Correspondence to: Shalini Gera, Howard Hughes Medical Institute, Stanford University Medical Center, Beckman Center, Room B177, Molecular and Cellular Physiology Department, Stanford, CA 94305-5428. Fax: 650-725-4463; E-mail:sgera{at}cmgm.stanford.edu.
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Abstract |
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Ca2+ channel inactivation in the neurons of the freshwater snail, Lymnaea stagnalis, was studied using patch-clamp techniques. In the presence of a high concentration of intracellular Ca2+ buffer (5 mM EGTA), the inactivation of these Ca2+ channels is entirely voltage dependent; it is not influenced by the identity of the permeant divalent ions or the amount of extracellular Ca2+ influx, or reduced by higher levels of intracellular Ca2+ buffering. Inactivation measured under these conditions, despite being independent of Ca2+ influx, has a bell-shaped voltage dependence, which has often been considered a hallmark of Ca2+-dependent inactivation. Ca2+-dependent inactivation does occur in Lymnaea neurons, when the concentration of the intracellular Ca2+ buffer is lowered to 0.1 mM EGTA. However, the magnitude of Ca2+-dependent inactivation does not increase linearly with Ca2+ influx, but saturates for relatively small amounts of Ca2+ influx. Recovery from inactivation at negative potentials is biexponential and has the same time constants in the presence of different intracellular concentrations of EGTA. However, the amplitude of the slow component is selectively enhanced by a decrease in intracellular EGTA, thus slowing the overall rate of recovery. The ability of 5 mM EGTA to completely suppress Ca2+-dependent inactivation suggests that the Ca2+ binding site is at some distance from the channel protein itself. No evidence was found of a role for serine/threonine phosphorylation in Ca2+ channel inactivation. Cytochalasin B, a microfilament disrupter, was found to greatly enhance the amount of Ca2+ channel inactivation, but the involvement of actin filaments in this effect of cytochalasin B on Ca2+ channel inactivation could not be verified using other pharmacological compounds. Thus, the mechanism of Ca2+-dependent inactivation in these neurons remains unknown, but appears to differ from those proposed for mammalian L-type Ca2+ channels.
Key Words: molluscs, cytochalasin B, intracellular Ca2+, Ca2+ buffering
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INTRODUCTION |
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Inactivation of Ca2+ channels is thought to be of two typesvoltage-dependent inactivation, like that of Na+ channels (as originally proposed by
In this paper, we have studied the inactivation of Ca2+ channels in Lymnaea neurons. Ca2+-dependent inactivation in molluscan neurons has received considerable attention; it was in these neurons that this phenomenon was first characterized (
In this study, we show that Ca2+ channel inactivation in Lymnaea neurons has both Ca2+- and voltage-dependent components, and that both of these components have a bell-shaped voltage dependence. From the kinetics of the development of and the recovery from inactivation, we infer that there are two distinct inactivation states, even in the absence of Ca2+-dependent inactivation, and an increase in Ca2+ causes a greater occupancy of the longer-lived inactivation state. We find that while Ca2+-dependent inactivation is influenced by Ca2+ influx, its magnitude does not depend linearly on the magnitude of the influx, as was shown previously (
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MATERIALS AND METHODS |
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Cell Preparation and Electrophysiology
Neurons were dissociated from the pedal, parietal, and visceral ganglia of adult Lymnaea stagnalis, and prepared for patch clamp experiments as previously described ( and tip diameters of 1216 µm. Series resistance (usually ~24 M
) was electronically compensated to >90%. Inactivation measurements were taken at least 10 min after entering the whole-cell configuration, unless otherwise noted, to allow for the diffusion of the electrode solution into the cell. Junction potential errors (described in
Internal Perfusion Experiments
Internal perfusion experiments were done following the method described by
Solutions
The Lymnaea saline used for dissociation and storage of cells contains 50 mM NaCl, 2.5 mM KCl, 4 mM MgCl2, 4 mM CaCl2, 10 mM HEPES (N-[2-hydroxyethyl] piperazine-N'-[2-ethane sulfonic acid]), adjusted to pH 7.4 with NaOH. The standard extracellular saline used for recording Ca2+ or Ba2+ currents is composed of 76 mM TrisCl and 10 mM CaCl2 or BaCl2, and is adjusted to pH 7.4. In some experiments where the concentration of Ca2+ in the external solution is reduced to 1 mM, 9 mM MgCl2 is added to keep the total concentration of divalent ions constant. All of the intracellular solutions contain 50 mM HEPES, 0.5 mM MgCl2, 312 mM CsCl, 1520 mM aspartic acid, and 2 mM Mg-ATP, with varying amounts of calcium buffers, adjusted to pH 7.3 with CsOH, thus making Cs+ the main intracellular cation. The different levels of intracellular Ca2+ buffers used in this study are 0.1 mM EGTA, 5 mM EGTA, 5 mM EGTA with 2.5 mM CaCl2, 5 mM EGTA with 4.5 mM CaCl2, and 11 mM 1,2-bis(2-amino phenoxy) ethane-N, N, N', N'-tetraacetic acid (BAPTA) with 1 mM CaCl2. These solutions adjusted to various free Ca2+ levels were used for calibrating Fura-2.
