Cross-coupling between voltage-dependent Ca²⁺ channels and ryanodine receptors in developing ascidian muscle blastomeres

Abstract

1. Ascidian blastomeres of muscle lineage express voltage-dependent calcium channels (VDCCs) despite isolation and cleavage arrest. Taking advantage of these large developing cells, developmental changes in functional relations between VDCC currents and intracellular Ca²⁺ stores were studied.

2. Inactivation of ascidian VDCCs is Ca²⁺ dependent, as demonstrated by two pieces of evidence: (1) a bell-shaped relationship between prepulse voltage and amplitude during the test pulse in Ca²⁺, but not in Ba²⁺, and (2) the decay kinetics of Ca²⁺ currents (I_Ca) obtained as the size of tail currents.

3. During replacement in the external solution of Ca²⁺ with Ba²⁺, the inward current appeared biphasic: it showed rapid decay followed by recovery and slow decay. This current profile was most evident in the mixed bath solution (2% Ca²⁺ and 98% Ba²⁺, abbreviated to ‘2Ca/98Ba’).

4. The biphasic profile of I_2Ca/98Ba was significantly attenuated in caffeine and in ryanodine, indicating that Ca²⁺ release is involved in shaping the current kinetics of VDCCs. After washing out the caffeine, the biphasic pattern was reproducibly restored by depolarizing the membrane in calcium-rich solution, which is expected to refill the internal Ca²⁺ stores.

5. The inhibitors of endoplasmic reticulum (ER) Ca²⁺-ATPase (SERCAs) cyclopiazonic acid (CPA) and thapsigargin facilitated elimination of the biphasic profile with repetitive depolarization.

6. At a stage earlier than 36 h after fertilization, the biphasic profile of I_2Ca/98Ba was not observed. However, caffeine induced a remarkable decrease in the amplitude of I_2Ca/98Ba and this suppression was blocked by microinjection of the Ca²⁺ chelator BAPTA, showing the presence of caffeine-sensitive Ca²⁺ stores at this stage.

7. Electron microscopic observation shows that sarcoplasmic membranes (SR) arrange closer to the sarcolemma with maturation, suggesting that the formation of the ultrastructural machinery underlies development of the cross-coupling between VDCCs and Ca²⁺ stores.

The process of excitation-contraction (E-C) coupling in vertebrate cardiac muscle depends on calcium-mediated coupling between ryanodine receptors on the sarcoplasmic reticulum (SR) and voltage-dependent calcium channels (VDCCs) on the sarcoplasmic membrane (Fabiato, 1983). Ca²⁺ influxes through VDCCs activate the opening of intracellular Ca²⁺ release channels, leading to Ca²⁺ release into the cytoplasm. This mechanism, called Ca²⁺-induced Ca²⁺ release (CICR), amplifies Ca²⁺ signals and enables a quick and potent rise of intracellular Ca²⁺ ([Ca²⁺]_i). On the other hand, Ca²⁺ release channels control the activity of VDCCs via Ca²⁺-mediated inactivation as a negative feedback loop. Such positive and negative interactions between ryanodine receptors and VDCCs through local changes in [Ca²⁺]_i underlie regenerative but graded E-C coupling. Similar properties have been described in crayfish muscle (Györke & Palade, 1992), suggesting that Ca²⁺-mediated E-C coupling is a common feature of invertebrate striated muscles and vertebrate cardiac muscles. However, little is known about how interactions between VDCCs and ryanodine receptors are established during embryogenesis, in particular, during the transition from the undifferentiated state to the mature state. This is, in part, due to changes in morphology and continuous cell division of immature muscle cells during the early embryonic stage. To circumvent these problems, we took advantage of a sessile marine chordate of the class Ascidiacea.

The ascidian larva is phylogenetically related to the vertebrates, as demonstrated by its body organization (Katz et al. 1983), recent identification of genes involved in early embryogenesis (Satoh et al. 1996) and amino acid sequences of various proteins for cell functions, including contractile proteins (MacLean et al. 1997) and ion channels (Okamura et al. 1994). The ascidian larval tail contains striated muscle cells which show an alternating contraction pattern during swimming (Mackie & Bone, 1977). The ascidian tail muscle cells show significant similarities to vertebrate cardiac muscle cells in several respects. First, ascidian larval muscle cells are mononuclear and connected with each other via gap junctions (Katz et al. 1983; Bone, 1989). Second, structures similar to the peripheral couplings, diads and feet of vertebrate cardiac muscle have been described (Cavey & Cloney, 1972; Schiaffino et al. 1976). Third, we have recently isolated an ascidian voltage-gated calcium channel cDNA which shows a higher homology to the vertebrate cardiac-type calcium channel, α_1C, than to the skeletal muscle calcium channel, α_1S (Y. Okamura & K. Nakajo, unpublished data). Analysis of the phylogenetic tree of Ca²⁺ channels from various species showed that the ascidian VDCC gene seems closely related to a common ancestor of vertebrate L-type Ca²⁺ channels. This gene is expressed in muscle cells and neuronal cells in ascidian larva. These similarities strongly suggest that essential mechanisms of E-C coupling in vertebrate cardiac muscle may be evolutionarily conserved in ascidian muscles.

Ascidians have frequently been used for studies on developmental biology (Conklin, 1905), because the developmental fate of each blastomere is precisely assigned early in embryogenesis (Satoh, 1994). In a short developmental period, isolated ascidian blastomeres express sets of proteins corresponding to their cell fates despite cleavage arrest (Whittaker, 1973; Takahashi & Yoshii, 1981; Hirano et al. 1984). Because of their large size and simple shape, these cells are suitable for stable recording of ion channel currents from the early stages of development (Takahashi & Okamura, 1998). Cleavage-arrested Halocynthia blastomeres of muscle lineage show phenotypes specific to muscle cells, including Ca²⁺-dependent action potentials similar to muscle potentials, an acetylcholine response and ultrastructural formation of myofilaments (Hirano et al. 1984).

