The ordinate for is the peak inward current and that for B is the half-time for current relaxation; the abscissa is the transmembrane voltage. Experiments were done in 6 Sr solution. Note that the current relaxation is fastest at those voltages for which the inward current is largest. Leakage correction was done by the linear extrapolation method described in the Methods. good condition. Based upon data obtained for longer pulses, we estimate that after 1 s, the current had always decayed to within 1% of the steady-state value, and the main effect of this error is to make the relaxation appear somewhat smaller than it should be at the more negative and positive potentials (this error never amounting to more than a reduction by -1 for the data points in Fig. 5B). The data of Fig. 5 are consistent with a current-dependent hypothesis. Concomitantly, the fact that little current relaxation takes place during very depolarized conditioning pulses is inconsistent with a voltage-dependent inactivation where one does not expect recovery from inactivation at very depolarized potentials. The kinetics of current relaxation also parallel the magnitude of the inward current as deduced from Fig. 6 and B. Fig. 6 plots the half-times of current decay as a function of voltage, and Fig. 6B plots the current-voltage relationship, both in 6 Sr solution. Examination of these Figures shows that the rate of current decay is not a function of the voltage but rather parallels total current.

12 52. P. FOX ND S. KRSNE Ca does not 'inactivate' the Ca(II) channel nor does it accumulate internally If the reason for the current decrease were a Ca-induced Ca inactivation or an internal accumulation of Ca ions, a reasonable expectation would be a speed-up in the relaxation kinetics as the current size increased. For the Ca(JI) current relaxation the converse seems to be true; at any given voltage, as the currents get larger due to an increase in the bath Ca concentration, the rate of current decrease gets slower. This slowing of the kinetics for a larger size of current can be seen in Fig. 7 B, which plots the time for the current to relax to a value halfway between its peak and steady-state values as a function of current size. Several different bath Ca concentrations were used; for any given concentration the current decrease speeds up as the size of the current increases. lso apparent, however, is that as the bath Ca concentration is increased, the current relaxation slows down, making both Ca-induced Ca inactivation and an internal accumulation very unlikely. The time-dependent current decrease is due to an external depletion of Ca It is clear that the Ca(II) current relaxation is a current- rather than voltagedependent phenomenon, and that it is not Ca-induced Ca inactivation. The data heretofore presented are also consistent with a change in driving force due to an external depletion of Ca, however. Specifically, the maximum amount of current relaxation is seen at the same potentials as the maximum inward current (Fig. 5); the kinetics of the relaxation process parallel the size of the inward current (Fig. 6), being most rapid at the same potentials as those at which the maximum Ca(II) currents are seen; also, the kinetics of the relaxation process are slowed by increasing the bath Ca concentration even though the size of the Ca(II) currents is increased (Fig. 7). final experiment carried out in order to examine whether external depletion of Ca could account for the current relaxations was suggested by S. Hagiwara. This experiment is aimed at determining whether the kinetics of relaxation are solely dependent upon the size of the Ca(II) current or whether the external Ca concentration itself influences these kinetics. In order to dissociate the current size from the external Ca concentration, 6 mm-co was added to a 5 Ca solution in order to reduce the currents. Fig. 7 Cshows the current-voltage relationships in several different bath Ca concentrations, including the one with Co. The currents in the 5 mm-ca with 6 mm-co added were suppressed to approximately the level seen in the 15 mm-ca solution. Even so, the half-times of current decay are similar for both 5 mm-ca experiments (with or without Co) at any given current size. This similarity of kinetics would indicate that the current relaxation depends both on the size of the inward current and the bath Ca concentration, in agreement with the hypothesis that the relaxation of the Ca(II) current is due to depletion of external Ca. DISCUSSION Ca(II) currents decrease with time under a maintained depolarization. The only mechanism consistent with all the results reported here is a change in driving force due to an external depletion of Ca ions. The most critical results are the following: the current decrease is due to a change in the size of the Ca current and is not due