H-7 [1-(5-isoquinolinylsulfonyl)-2-methyl piperazine], cyclosporin A, and colchicine are readily soluble in water and their stock solutions were made in distilled water. Although phalloidin is not highly soluble in water, its aqueous solubility is adequate to form a 5-mM stock solution in distilled water. The stock solution for okadaic acid (K+ salt) was only three times more concentrated than the final concentrations required and was made directly in the extracellular saline. Stock solutions for calmidazolium, cytochalasin B, and cytochalasin D were made in DMSO. Appropriate volumes of these stock solutions were then added to the external solution in the bath to bring the bath concentration of these compounds to the required levels.
Fura-2 Measurements of Free Ca2+ in Cells
Free Ca2+ levels in cells were measured by loading the cells with an intracellular solution containing 10 µM Fura-2, a Ca2+-sensitive ratiometric dye (
Measurement of Inactivation
In the studies reported in this paper, inactivation has been measured using mainly a three-pulse protocol (see Figure 1 A). First a short test pulse (10 ms) to +40 mV is applied, then a conditioning pulse (150 ms) of variable amplitude, followed by a gap (20 ms) at the holding potential (-60 mV), and, finally, a second test pulse to +40 mV is applied. The inactivation caused by the conditioning pulse is calculated as the percent reduction in the test pulse current after the conditioning pulse. In an earlier study (
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In some experiments, tail currents have been used to measure inactivation (e.g., see Figure 2 D, 4 C, and 5), using a protocol similar to the one described above. A short test pulse (3 ms) to +120 mV is applied before and after the conditioning pulse, and inactivation is calculated as the percent reduction in the tail current (measured at -40 mV) elicited by the termination of the test pulse. Tail currents typically reached their peak magnitude in 100 µs. We have previously shown (
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Analysis
Statistical analyses were performed using Systat software (Version 6; SPSS Inc.) and the corrected R2 parameter was used to determine the quality of fit of a model to the experimental data.
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RESULTS |
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Ca2+ Channel Inactivation in Cells Containing 5 mM EGTA Is Independent of Ca2+ Influx
Inactivation is measured using a three-pulse protocol (Figure 1 A). In this protocol, a short test pulse is applied before and after a conditioning pulse to variable potentials, and inactivation is measured as the percentage reduction in the test pulse current due to the conditioning pulse. There is a 20-ms gap at the holding potential between the conditioning pulse and the second test pulse, which is necessary to allow background currents activated during the conditioning pulse to completely deactivate. However, it also results in some Ca2+ channels recovering from inactivation during the gap. Thus, our measurement of inactivation relates to the fraction of Ca2+ channels that are still inactivated at the end of a 20-ms gap. Inactivation measured this way in cells containing a high level of intracellular Ca2+ buffer (5 mM EGTA) is relatively small (the peak inactivation being ~0.25), and exhibits a bell-shaped voltage dependence (Figure 1 B). We show below that the inactivation measured in these conditions is independent of Ca2+ influx.