Using such ascidian cleavage-arrested blastomeres of muscle cell lineage, we studied two aspects of CICR. First, we tested ascidian muscle cells for cross-coupling between ryanodine receptors and VDCCs similar to that in vertebrate cardiac cells. Interestingly, VDCCs from ascidian muscle cells showed an anomalous biphasic profile of the inward current in a mixture of Ba²⁺ and Ca²⁺, which was dependent on Ca²⁺ release from caffeine- and ryanodine-sensitive Ca²⁺ stores. This provided evidence that both Ca²⁺ release activated by Ca²⁺ influx via opening of VDCCs and inhibition of VDCC activity by Ca²⁺ release occur in large cleavage-arrested ascidian blastomeres, and also suggested that formation of complexes containing VDCCs and Ca²⁺ release channels does not require an actin-based cytoskeleton. Second, we took advantage of cleavage-arrested blastomeres to examine developmental changes in this cross-coupling, by utilizing the biphasic current profile of VDCCs as a marker of interaction with ryanodine receptors. We found that caffeine-sensitive stores were already present in muscle at early stages when the current amplitude of VDCCs was not large, and that, as development progressed, the proximity between calcium channels and internal calcium stores increased.

METHODS

Cell preparation

Embryos were obtained from Halocynthia roretzi collected in northern Japan from November to January. They were maintained in sterilized, circulating sea water at 5°C, which ensured that eggs and sperm were not released until July. For use in this study, the animals were transferred into a spawning aquarium maintained at 10°C. They typically spawned eggs and sperm within 2 days. Experiments were performed from November to June.

Cleavage-arrested embryos were prepared as previously described (Okamura & Shidara, 1990). The large posterior-vegetal blastomere B_5.1 from each cleavage-arrested 16-cell embryo was used because it is predetermined to differentiate into a muscle cell (Nishida & Satoh, 1985) and consistently differentiates into a muscle cell in isolation (Shidara & Okamura, 1991). Isolated blastomeres were cultured at 10–11°C in seawater containing 2.0 μg ml⁻¹ cytochalasin B (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) for 1–2 h to arrest cleavage completely and then cultured in seawater containing 0.3 μg ml⁻¹ cytochalasin B and ascidian egg extract as previously reported (Nishida, 1987). The egg extract enhanced the expression of ion channels, but did not affect cell fate. Untreated sister embryos from the same batch hatched as larvae at about 48 h after fertilization. In some experiments, neuronal blastomeres were also used as a reference for functional coupling of VDCCs with Ca²⁺ stores, because they express the same class VDCCs as in muscle cells but do not have well developed caffeine-sensitive Ca²⁺ stores. Neuronal blastomeres were treated as previously described (Okamura & Takahashi, 1993).

Electrophysiological recordings

The VDCC currents of cleavage-arrested blastomeres were recorded with the two-microelectrode voltage clamp method using Axoclamp-2B (Axon Instruments), as previously reported (Okamoto et al. 1976; Okamura & Shidara, 1990). The following were used as external solutions. The calcium-rich solution contained (mM): 100 CaCl₂, 200 tetraethylammonium chloride (TEACl), 200 tetramethylammonium chloride (TMACl), 10 KCl and 5 Pipes, (pH 7.0); the barium-rich solution contained (mM): 100 BaCl₂, 200 TEACl, 200 TMACl, 10 KCl and 5 Pipes (pH 7.0); the 2 % Ca²⁺-98 % Ba²⁺ (2Ca/98Ba) solution contained (mM): 2 CaCl₂, 98 BaCl₂, 200 TEACl, 200 TMACl, 10 KCl and 5 Pipes (pH 7.0). Microelectrodes containing 3 M KCl and 5 mM EGTA with a resistance of 7–12 MΩ were used for recording membrane potential. Microelectrodes with 2–5 MΩ resistance filled with 1 M CsCl and 5 mM EGTA were used to inject current. Most outward K⁺ currents were suppressed by external TEA and Ba²⁺ and intracellular diffusion of Cs⁺ and EGTA from the current-passing electrode. In some experiments, however, we injected CsCl and EGTA or BAPTA from the current-passing microelectrodes by air pressure. Pressure injection reduced Ca²⁺ channel currents significantly (by 20 −60 %) in fully differentiated muscle blastomeres. This was probably due to cell damage by positive pressure. Therefore, we avoided using this manipulation in most fully differentiated blastomeres. For the experiments shown in Fig. 13, EGTA in the current electrode was replaced with 5 or 50 mM BAPTA. In early blastomeres, which do not express much outward potassium channel current, 3 M KCl and 5 mM EGTA were used in the current electrodes instead. The reference electrode was in contact with the bath through an agar bridge containing 3 M KCl.

Figure 13. I_2Ca/98Ba and caffeine sensitivity at the early developmental stage.

A, representative traces of I_2Ca/98Ba from the muscle blastomere at 28 h after fertilization. The blastomere was activated by depolarizing pulses to test potentials between −30 and +10 mV, given once every 10 s in 10 mV increments. B, the change in current amplitude of the 28 h muscle blastomere during application of 1 mM caffeine (bar) in the recording bath. C, effect of BAPTA injected during caffeine application on the current amplitude. A 28 h muscle blastomere was repetitively depolarized to 0 mV every 30 s. Then the blastomere was exposed to 1 mM caffeine and injected with BAPTA. The concentration of BAPTA in the current-recording pipette was 50 mM. D, changes in the current amplitude of the cell shown in C. Arrow indicates the timing of pressure injection.

For all recordings, the holding potential was −80 mV and the recordings were performed at 9–11°C. Data were filtered at 3.3 kHz and acquired at 10 kHz or 20–100 kHz (for tail current) using pCLAMP software (Axon Instruments). Data were analysed with IGOR Pro software (Wave Metrics, Inc., Lake Oswego, OR, USA) on a Macintosh personal computer.

Drugs

Intracellular injections of BAPTA and EGTA were performed by using air pressure pulses through the current-injecting microelectrode for voltage clamping. Caffeine (Sigma Chemical Co., St Louis, MO, USA) was dissolved in water to make a 100 mM stock solution. Cyclopiazonic acid (CPA; Sigma Chemical Co.) was dissolved in methanol to make a 10 mM stock solution. Ryanodine (Wako Pure Chemical Industries, Osaka, Japan) was dissolved in dimethylsulfoxide (DMSO) to make a 10 mM stock solution. Thapsigargin (Calbiochem, San Diego, CA, USA) was dissolved in DMSO to make a 1 mM stock solution. Cytochalasin B was dissolved in DMSO to make a 2 mg ml⁻¹ stock solution. These drugs were diluted in the recording solution or seawater just before use.