13 TWO Ca CURRENTS IN EGG CELL MEMBRNES 53 Solutions (mm) B - I n 1 Ca, 5 Mg 3 15 Ca, 45 Mg - 25 E * --_- 5 Ca, 6 Co, 4Mg E 2-,- 15 5Ca, 1 Mg / Current (n) Solutions (mm) 1 Ca, 5 Mg n 15n C o 15Ca, 45 Mg *5 s * 5 Ca, 1 Mg -7- c ? X5OCa, 6 Co, 4 Mg Voltage (mv) Fig. 7. The kinetics of the Ca(II) current decline are a function of the current size and the bath Ca concentration., the current records obtained in all the different solutions for a depolarization to + 2 mv. B, half-times for current relaxation V8. the peak inward current size. This Figure illustrates that for different bath Ca concentrations the currents relax at different rates for the same size of inward current (i.e. for Ca(II) the rate of current relaxation is a function of bath Ca concentration, even when the sizes of the peak currents are the same). The straight lines through the points are intended for illustration only and do not imply any model. For very small pulses the currents deviate from these straight lines, but they are difficult to measure as the half-times tend to infinity and have therefore not been included. C, the peak inward current for the Ca(II) channel plotted as a function of voltage in different Ca concentrations. Note that there is a trace with 5 mm-ca in which Co is present, partially blocking the inward Ca current. This solution produces almost the same size inward Ca current at any given voltage as is seen when the solution contains 15 mm-ca. These data have been leak-corrected by subtracting the steady-state current observed in 1 Ca, 5 Mg (which was the same between -15 and + 4 mv as that observed in the presence of Cd). to the development of net outward currents. The relaxations of Ca(IJ) currents become smaller as potentials became very positive (more positive than + 4 mv), the maximum magnitude of current relaxation occurring at those potentials where the largest inward currents are seen. pproximately the same kinetics are observed in Ca, Ba, or Sr, and the currents relax to approximately the same levels in these three solutions, in contrast to previous observations for Ca-induced Ca inactivation.

14 54. P. FOX ND S. KRSNE Finally, as the amount of Ca in the bath is increased, the absolute magnitude of the currents increases while the relaxation slows down. Similar current dependences (as opposed to voltage dependences) of Ca current relaxations have been observed in other preparations (Brehm, Eckert & Tillotson, 198; Tillotson, 1979). In most cases these have been shown to be due to a blocking or inactivation of the Ca channel by Ca itself. In at least one case, however, that of frog skeletal muscle fibres, the Ca current relaxation has been attributed to depletion of Ca from the restricted extracellular space of the transverse tubules (lmers et al. 1981). If such an external depletion exists in Neanthes eggs, the question arises as to whether any physical structures or membrane geometry are present which could limit free diffusion of divalent ions to the Ca(II) channels. One possibility is the presence of infoldings such as have been used- to account for similar phenomena in skeletal muscle fibres. However, electron microscopy done in our laboratory does not show any such infoldings; in addition, the capacitance measured from the time dependence of the voltage change following small current steps, when divided by the 'apparent' membrane surface area (assuming that the eggs are perfectly spherical and without infoldings), yields unit capacitance values of 1-2 uf/cm2, indicating that there is no extra membrane available for infoldings. Obviously then the depletion effect must arise from another source. Neanthes eggs have a second, membranous layer, the 'vitelline membrane', which lies in close apposition to the cell's plasma membrane. The vitelline membrane could be the origin of the depletion effect by somehow restricting diffusion of divalent cations. That the diffusion barrier does not affect the Ca(I) currents at all is probably due to the fact that Ca(I) currents undergo a voltage-dependent inactivation much faster than Ca would deplete near the channel. Two inward currents have been found previously in tunicate eggs as well as in starfish eggs (Hagiwara, Ozawa & Sand, 1975; Okamoto et al. 1976). The total current-voltage relationship of the two inward-going currents in each of these eggs is similar to those of Neanthes. The potentials at which the currents activate, as well as those potentials where the peak inward current is seen, are almost identical among all three eggs. Interestingly, whereas the currents corresponding to the Ca(II) channel appear similar in each egg, those corresponding to the Ca(I) channel are different: in tunicate eggs the current whose current-voltage relationship resembles that of the Ca(I) channel is carried by Na ions, whereas in the starfish egg there is a dependence on Na of the size of the inward current for this channel, although the current itself appears to be carried by Ca. It is unclear whether the differences in the starfish and tunicate currents, whose current-voltage relationships resemble that of the Ca(I) channel in Neanthes, are due to different stages ofdevelopment of a fully differentiated channel present in the adult forms of these animals or whether these channels represent true evolutionary differences. We would like to thank Dr S. Hagiwara for many helpful discussions. This work was supported by grants from NIH (HL2254), the Muscular Dystrophy ssociation and a pre-doctoral fellowship to. P. Fox from the Canadian Muscular Dystrophy ssociation.