A common test for the presence of Ca2+-dependent inactivation is to compare inactivation of Ca2+ currents with that of Ba2+ currents. The underlying assumption is that the intracellular site, which mediates Ca2+-dependent inactivation, is less sensitive to Ba2+ ions than to Ca2+ ions. Therefore, the current-dependent component of inactivation during Ba2+ influx would be reduced, as compared with that during Ca2+ influx. Exchanging external Ca2+ with Ba2+ does not cause a reduction in the levels of peak inactivation measured in Lymnaea neurons containing 5 mM EGTA (Figure 2 A), suggesting that there is very little Ca2+-dependent inactivation in this case. The leftward shift in the inactivation curve observed with external Ba2+ can be explained on the basis of the shift of Ba2+ current activation to potentials lower than those for Ca2+-current activation.
Ca2+-dependent inactivation also implies that inactivation should depend on the amount of Ca2+ influx. This is not true for Lymnaea neurons containing 5 mM EGTA. The standard extracellular solution in our experiments contains 10 mM Ca2+; reducing this concentration 10-fold to 1 mM does not cause a significant reduction in the amount of inactivation measured (Figure 2 B), even though the current magnitude decreases fourfold.
We also compared inactivation in cells dialyzed with a 5 mM EGTA solution to that in cells dialyzed with an 11 mM BAPTA solution. In the second case, not only is there a higher concentration of a Ca2+ buffer, but the buffer used (BAPTA) is also substantially faster. Consequently, the Ca2+ transients in cells with 11 mM BAPTA should be substantially smaller than those in cells with 5 mM EGTA (
Another way of assessing the contribution of Ca2+ influx in Ca2+-channel inactivation is to eliminate all Ca2+ influx, and measure the inactivation of the Ca2+ channel current carried by monovalents (
We conclude from the four types of experiments described above (Figure 2, AD) that Ca2+-channel inactivation in cells containing 5 mM EGTA is entirely voltage dependent and is independent of Ca2+ influx. Ca2+ channels in Lymnaea neurons are capable of exhibiting Ca2+-dependent inactivation when the intracellular Ca2+ buffering is lowered (shown below). Thus, we conclude that 5 mM EGTA reduces intracellular Ca2+ transients to a size where they are incapable of activating the site that mediates Ca2+-dependent inactivation.
Ca2+ Channel Inactivation in Cells Containing 0.1 mM EGTA Has a Current-dependent Component
To demonstrate that Ca2+ channels in Lymnaea neurons are capable of exhibiting Ca2+-dependent inactivation, we compared Ca2+-channel inactivation in two identical populations of neurons loaded with different amounts of intracellular Ca2+ buffer. Cells containing 0.1 mM EGTA show substantially more inactivation than those containing 5 mM EGTA (Figure 3 A), indicating that increased levels of intracellular Ca2+ lead to an increase in Ca2+ channel inactivation. This result was confirmed with internal perfusion experiments in which intracellular solutions were changed while recording from one cell. Inactivation was first measured when the cells were perfused with a solution containing 5 mM EGTA, and was found to increase if the intracellular solution was changed to one containing only 0.1 mM EGTA (Figure 3 B). In control experiments, in which the second intracellular solution perfused into the cells was the same as the first, inactivation was not affected by the exchange (data not shown).
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To ensure that it is indeed the Ca2+-buffering properties of EGTA and not some other pharmacological property that contributes to Ca2+-channel inactivation, we measured inactivation in cells where the intracellular solution contained 5 mM EGTA, loaded with different amounts of CaCl2 (Figure 3 C). Cells containing 5 mM EGTA alone show smaller magnitudes of inactivation than those in which 5 mM EGTA has been loaded with 2.5 mM CaCl2 or 4.5 mM CaCl2, suggesting that it is the concentration of free Ca2+ buffer that is important for determining the amount of inactivation. These solutions of a fixed amount of Ca2+ buffer and variable amounts of CaCl2 also contain slightly different levels of free Ca2+. But the free Ca2+ levels in all these solutions are low (<10-6 M) and do not appear to determine the amount of Ca2+-dependent inactivation (Figure 3 D, discussed later).
The increased Ca2+ channel inactivation in Lymnaea neurons containing 0.1 mM EGTA is dependent upon Ca2+ influx. In these cells, exchange of an external Ca2+-containing solution with a Ba2+-containing solution causes a significant decline in peak inactivation (Figure 4 A), even though the magnitude of Ba2+ current is two to three times larger than that of Ca2+ current. Similarly, changing the external Ca2+ concentration from 10 to 1 mM also leads to a decrease in the total Ca2+-channel inactivation measured in cells containing 0.1 mM EGTA (Figure 4 B). Furthermore, 100 µM Cd2+ in the extracellular solution causes a substantial decrease in inactivation measured in cells containing 0.1 mM EGTA (Figure 4 C); the remaining inactivation is not significantly different from that in cells with 5 mM EGTA under similar conditions (Figure 2 D).