Electron microscopy

Embryos at 28, 48 and 72 h after fertilization were fixed with 2.5 % glutaraldehyde and 0.1 M cacodylate buffer (pH 7.4) for 2 h. The 72 h larvae were anaesthetized in seawater on ice for about an hour before fixation. They were then fixed with 1 % OsO₄, 0.5 % potassium ferrocyanide and 0.1 M cacodylate buffer for 1 h, dehydrated through a graded ethanol series, and embedded in Epon 812 (TAAB Laboratories Equipment Ltd, Berks, UK) following a standard protocol. Ultrathin sections (about 100 nm in thickness) were cut with an ultramicrotome and double-stained with 5 % uranium acetate and 0.1 % lead citrate.

Pictures were scanned into a personal computer by an image scanner. Areas of intracellular membranes were measured by NIH image software. All the membrane structures except mitochondrial membranes were taken as endoplasmic reticulum (ER). The ratio of these areas against a total area of cytoplasm was calculated.

RESULTS

Currents from muscle blastomeres show Ca²⁺-dependent inactivation

Figure 1A shows representative traces of currents recorded in calcium (I_Ca) and in barium (I_Ba) from differentiated muscle blastomeres at 54 h after fertilization. This stage corresponds to the stage when most of the control embryos from the same batch had hatched. The I_Ca showed more rapid decay than the I_Ba, which is consistent with Ca²⁺-dependent inactivation. Normalized current amplitude plotted against the membrane potential (Fig. 1B) shows that the current-voltage relation is shifted leftward by about 10 mV for I_Ba, probably reflecting the fact that Ba²⁺ is less efficient than Ca²⁺ at cancelling surface charge, which is one of the common properties of VDCCs (Ohmori & Sasaki, 1977).

Figure 1. Comparison of I_Ca and I_Ba from the same muscle blastomere.

A, representative traces of I_Ca and I_Ba from the same muscle blastomere. At 54 h after fertilization, the blastomere was activated with depolarizing pulses (400 ms) to test potentials between −40 and +10 mV, given once every 10 s in 10 mV increments. Top: Ca²⁺-containing solution. Bottom: Ba²⁺-containing solution. B, normalized current-voltage (I-V) relations of I_Ca (^; n = 4) and I_Ba (•; n = 5). Data are expressed as means ±s.e.m.

To examine the inactivation of VDCCs in more detail, I_Ca and I_Ba were recorded with the two-pulse protocol (Fig. 2A): the current amplitude during the fixed test pulse was plotted against the current amplitude during the prepulse to a variable potential level (Brehm & Eckert, 1978). In this experiment, the current amplitude of the inward current with a larger prepulse could be slightly underestimated, since some outward K⁺ currents contaminate the VDCC currents in the Ca²⁺ solution. In the calcium-rich solution, however, when I-V relations were examined, the I_Ca test pulse currents had the smallest amplitude when the pre-pulse I_Ca had the largest amplitude, showing a bell-shaped profile. The Ca²⁺ currents were sensitive to low activation during prepulse: even a prepulse to −40 mV, which gives less than 3 % of the maximum inward current, can decrease the current amplitude during the test pulse (Fig. 2B). In contrast to the profile in the calcium-rich solution, only a minor decrease in test pulse I_Ba was found with increased depolarization of the prepulse, probably due to voltage-dependent inactivation. This difference in I-V profile between Ca²⁺ and Ba²⁺ solutions is consistent with the idea of Ca²⁺-dependent inactivation of the ascidian muscle VDCCs.

Figure 2. Evidence for Ca²⁺-dependent inactivation of ascidian VDCCs.

A, representative traces of I_Ca (left) and I_Ba (right) using with the two-pulse protocol. The muscle blastomere was activated with prepulses to test potentials between −120 and +90 mV in 10 mV increments followed by a test pulse to +15 mV every 10 s. Left traces recorded in Ca²⁺-containing solution: trace 1 at −120 mV; trace 2 at +20 mV; trace 3 at +90 mV. Right traces recorded in Ba²⁺-containing solution: trace 4 at −120 mV; trace 5 at 0 mV; trace 6 at +90 mV. B, current-voltage (I-V) relations of I_Ca (left; n = 5) and I_Ba (right; n = 4): normalized currents at prepulse (^) and at test pulse (•) versus prepulse voltage. Data are expressed as means ±s.e.m.C, normalized tail currents of I_Ca recorded from a muscle blastomere. Tail currents of +8 mV (•), +17 mV (▪), +30 mV (^), and +48 mV (□) are plotted from 6 to 60 ms in 6 ms increments. Values of tail currents were normalized by the value at 6 ms of each test potential. Representative trace at +8 mV (12 ms) and fitted curves (thick curved line) are shown in the inset.

Another important condition of Ca²⁺-dependent inactivation is that the decay kinetics become slower with greater depolarization, which is the opposite of the profile expected from a voltage-dependent inactivation (Gutnick et al. 1989). However, in the differentiated muscle blastomeres, as depolarization increased, Ca²⁺ currents overlapped with outward K⁺ channel currents, making such a test difficult. To avoid this problem, tail currents of I_Ca were recorded from muscle blastomeres by applying test pulses of variable duration to a fixed potential (Fig. 2C). Tail currents were fitted with a single exponential and the current amplitude at the start time of repolarization was plotted against the duration of depolarization. The kinetics of the VDCC currents, obtained as the tail current amplitude with time of test pulse were slower at +48 mV than at +8 mV, fulfilling the above condition.

The biphasic profile of VDCC current in mixed Ca²⁺-Ba²⁺ solutions

In recording I_Ca and I_Ba from muscle blastomeres, we observed an unexpected change in the inward currents. When the external solution was changed from Ca²⁺ to Ba²⁺, the inward current gradually increased by at least 2-fold and by as much as 5-fold and the decay kinetics became slower, events consistent with the general properties of L-type calcium channels of many animals. However, just before the current amplitude reached a steady state, the muscle I_Ca showed an anomalous profile which deviated from an exponentially increasing current shape (Fig. 3). The inward current attained a peak between 10 and 20 ms, then declined between 20 and 80 ms, and finally slowly increased. The profile had a notch between an initial sharp peak and a later broad peak. This notch was especially prominent between −30 and 0 mV (Fig. 3, left). The notch attenuated at more depolarizing membrane potentials, particularly at levels more positive than the potential level which gave the maximum current amplitude (Fig. 3, right). The decay phase following the second peak had a time course similar to the decay phase of I_Ba, suggesting that voltage-dependent inactivation underlies this slow decay.