15 TWO Ca CURRENTS IN EGG CELL MEMBRNES LIM REFERENCES DMS, D. J. & GGE, P. W. (1979). Characteristics of sodium and calcium conductance changes produced by membrane depolarization in an plygia neurone. Journal ofphy8iology 289, KIKE, N., LEE, K. S. & BROWN,. M. (1978). The calcium current of Helix neuron. Journal of General Phyeioy 71, LMERS, W., FINK, R. & PLDE, P. T. (1981). Calcium depletion in frog muscle tubules: the decline of calcium currents under maintained depolarization. Journal of Physiology 312, BREHM, P. & ECKERT, R. (1978). Calcium entry leads to inactivation of calcium channels in Paramecium. Science 22, BREHM, P., ECKERT, R. & TILLOTSON, D. (198). Calcium-mediated inactivation of calcium current in Paramecium. Journal of Physiology 36, COT, G., NICoI SiRi, L. & STEFNI, E. (1982). Current independent inactivation of the calcium channel in frog skeletal muscle. Biophysical Journal 37, 316. Fox,. (1981). Voltage-dependent inactivation of a calcium channel. Proceedings of the National cademy of Sciences of the U.S.. 78, HGIWR, S. & BYERLY, L. (1981). Calcium channel. nnual Review of Neuroscience 4, HGIWR, S. & NKJIM, S. (1966). Effects of the intracellular Ca ion concentration upon the excitability ofthe muscle fiber membrane ofa barnacle. Journal ofgeneral Physiology 49, HGIWR, S., OZW, S. & SND,. (1975). Voltage-clamp analysis of two inward current mechanisms in the egg cell membrane of a starfish. Journal of General Physiolrxy 65, HENCEK, M. & ZCHR, J. (1977). Calcium currents and conductances in the muscle membrane of the crayfish, Journal of Physiology 268, HERMNN,. & GORMN,. L. F. (1979). Blockade of voltage dependent K current component by internal Ba in molluscan pacemaker neurones. Experientia 35, HODGKIN,. L. & HUXLEY,. F. (1952a). Currents carried by sodium and potassium ions through the membrane of the giant axon of Loligo. Journal of Physiology 116, HODGKIN,. L. & HUXLEY,. F. (1952b). The components of membrane conductance in the giant axon of Lolgo. Journal of Phy8iology 116, HODGKIN,. L. & HUXLEY,. F. (1952c). The dual effect of membrane potential on sodium conductance in the giant axon of Loligo. Journal of Physiology 116, HODGKIN,. L. & HUXLEY,. F. (1952d). quantitative description of membrane current and its application to conduction and excitation in nerve. Journal of Physiology 117, KOSTYUK, P. G. & KRISHTL,.. (1977a). Separation of sodium and calcium currents in the somatic membrane of mollusc neurones. Journal of Phy8iology 27, KOSTYUK, P. G & KRISHTL,.. (1977b). Effects of calcium and calcium chelating agents on the inward and outward current in the membrane of mollusc neurones. Journal of Physiology 27, MEECH, R. W. & STNDEN, N. B. (1975). Potassium activation in Helix aspersa neurones under voltage clamp: a component mediated by Ca influx. Journal of Physiology 249, OKMOTO, H., TKHSHI, K. & YOSHI, M. (1976). Membrane currents of the tunicate egg under the voltage-clamp condition. Journal of Physiology 254, REUTER, H. (1973). Divalent cations as charge carriers in excitable membranes. Progress in Biophysics & Molecular Biology 26, STNDEN, N. B. (1974). Properties of a calcium channel in snail neurones. Nature 25, TKHSHI, K. & YOSHII, M. (1978). Effects of internal free calcium upon the sodium and calcium channels in the tunicate egg analysed by the internal perfusion technique. Journal of Physiology 279, TTLLOTSON, D. (1979). Inactivation of Ca conductance dependent on entry of Ca ions in molluscan neurons. Proceedings of the National cademy of Sciences of the U.S.. 76,