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From these experiments, we conclude that Lymnaea neurons containing 0.1 mM EGTA have both Ca2+- and voltage-dependent components, while those with 5 mM EGTA exhibit only the voltage-dependent component of Ca2+ channel inactivation. For the purposes of this study, we define Ca2+-dependent inactivation in these cells as the difference in inactivation observed with cells containing 0.1 and 5 mM EGTA.
Recovery from and Development of Inactivation
We measured the rates with which the Ca2+ channels recover from inactivation in cells containing 5 or 0.1 mM EGTA. This was done by varying the lengths of the gap after the conditioning pulse in a protocol that measures inactivation using tail currents (see MATERIALS AND METHODS). Using this protocol, we find that the recovery of Ca2+ channels from inactivation has a biexponential time course at -60 mV (fast = 15 ms,
slow = 600 ms; Figure 5 A). After a conditioning pulse to +120 mV, the rate of recovery in 0.1 mM EGTA is not substantially different from that in 5 mM EGTA. However, after conditioning pulses to +40 and +60 mV (which cause maximal inactivation) the slow component of recovery is much larger for 0.1 mM EGTA compared with that for 5 mM EGTA. This is accompanied by a modest decrease in the magnitude of the fast component of recovery in 0.1 mM EGTA. The fast component of recovery decays rapidly in the first 20 ms; thus, most of the difference observed between inactivation measured in 0.1 and 5 mM EGTA measured using the 20-ms gap (as in Figure 3 A) is due to the differences in the magnitude of the slow component of recovery in the two conditions. The two separate time constants for the rate of recovery from inactivation indicate that there are two different inactivated states from which the channels are recoveringat negative potentials, recovery from one inactivated state takes place at a considerably faster rate than from the other inactivated state. The effect of Ca2+ is to increase the occupancy of the latter state.
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We also measured the development of inactivation during conditioning pulses to +60 and +120 mV. The inactivation measurements were made after allowing the channels to recover for 20 ms after conditioning pulses of variable length, and thus are not independent of the recovery rates. During a pulse to +120 mV, inactivation develops with a single time constant of 250 ms in both 0.1 and 5 mM EGTA (Figure 5 B). However, when the conditioning pulse is to +60 mV, there is an additional faster component ( = 50 ms), and the amplitude of this component is three times as large in 0.1 as in 5 mM EGTA. These experiments also indicate that the Ca2+ influx during the tail currents at the end of the conditioning pulse does not contribute significantly towards inactivation, since inactivation approaches zero as the conditioning pulse becomes very short. In the DISCUSSION, we develop a model of Ca2+-induced inactivation that can account for our observations regarding the kinetics of inactivation in 5 and 0.1 mM EGTA.
Ca2+-dependent Inactivation Does Not Depend Linearly on Ca2+ Influx
We have shown that for cells containing 0.1 mM EGTA, the Ca2+-dependent component of inactivation can be reduced by reducing Ca2+ influx (Figure 4). The magnitude of Ca2+-dependent inactivation, however, is not linearly related to the amount of Ca2+ influx. This conclusion is demonstrated by the experiment in which the Ca2+ channel blocker Co2+ was used to reduce the influx of Ca2+ during conditioning pulses. This experiment is analogous to the one described above using Cd2+ to block the Ca2+ current, but Co2+ is a weaker Ca2+ channel blocker than Cd2+, and exerts a simpler, nonvoltage-dependent block of current (
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Our conclusion that the amount of Ca2+-dependent inactivation is not simply related to the magnitude of Ca2+ influx during the conditioning pulse is also supported by other observations. In cells perfused with 0.1 mM EGTA, the decrease in Ca2+ current over time (due to rundown) is not accompanied by any significant changes in inactivation. Also, there is no obvious correlation between the peak inactivation and the current density measured in the 53 cells containing 0.1 mM EGTA that we studied (Figure 6 B). It should be noted, however, that peak inactivation is the sum of voltage- and Ca2+-dependent inactivation. It is possible that in the absence of a strong dependence of Ca2+-dependent inactivation on Ca2+ current density, the random variation in voltage-dependent inactivation obscures any weaker correlation between the two quantities in Figure 6 B.