Figure 3. Anomalous biphasic profile of inward currents during solution exchange from Ca²⁺ to Ba²⁺.

Representative traces of I_2Ca/98Ba. The blastomere was activated by depolarizing pulses (400 ms) to test potentials between −60 and +90 mV, given once every 10 s in 10 mV increments in the 2Ca/98Ba solution. The traces shown here are divided into two groups, from −60 to 0 mV and from +10 to +90 mV.

This anomalous current profile was most prominently observed just before solution exchange was complete. To identify conditions which give the most sharply defined biphasic profile of inward current, we measured Ca²⁺ channel currents by systematically changing the ratios of Ca²⁺ and Ba²⁺ with a constant total concentration of 100 mM (Fig. 4A). In the solution containing 2 % (2 mM) Ca²⁺ and 98 % (98 mM) Ba²⁺ (abbreviated to ‘2Ca/98Ba’ solution), the VDCC current of the muscle blastomere appeared most sharply biphasic (upper right). Similar results were obtained in all the cells tested (n = 5). This was not due to uncontrolled voltage. In fact, this pattern was not observed in the 100 % Ba²⁺ solution in which current amplitude equalled that in the 2Ca/98Ba solution. All the differentiated muscle blastomeres showed the biphasic profile in the 2Ca/98Ba solution. In contrast, currents from neuronal blastomeres never showed a similar pattern in any solution containing a mixture of Ca²⁺ and Ba²⁺(Fig. 4B), even when they expressed VDCC currents with amplitudes similar to those of muscle blastomeres.

Figure 4. VDCC currents in mixed Ca²⁺-Ba²⁺ solutions.

A, representative traces of VDCC currents in the muscle blastomere (51 h) in external recording solution containing varying proportions of Ca²⁺ and Ba²⁺. Representative currents recorded in 20 mM Ca²⁺ and 80 mM Ba²⁺, 2 mM Ca²⁺ and 98 mM Ba²⁺ (2Ca/98Ba) and 0.2 mM Ca²⁺ and 99.8 mM Ba²⁺ are shown. B, representative traces of VDCC currents from a neuronal blastomere (51 h) in 2Ca/98Ba solution.

Despite the presence in the external solution of 200 mM TEA and 100 mM Ba²⁺ (Hille, 1992) and of CsCl in the current-measuring electrode, it is possible that this biphasic profile might be caused by residual outward K⁺ current. To test whether the anomalous biphasic profile of I_2Ca/98Ba was caused by overlapping currents through K⁺ channels, Ca²⁺ tail currents were measured from −30 to −50 mV at which potentials the driving force for K⁺ ions was reduced. Ideally repolarization should be to less than −60 mV in order to minimize overlapping K⁺ channel currents. However, we used the −30 to −50 mV voltage range, because VDCC tail currents decay too rapidly to measure at less than −50 mV. Tail currents were fitted with a single exponential and the tail current amplitude was obtained as the extrapolated value at the onset of repolarization. The current amplitude of the tail current thus obtained was plotted against the duration of depolarization. Examples of such an analysis (Fig. 5) show that tail currents in the 2Ca/98Ba solution produce a biphasic profile, suggesting that the biphasic profile of I_2Ca/98Ba was not derived from contamination with K⁺ currents but rather based on VDCC current kinetics. Similar results were obtained from all the cells examined (n = 4).

Figure 5. Tail currents of I_2Ca/98Ba.

A, the time course of VDCC currents in 2Ca/98Ba solution estimated by measurement of the tail current. Two representative traces are shown here. Blastomeres were depolarized to −30 mV (a) or −15 mV (b) for varying durations and then hyperpolarized to −50 mV (a) or −40 mV (b) for 15 ms. The duration of the first pulse was varied from 3 to 133 ms in 10 ms increments. Each point (• in a and ▴ in b) indicates the magnitude of tail current obtained by curve fitting (shown in B). B, tail currents were fitted by single exponential curves (thin curved line) and the values of tail currents are determined by extrapolation. Time constants were between 2.6 and 3.4 ms. One representative tail current (the 6th sweep of Ab) and its fitted curve are shown.

The biphasic profile depends on Ca²⁺ release from caffeine-sensitive Ca²⁺ stores

The absence of the biphasic profile of inward currents in neuronal blastomeres (see Fig. 4B) suggests that some mechanism unique to muscle cells must mediate this phenomenon. Ascidian muscle cells share several ultrastructures with vertebrate cardiac muscle cells, including gap junctions between muscle cells (Katz et al. 1983; Bone, 1989) and peripheral coupling (Cavey & Cloney, 1972). Calcium channel inactivation is modified by CICR in mammalian cardiac muscle cells through Ca²⁺-dependent inactivation (Balke & Wier, 1991; Sham et al. 1995). This led us to hypothesize that the fast and transient decay of I_2Ca/98Ba reflects Ca²⁺-dependent inactivation mediated by Ca²⁺ release by activation of ryanodine receptors.

To test this hypothesis, we examined the effect of caffeine, which is known to induce Ca²⁺ release from the intracellular Ca²⁺ stores by activating ryanodine receptors, on the biphasic profile of I_2Ca/98Ba. Perfusing the blastomere with 2Ca/98Ba solution containing 1 mM caffeine first decreased the current amplitude while attenuating the biphasic profile. The current amplitude recovered in several minutes, probably due to the removal of cytoplasmic Ca²⁺ by pumping or resequestration into organelles. In this state, the biphasic profile was no longer discerned (Fig. 6). This phenomenon was observed in all the cells examined (n = 14).

Figure 6. Effect of caffeine on the biphasic profile of I_2Ca/98Ba.

A, the change in current by addition of 1 mM caffeine to the 2Ca/98Ba solution. Traces before and at 1.5 and 3.5 min after application of caffeine are shown. Time zero corresponds to the time at which caffeine was added to the bath. The blastomere was depolarized by a −15 mV pulse every 30 s. B, the peak current amplitude upon application of 1 mM caffeine (bar) plotted against the time after caffeine application. Data were taken from the same cell shown in A.

This effect of caffeine proved to be reversible. After Ca²⁺ stores were depleted with caffeine, the blastomere was washed with calcium-rich solution, depolarized to facilitate the refilling of Ca²⁺ stores, and finally perfused with 2Ca/98Ba solution again. As a result, the biphasic profile reappeared (Fig. 7; n = 4). The disappearance and reappearance of the biphasic profile could be reproduced.

Figure 7. Reversible effect of caffeine on the biphasic profile of I_2Ca/98Ba.

Traces show the change in current by addition of 1 mM caffeine to the 2Ca/98Ba solution. First, the blastomere in the 2Ca/98Ba (a) was exposed to 1 mM caffeine (b). Then the blastomere was washed with calcium-rich solution and depolarized several times to refill the depleted Ca²⁺stores. The biphasic profile reappeared when the blastomere was exposed to the 2Ca/98Ba solution again (c). This pattern could be repeated (d and e). Similar results were obtained from 3 different cells.

Ryanodine, a specific agonist of the Ca²⁺ release channel, also attenuated the biphasic profile. Perfusion with 2Ca/98Ba solution containing ryanodine (10 μM) transiently decreased the VDCC currents, probably due to an increase in [Ca²⁺]_i after opening of ryanodine receptor (Ca²⁺ release) channels on the sarcoplasmic reticulum (Fig. 8). The current amplitude recovered slowly over about 10 min as was in the case with caffeine. The change in current profile during application of ryanodine was also similar to that seen with caffeine: the biphasic profile was attenuated and decay following the first peak became slower. The only difference was that the attenuation of the biphasic profile by ryanodine was more moderate than that by caffeine.

Figure 8. Effect of ryanodine on the biphasic profile of I_2Ca/98Ba.

A, the change in I_2Ca/98Ba induced by application of 10 μM ryanodine. Traces before and at 2.5 and at 15 min after ryanodine application are shown. The blastomere was depolarized to −15 mV every 30 s. B, the change in peak current amplitude upon application of 10 μM ryanodine (bar) plotted against the time after ryanodine application. Data are from the same cell shown in A. Similar results were obtained from other 2 cells.

Involvement of Ca²⁺ stores in the biphasic kinetics of decay was also tested by depletion of Ca²⁺ stores by repetitive stimulation. Blastomeres were depolarized repeatedly (a 400 ms depolarizing pulse every 3 s, 80 times) and VDCC currents at each stimulation were monitored. As shown in Fig. 9, I_2Ca/98Ba was reduced and the biphasic profile attenuated gradually, compatible with the idea that the biphasic kinetics of I_2Ca/98Ba requires the presence of Ca²⁺ in the Ca²⁺ stores. Similar results were obtained from all the cells examined (n = 2).

Figure 9. Effect of depletion of the Ca²⁺ stores by repetitive depolarizing stimuli.

A, the change in I_2Ca/98Ba with repetitive depolarization is shown. The blastomere was depolarized to −15 mV every 3 s. Traces of the 1st, 20th, 40th, 60th and 80th depolarization are shown. B, the change in amplitude of I_2Ca/98Ba with repetitive stimuli. Data were taken from the same cell shown in A. A similar result was obtained from another cell.

Effects of inhibitors of Ca²⁺-ATPases on the biphasic profile

Cytosolic Ca²⁺ is taken up into intracellular Ca²⁺ stores by Ca²⁺-ATPases, and these Ca²⁺-ATPases (SERCAs) are known to be blocked by cyclopiazonic acid (CPA) reversibly and by thapsigargin irreversibly (Pozzan et al. 1994). Attenuation of the biphasic profile by low-frequency repetitive depolarization was tested in 30 μM CPA or 3 μM thapsigargin (Fig. 10). Control cells still showed the biphasic profile of inward currents until the 21st stimulation. However, CPA-treated cells showed remarkable attenuation of the biphasic profile form by the 11th stimulation. This attenuation seemed to be already saturated at the 11th stimulation, because the current profile was almost superimposable on the trace at the 21st stimulation. Cells treated with thapsigargin showed milder attenuation of the biphasic profile by the 21st stimulation. Attenuation of the biphasic profile was significant as shown by the normalized superimposition (Fig. 10C, right).

Figure 10. Effect of depletion of the Ca²⁺ stores by blockers of SERCAs.

The muscle blastomeres in 2Ca/98Ba solution were exposed to the pump inhibitors CPA and thapsigargin 5 min before the first depolarization was applied and were depolarized to −15 mV every 10 s. Only traces of the 1st, 11th and 21st stimuli are shown. Right-hand traces show normalized currents to indicate the changes of kinetics. A, traces without inhibitor. B, traces in the presence of 30 μM CPA. C, traces in the presence of 3 μM thapsigargin. Each blocker was tested in another cell and similar results were obtained.

The reversible effects of caffeine on the biphasic profile of I_2Ca/98Ba were again tested in the presence of these blockers. Following caffeine-induced depletion of the Ca²⁺ stores, 30 μM CPA was introduced into the bath solution to see if it could prevent recovery of the biphasic profile. Cyclopiazonic acid prevented the recovery of the biphasic profile even after depolarization in calcium-rich solution (data not shown). Taken together with the results of the experiments with caffeine and ryanodine, these observations strongly suggest that Ca²⁺ release from caffeine- and ryanodine-sensitive Ca²⁺ stores underlies the biphasic profile of the VDCC currents in 2Ca/98Ba solution.

Ca²⁺ release significantly contributes to inactivation of Ca²⁺ currents

The L-type VDCCs are inactivated by the binding of Ca²⁺ ions (Imredy & Yue, 1994), and the appearance of the biphasic profile in 2Ca/98Ba solution seems to be due to the local elevation of [Ca²⁺]_i by Ca²⁺ release from the Ca²⁺ stores. In rat ventricular myocytes, the rate of inactivation of I_Ca is slowed by depletion of the Ca²⁺ content of the sarcoplasmic reticulum by caffeine (Sham et al. 1995), proving that Ca²⁺ release contributes to I_Ca inactivation.