Intracellular Sources of Calcium Do Not Contribute Towards Ca2+-dependent Inactivation
Effects of basal Ca2+ levels on inactivation.
We investigated the possibility that the basal (i.e., steady state) levels of Ca2+ within the cell may affect Ca2+-channel inactivation. Fura-2 measurements indicated that steady state Ca2+ levels in cells containing 0.1 mM EGTA are much higher (70300 nM) than those in cells containing 5 mM EGTA (520 nM). This difference occurs despite the fact that the two intracellular solutions, containing 5 and 0.1 mM EGTA, respectively, have similar low levels of free Ca2+ (210 nM, according to Fura-2 measurements in microcuvettes). The increase in free Ca2+ levels in cells perfused with a poorly buffered solution is probably due to a high rate of Ca2+ influx through the plasma membrane. Thus, it is possible that increased Ca2+-channel inactivation observed in cells with 0.1 mM EGTA results from higher basal levels of free Ca2+ inside the cells.
To examine whether differences in basal Ca2+ levels can account for the variations in inactivation measured in different cells, we measured free Ca2+ levels (using Fura-2) and inactivation in cells perfused with an intracellular solution containing either 0.1 mM EGTA or 5 mM EGTA /2.5 mM Ca2+. These two intracellular solutions were chosen since they result in comparable values of free intracellular Ca2+ levels, but have very different Ca2+-buffering capacities. We find that while cells with 0.1 mM EGTA consistently show more inactivation than cells with 5 mM EGTA/2.5 mM Ca2+, there is no correlation between peak inactivation and steady state Ca2+ levels for the 0.1 mM EGTA data (R2 = 0.021, Figure 3 D). This leads us to believe that the site that mediates Ca2+-dependent inactivation is not sensitive to resting levels of Ca2+ (300 nM); instead, intracellular domains of high Ca2+ that are transiently set up when Ca2+ channels are activated must be mediating Ca2+-channel inactivation.
Intracellular sources of calcium ions do not contribute to Ca2+-dependent inactivation.
Recent studies have shown that, in ventricular myocytes, an influx of Ca2+ through voltage-gated Ca2+ channels triggers a release of Ca2+ from the sarcoplasmic reticulum, and it is this release of Ca2+ from intracellular stores that is largely responsible for Ca2+ channel inactivation in these cells (
Intracellular Proteins Involved In Ca2+-dependent Inactivation
Different mechanisms for Ca2+-dependent inactivation have been proposed in the literature. Some researchers have concluded that Ca2+ may bind directly to the Ca2+ channel (
Serine-threonine phosphorylation is not involved in Ca2+-channel inactivation.
It has been proposed that an increase in cytoplasmic Ca2+ levels may lead to an activation of a Ca2+-dependent phosphatase (or a kinase) that may alter the phosphorylation state of the Ca2+ channel leading to an increase in inactivation (
Cytochalasin B greatly enhances inactivation of Ca2+ channels.
Previous experiments done in our lab have shown that the rundown process of Ca2+ channels in giant inside-out patches is accelerated by the disruption of cytoskeleton (). The effect of cytochalasin B is selective for inactivation, since it has very little effect on the magnitude of Ca2+ current (Figure 7 B,
), and its rate of rundown. Furthermore, the recovery of Ca2+ channels from inactivation is also much slower in the presence of cytochalasin B, primarily because of the increased amplitude of the slow component of recovery (Figure 7 C), which is similar to the effect of reducing intracellular EGTA concentration. Surprisingly, the effect of cytochalasin B is readily reversible; the Ca2+-channel inactivation returns to its precytochalasin B levels after a few minutes of perfusion with the control external saline.