To examine whether Ca²⁺ release contributes to the decay kinetics of ascidian VDCCs not only in 2Ca/98Ba solution but also in calcium-rich solution, the decay rate of I_Ca was examined before and after the application of 1 mM caffeine in the calcium-rich solution. The trace after the application of caffeine had slower decay kinetics than that before the application (Fig. 11). Because caffeine is expected to deplete the Ca²⁺ stores, the difference in the traces before and after the application of caffeine corresponds to the degree of contribution of the Ca²⁺ release-induced decay. Thus, a significant part of VDCC inactivation is dependent on Ca²⁺ release.

Figure 11. Change in the degree of Ca²⁺-dependent inactivation by depletion of the Ca²⁺ stores.

The muscle blastomere in Ca²⁺-rich solution was depolarized by a +10 mV pulse before (^) and after (•) the application of 1 mM caffeine. Similar results were obtained from other two cells.

The change in VDCC currents with muscle differentiation

The results described above show that the biphasic profile of I_2Ca/98Ba reflects interaction between VDCCs and Ca²⁺ release from Ca²⁺ stores. Although developmental changes in VDCC currents in cleavage-arrested blastomeres have been previously described (Takahashi & Yoshii, 1981), quantitative estimation of VDCC currents from isolated, cleavage-arrested blastomeres has yet to be done. We therefore recorded VDCC currents from the muscle blastomeres at early stages and also examined when such coupling between two regulators for CICR is established during muscle differentiation.

Maximum amplitudes of I_Ca and I_Ba were plotted against time after fertilization in Fig. 12. Persistent inward Ba²⁺ currents began to appear at about 24 h after fertilization, which corresponds to the neurula stage. This timing of the first appearance of VDCCs is consistent with that in muscle lineage cells of Boltenia (Simoncini et al. 1988). However, it is earlier than that previously reported (Takahashi & Yoshii, 1981) for Halocynthia, probably due to a difference in the composition of the external recording solution. The stage at which I_Ca is first detected precedes the appearance of TEA-insensitive outward currents by about 10 h (Shidara & Okamura, 1991). Up until 40 h after fertilization, the increase in I_Ca was not drastic and no significant outward K⁺ currents were recorded (data not shown). About 40 h after fertilization, VDCC currents increased more rapidly, and delayed rectifier K⁺ currents appeared (Shidara & Okamura, 1991).

Figure 12. Developmental changes in I_Ca and I_Ba.

A, changes in the current amplitudes of I_Ca (_^) and I_Ba (•) with muscle development. The number of replicates in each experiment (n) is indicated in parentheses. Data are expressed as means ±s.d.B, representative traces of I_Ba from an immature cell recorded at 29 h and a differentiated cell recorded at 50 h. The blastomeres were activated by depolarizing pulses (400 ms) to test potentials between −60 and +90 mV, given once every 10 s in 10 mV increments.

In the 2Ca/98Ba solution, the biphasic profile of the inward current was not observed before 36 h after fertilization (Fig. 13A; n = 11). This suggests that coupling of Ca²⁺ channels with Ca²⁺ stores does not occur earlier than 36 h after fertilization. One cell recorded at 36 h showed the biphasic current. To see whether Ca²⁺ release from caffeine-sensitive Ca²⁺ stores occurred before 36 h, we examined the effect of caffeine on I_2Ca/98Ba from blastomeres between 28 and 36 h after fertilization (Fig. 13B; n = 11). In most cases (9 out of 11), the current amplitude of I_2Ca/98Ba of an immature blastomere first decreased and then increased gradually in 1 mM caffeine, as has been found in the differentiated blastomere (see Fig. 6). This change in the amplitude of I_2Ca/98Ba was blocked by injection of 5 or 50 mM BAPTA (Fig. 13C and D; n = 5), indicating that calcium channel activity at an early stage is also sensitive to [Ca²⁺]_i and that the decrease in current amplitude produced by caffeine is mediated by an increase in [Ca²⁺]_i. Recovery of the current amplitude in the immature blastomere was much slower than that in the differentiated one. In some cases (2 out of 11), currents did not recover at all after caffeine treatment. We interpret this as the relatively low ability of the immature blastomere to clear Ca²⁺ from the cytoplasm, probably as a result of insufficient expression of the Ca²⁺ pumps. The sensitivity of VDCC current to caffeine suggests that the mechanisms of CICR are already present at this stage.

In conclusion, caffeine-sensitive Ca²⁺ stores are already present at 28 h after fertilization when VDCCs start to appear. However, at this stage, coupling between Ca²⁺ stores and VDCCs is not tight enough to regulate VDCC current kinetics.

Electron microscopic analysis of differentiation of ascidian muscle

One possible mechanism underlying changes in the proximity of VDCCs to Ca²⁺ release channels during development may be the changes in the physical distance between the two elements. To validate this idea, electron microscopic analysis was performed on intact ascidian muscle cells (Fig. 14). Ultrathin sections were obtained from ascidian embryos at three distinct stages, corresponding to the tail-bud stage, the young tadpole stage and the swimming larva stage.

Figure 14. Electron micrographs of ascidian larval muscle cells at three developmental stages.

Electron micrographs of ascidian larval muscle cells from the tail-bud stage (A, 28 h post fertilization), young tadpole larva (B, 48 h post fertilization) and swimming larva (C, 72 h post fertilization). Plasma membranes of two neighbouring cells are indicated by arrowheads. Arrows indicate putative sarcoplasmic reticulum. Mf, myofilament. Mt, mitochondria. Scale bars in A−C, 1 μm. D, ratio of area of endoplasmic reticulum (ER) in the three cytoplasmic regions is compared between 28- and 72-h-old larva. The cytoplasmic area is divided into three regions by the distance from the cell surface (0≈200 nm, 200≈1000 nm and 1000≈2500 nm). Electron micrographs shown in A and C were scanned into a personal computer by an image scanner. All intracellular membranes except mitochondrial membranes were measured as putative ERs. Areas of putative ERs were calculated using NIH image software.

In the embryos of the tail-bud stage, when muscle blastomeres showed caffeine-sensitive VDCC currents but not the biphasic profile of I_2Ca/98Ba, large membrane structures similar to SR are not found, and instead, vesicular membranes are sparsely distributed in the cytoplasm. Quantification of membrane structures in two distinct regions (the region down to 200 nm from the surface and the deeper region between 200 and 1000 nm from the surface) demonstrates that membrane structure is homogeneously distributed in the cytoplasm (Fig. 14A and D).

At the young tadpole stage, when muscle blastomeres show a slight biphasic I_2Ca/98Ba profile, myofilaments are detected in the cortical area of the cytoplasm, although they do not form sarcomeres. A membrane structure similar to endoplasmic reticulum is still not especially prevalent but is more concentrated in the vicinity of the sarcolemma than in earlier embryos. In the fully differentiated muscle cells of the swimming tadpole larva, myofilaments form sarcomeres in the cortical area of the cytoplasm, thus shifting mitochondria to the middle of the cell. Membrane structures, the putative sarcoplasmic reticulum, develop well beneath the sarcolemma, forming an arrangement similar to the peripheral junctions of chicken cardiac muscle cells (Sun et al. 1995). Maturation of the SR membrane structure beneath the sarcolemma was quantified in Fig. 14D by measuring the rate of putative SR per unit. By contrast with the homogenous distribution of membrane structures present 28 h after fertilization, by 72 h after fertilization most of SR membranes developed in the vicinity of the sarcolemma.

These morphological findings in the muscle cell are compatible with the idea that interaction between VDCCs and Ca²⁺ release is controlled by spatial profiles between the two membrane components during muscle differentiation.

DISCUSSION

In the cleavage-arrested ascidian muscle blastomere, VDCC currents show Ca²⁺-dependent inactivation as one of the general features of the high-threshold, long-lasting Ca²⁺ channels. In a mixed solution of Ca²⁺ and Ba²⁺, they showed an anomalous biphasic profile of decay kinetics occurring dependent on Ca²⁺ release from the caffeine-sensitive Ca²⁺ stores. Using this pattern as an indication of functional coupling between VDCCs and Ca²⁺ stores, we were able to trace maturation of cross-coupling between the two regulators of [Ca²⁺]_i during the muscle differentiation.

Mechanisms underlying the biphasic profile of inward currents

Three types of outward K⁺ currents exist in differentiated muscle blastomeres: two delayed rectifier K⁺ currents with distinct TEA sensitivity and kinetics (Shidara & Okamura, 1991), and at least one class of Ca²⁺-induced K⁺ current (Hirano et al. 1984; Greaves et al. 1996). However, results from tail current recordings suggest that it is unlikely that the biphasic profile was caused by contamination by the outward K⁺ currents.

In artificial seawater, the transient outward current is activated between −40 and −20 mV; this voltage range also slowly activates delayed rectifier K⁺ currents (Shidara & Okamura, 1991). The transient outward current seems to be due to K⁺ currents activated by Ca²⁺, probably through CICR, because this component is also sensitive to caffeine. However, this type of outward K⁺ current is sensitive to TEA which was present in the recording solution for I_2Ca/98Ba (Y. Okamura & K. Nakajo, unpublished data). In addition, current kinetics measured from the magnitude of tail currents were also biphasic at −50 mV, a potential at which the transient outward current is not activated (Fig. 5Aa). It is therefore unlikely that this type of K⁺ channel contributes to the biphasic profile of I_2Ca/98Ba. Although we cannot neglect the possibility that some other uncharacterized K⁺ currents may partly contribute to the biphasic profile of I_2Ca/98Ba, we conclude that the biphasic profile of I_2Ca/98Ba is mainly based on VDCC kinetics.

The biphasic pattern is also not due to the presence of rapidly inactivating T-type channels. A transient type of VDCC similar to vertebrate T-type VDCCs has been reported in immature ascidian muscle cells (Greaves et al. 1996). The T-type VDCCs are known to be permeable to Ca²⁺ as well as Ba²⁺ in ascidian eggs and in other preparations (Okamoto et al. 1976). However, we could not detect the transient type of VDCC in cleavage-arrested muscle blastomeres. The discrepancy between these observations and a previous report (Greaves et al. 1996) may be due to a difference in species or, more probably, to different culture conditions. In addition, a change in holding potential decreased the earlier peak and the later plateau of I_2Ca/98Ba equally, and did not lead to separation into two components (data not shown).

Inactivation of VDCCs in ascidian muscle cells is dependent on Ca²⁺ as seen from two pieces of evidence: (1) the bell-shaped relation between the prepulse and the current amplitude during the test pulse; and (2) the slower decay kinetics at more depolarized potentials which give lower current amplitudes. Similar properties were reported in VDCCs in another species of ascidian (Davis et al. 1995). This is consistent with the deduced primary structure of a recently cloned Halocynthia Ca²⁺ channel (Y. Okamura & K. Nakajo, in preparation) in which the C-terminal region flanking the fourth repeat, a site responsible for Ca²⁺-dependent inactivation in mammalian L-type VDCCs (de Leon et al. 1995), is highly conserved.

Our interpretation of the anomalous biphasic profile in the 2Ca/98Ba solution is that it reflects inactivation of Ca²⁺ channels by a transient increase in the local Ca²⁺ concentration caused by Ca²⁺ release. In the 2Ca/98Ba solution, activation of VDCCs during the early part of the voltage step causes considerable Ca²⁺ influx, and then triggers a transient Ca²⁺ release from internal Ca²⁺ stores which are localized in the vicinity of the VDCCs. This leads to a significant increase in [Ca²⁺]_i in the space between the Ca²⁺ stores and the VDCCs. Calcium release seems to require only a small fraction of Ca²⁺ influx. This idea is evidenced by a result showing that even the small Ca²⁺ influx through VDCCs elicited by weak depolarization to −40 mV, corresponding to only about 3 % of the maximum current amplitude of the I-V curve, causes a significant degree of inactivation of VDCCs (Fig. 2). Calcium release occurs only transiently, probably because of the intrinsic properties of the Ca²⁺ release channels in the sarcoplasmic reticulum or the fast Ca²⁺ clearing mechanism of cytoplasm. A transient increase in [Ca²⁺]_i suppresses the activity of VDCCs through Ca²⁺-sensitive inactivation. In the external high-Ca²⁺ solution, Ca²⁺ continuously flows into the channels, thus leading to the Ca²⁺-dependent inactivation. In contrast, in the high-Ba²⁺ solution (2Ca/98Ba solution), influx does not directly lead to significant inactivation of VDCCs, as expected from the current profile of Ba²⁺. However, the influx of Ca²⁺ is enough to provide an increase in [Ca²⁺]_i which can activate Ca²⁺ release. As a result, inhibition of VDCC activity by Ca²⁺-dependent inactivation mostly correlates with a transient increase in [Ca²⁺]_i rather than with the influx of Ca²⁺ itself in 2Ca/98Ba solution.