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To quantify the effect of cytochalasin B on inactivation, we calculated an f parameter, the fractional increase in inactivation. The f parameter is defined as:
where I is the change in inactivation upon the addition of cytochalasin B, and
Imax is the maximal possible change in inactivation. We chose f, calculated for a conditioning pulse to +40 mV, f40 mV, to measure the effect of different concentrations of cytochalasin B upon inactivation in cells with 5 mM EGTA, and obtain the doseresponse curve. The concentration of cytochalasin B that causes half the maximal effect on inactivation is ~100 µM. The effect of cytochalasin B in increasing inactivation in cells with 5 mM EGTA was compared with that for cells with 0.1 mM EGTA. Surprisingly, f40mV, which is measured at +40 mV, where Ca2+ influx is maximal, is not significantly different in the two cases (Figure 8), suggesting that the effect of cytochalasin B in increasing inactivation is independent of the Ca2+-dependent inactivation.
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While cytochalasin B is known to disrupt the actin cytoskeleton (
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DISCUSSION |
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In this study, we have characterized the Ca2+ and voltage-dependent inactivation of Ca2+ channels in Lymnaea neurons. Many of the earliest studies describing Ca2+-dependent inactivation of Ca2+ channels were done in molluscan neurons (
Much of the recent work in Ca2+-channel inactivation has focussed on the study of recombinant channels expressed in heterologous systems (
Bell-shaped Inactivation Curve in the Absence of Ca2+-dependent Inactivation
Ca2+-channel inactivation in Lymnaea neurons perfused with 5 mM EGTA solution is not affected by replacing external Ca2+ by Ba2+ (Figure 2 A), reducing the Ca2+ influx (Figure 2B and Figure D), or by increasing the level of intracellular Ca2+ buffering (Figure 2 C). We conclude from this that Ca2+ channels in Lymnaea neurons containing 5 mM EGTA exhibit only voltage-dependent inactivation, even though the inactivation curve in 5 mM EGTA is bell shaped. A bell-shaped inactivation curve has often been taken as evidence for the presence of Ca2+-dependent inactivation, though previous studies of native Ca2+ channels in bullfrog sympathetic neurons (
Kinetics of Ca2+-channel Inactivation
We show that increased intracellular Ca2+ concentration increases the time that Ca2+ channels require to recover from inactivation. The effect of Ca2+ in inhibiting the recovery of channels from inactivation at negative potentials has been observed before (
It is more difficult to interpret our results pertaining to the development of inactivation (shown in Figure 5 B) since the measurements of inactivation are dependent on the rate of recovery. To explain these observations, we have developed a simple model of the inactivation kinetics of Ca2+ channels at different voltages that fits the data shown in Figure 5A and Figure B (the model is shown in Figure 9, with rate constants at three different voltages given in Table 1). In this model, there are two inactivated states, IFR and ISR, that can be reached from the noninactivated states (which have been lumped together as NI). At negative potentials, recovery from IFR is considerably faster than that from ISR. Ca2+ influx during a conditioning pulse increases the occupancy of the inactivated state ISR, and thus increases the amplitude of the slow component of recovery at negative potentials. Our data can be fit by making only the forward rate constant (SR) Ca2+ dependent, but we cannot rule out the possibility that the reverse rate constant (ßSR) may also be dependent on Ca2+ influx. After repolarization, IFR is depleted rapidly, while ISR is not greatly affected within the first 20 ms. Hence, the difference in inactivation that we measure between 0.1 and 5 mM EGTA with the three-pulse protocol is largely the difference in the ISR components under the two conditions. Also, the model predicts that the actual difference in inactivation between 0.1 and 5 mM EGTA (Figure 9 B, continuous curves) during the course of a conditioning pulse is smaller than the difference we measure at the end of the 20-ms gap (Figure 9 B, points and dashed curves). This is so because the proportion of channels in IFR is larger in 5 than in 0.1 mM EGTA; however, the difference measured after a 20-ms gap reflects primarily the difference in the ISR components in the two cases.
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Since inactivation in 5 mM EGTA is completely voltage dependent, these results imply that SR is not zero at positive voltages even in 5 mM EGTA, and a conditioning pulse to +60 mV causes some occupancy of ISR.