The biphasic profile of I_2Ca/98Ba could be based on two populations of VDCCs, one coupled with CICR and the other not linked to it. Only VDCCs tightly coupled with Ca²⁺ stores may inactivate via a transient increase in [Ca²⁺]_i by CICR, showing a fast decay of I_2Ca/98Ba. The other population of VDCCs may not be significantly affected by Ca²⁺ release because of their remoteness from Ca²⁺ release sites. These channels inactivate slowly due to voltage-dependent inactivation as seen in 100 mM Ba²⁺ solution. In fact, the ultrastructure of ascidian muscle cells showed that the SR is well developed just beneath the sarcolemma, but is not completely continuous along the sarcolemma (see Fig. 14). If VDCCs distribute homogeneously over the sarcolemma, there could be two populations of VDCCs at distinct distances from Ca²⁺ release sites.

Recordings of [Ca²⁺]_i are needed to discriminate between the above two possibilities.

Maturation of coupling between VDCCs and ryanodine receptors

The absence of the biphasic I_2Ca/98Ba profile in 2Ca/98Ba solution in immature cells suggests that early VDCCs in ascidian muscles are not as tightly coupled with caffeine-sensitive stores as they are in the differentiated cells. The absence of the biphasic profile of I_2Ca/98Ba is not due to the lower sensitivity of VDCCs to [Ca²⁺]_i, because microinjection of BAPTA or EGTA restores inhibition of VDCC currents by caffeine. The biphasic profile in the 2Ca/98Ba solution was first detected in cells around 36 h after fertilization. Correspondingly, electron microscopic pictures from muscle cells at 48 h showed the presence of structures similar to peripheral couplings. The developmental profile of the biphasic pattern of I_2Ca/98Ba agrees well with the ultrastructural maturation of an SR-like structure, thus supporting the idea that the development of the ultrastructural organization of the E-C coupling machinery may account for changes in local interactions between ryanodine receptors and VDCCs (Fig. 15).

Figure 15. Model for the relationship between VDCCs and Ca²⁺ stores during ascidian muscle development.

The intracellular Ca²⁺ stores are distant from the plasmalemma and the Ca²⁺ channels in the early embryonic stage (left). Only the basal level of VDCC activity but not the VDCC kinetics are affected by CICR. As the muscle cell matures, the Ca²⁺ stores are closer to the plasmalemma and cross-couple with the VDCCs (right). Ca²⁺ entry through the VDCCs causes CICR and the Ca²⁺-dependent inactivation of I_Ca is significantly affected by CICR. RyR: ryanodine receptor.

Despite the fact that ultrastructural changes match well with progression of functional coupling between VDCCs and ryanodine receptors, however, we cannot neglect the possibility that the sensitivity of the Ca²⁺ release channel to Ca²⁺ concentration also changes with development. If a Ca²⁺ release channel is less sensitive to Ca²⁺, the extent of transient Ca²⁺ release triggered by Ca²⁺ influx via the VDCCs will be less even if the spatial relation between VDCCs and ryanodine receptors is constant. Characterization of ryanodine receptors in ascidian muscles is required to test this possibility.

Cross-coupling of VDCC and Ca²⁺ stores appears after 36 h post fertilization. This timing precedes the appearance of spontaneous synaptic activities in muscle cells but matches well with the appearance of sensitivity to acethylcholine (Ohmori & Sasaki, 1977). This suggests that development of cross-coupling between the two regulators of intracellular calcium occurs as one of the early events for establishment of intrinsic functional properties of muscle cells. Later development of cross-coupling between VDCC and Ca²⁺ stores may correlate with maturation of swimming behaviour. The amplitudes of tail movements increase with time. Although changes in the neural input are one of the critical factors for maturation of swimming (Ohmori & Sasaki, 1977; Sun & Dale, 1998), changes in the proximity between VDCCs and Ca²⁺ stores may also contribute to this development.

Why, then, are both of VDCCs and caffeine-sensitive Ca²⁺ stores already detectable at 28 h after fertilization which is much earlier than the emergence of motility? The presence of VDCCs in tail-bud embryos has also been reported in another ascidian, Boltenia (Simoncini et al. 1988). What are the functional roles of VDCCs and caffeine-sensitive Ca²⁺ stores in early immature muscle cells? The difference in the amplitudes of I_Ca and I_Ba was larger in immature cells than in differentiated cells. In the early stages (24–32 h after fertilization; see Fig. 12A), many cells showing significant Ba²⁺ currents did not show any detectable inward Ca²⁺ current, suggesting that channel activity of the VDCCs is controlled by a higher global [Ca²⁺]_i in immature muscle cells. It is possible that Ca²⁺ entry via VDCCs activates Ca²⁺ release through the global cytoplasmic Ca²⁺ concentration in muscle cells at an early stage. The presence of spontaneous electrical activity and its critical developmental role in the maturation of muscle phenotypes has been shown in muscle precursors of Boltenia embryos (Greaves et al. 1996; Dallman et al. 1998). A loose coupling between VDCCs and Ca²⁺ stores may provide a mechanism for changes in [Ca²⁺]_i, which may be important for cellular events such as morphogenesis and the gene regulation of muscle phenotypes.

Mechanism of formation of complexes involved in E-C coupling

The molecular mechanism for forming a functional apparatus for E-C coupling during muscle development still remains unknown (Flucher & Franzini-Armstrong, 1996). In our experiments, cytokinesis of muscle lineage cells was perturbed with cytochalasin B and, nevertheless, cross-coupling between VDCCs and ryanodine receptors was detected. The formation of actin filaments is disrupted under this condition, as demonstrated by electron microscopic analysis (data not shown). This suggests that the actin-based cytoskeleton does not play a crucial role in the assembly of the ultrastructural machinery for E-C coupling. Probably membrane compartments containing ryanodine receptors autonomously form in the vicinity of VDCCs via some unknown mechanism for specific interactions. Cleavage-arrested ascidian muscle blastomeres will provide a simple model for studying not only early muscle differentiation but also the cell biological question of what controls the co-ordinated arrangement of the two key regulators of E-C coupling, VDCCs and ryanodine receptors.