In this analysis, we have assumed that there is only one class of Ca2+ channels in Lymnaea neurons. Pharmacological and kinetic studies done in our lab have failed to resolve more than one component of Ca2+ current, though multiple types of Ca2+ channels cannot be ruled out. In such a case, is it possible that Ca2+- and voltage-dependent inactivation are due to different channel types that gate independently of each other? The results from our kinetic analyses (Figure 5 A) show that the magnitude of voltage-dependent inactivation is reduced in the presence of Ca2+-dependent inactivation (since the fast component of recovery is smaller in 0.1 than in 5 mM EGTA), which indicates that these phenomena are not independent of each other. It is, therefore, unlikely that different channel types underlie voltage- and Ca2+-dependent inactivation.
Saturation of Ca2+-dependent Inactivation
Our results with the Ca2+ channel blocker Co2+ indicate that just half of the Ca2+ influx under standard conditions may be adequate to almost saturate the Ca2+-dependent component of inactivation (Figure 6 A). To illustrate the relation between Ca2+-dependent inactivation and Ca2+ influx, we plotted Ca2+-dependent inactivation against Ca2+ influx for a typical cell in Figure 10. Figure 10 (, connected by a continuous line) shows the relation between these two quantities for each of the conditioning pulse potentials with 10 mM external Ca2+. The general shape of these points shows that the Ca2+-dependent inactivation is not linearly related to the Ca2+ influx, and that Ca2+ influx during pulses from +30 to +80 mV (with 10 mM external Ca2+) causes considerable saturation of the Ca2+-dependent component of inactivation. The shape of this curve is in good agreement with the 20% reduction in peak Ca2+-dependent inactivation that is caused by a 50% reduction in Ca2+ influx with 1 mM Co2+ (Figure 10,
). The result that a fourfold reduction in Ca2+ influx, caused by reducing extracellular Ca2+ from 10 to 1 mM, only blocks half of the peak Ca2+-dependent inactivation also fits this same relationship (Figure 10,
).
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Mechanisms of Ca2+-dependent Inactivation
Several researchers have proposed that Ca2+ may cause inactivation by binding to a site on the channel itself (
Studies on vertebrate L-type Ca2+ channels have led several researchers to conclude that Ca2+-dependent inactivation in these channels is caused by Ca2+-ion binding directly to the channel or to a site closely associated with it (1C subunit of the Ca2+ channel that controls the Ca2+-dependent inactivation of these channels (
1C subunit suggest that the channel is stably complexed with calmodulin, and it is the binding of Ca2+ ions to calmodulin that causes inactivation of the Ca2+ channel (
Effect of Cytochalasin B on Ca2+-channel Inactivation
Several studies have shown that voltage-gated Ca2+ channels are sensitive to the state of the cytoskeleton (
In this study, we have shown that cytochalasin B, an actin microfilament disrupter, increases the inactivation of Ca2+ channels; however, there is some suggestion in our results that actin filaments may not be involved in this effect of cytochalasin B on inactivation. First, the concentration of cytochalasin B that results in half-maximal increase in inactivation is ~100 µM, which is very high for an effect on actin filaments. Second, cytochalasin D does not yield the same affect as cytochalasin B (Figure 8). Also, cytochalasin B applied intracellularly does not increase the amount of inactivation measured, and phalloidin applied intracellularly cannot block the effect of extracellularly applied cytochalasin B (Figure 8). (Although, in the last two instances, it is questionable whether these drugs can effectively diffuse through the cytoplasm to reach the cytoskeleton close to the membrane.) Additionally, the time course of the onset of the cytochalasin B effect is very fast, limited only by the delay in application, and the effect is readily and completely reversible, inactivation returning to its original levels within minutes of perfusion with the control solution. This suggests that cytochalasin B may have only an extracellular effect.
The mechanism by which cytochalasin B increases inactivation currently remains unresolved. Cytochalasin B is known to inhibit the glucose transporter (
These studies leave unknown the mechanisms of Ca2+ channel inactivation in Lymnaea neurons. While we conclude that serine/threonine phosphorylation does not play any role in Ca2+ channel inactivation, it is possible that tyrosine phosphorylation may be involved. Indeed, tyrosine phosphorylation has been shown to modulate excision-activated Ca2+ channels in Lymnaea (1 subunits in a Ca2+-dependent manner (
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Acknowledgements |
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We thank Dr. John P. Walsh and Dr. Barry D. Johnson for helpful comments on an early version of this manuscript.
This work was supported by the National Institutes of Health grant NS-28484.
Submitted: 28 January 1999
Revised: 28 July 1999
Accepted: 28 July 1999
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