Adenosine 5′-cyclic diphosphoribose [cyclic ADP-ribose (cADPR)], a metabolite of NAD+ that promotes Ca2+ release from sea urchin egg homogenates and microsomal fractions, has been proposed to act as an endogenous agonist of Ca2+ release in sea urchin eggs. We describe experiments showing that a microsomal fraction isolated from Tetrapigus nyger sea urchin eggs displayed Ca2+-selective single channels with conductances of 155.0 ± 8.0 pS in asymmetric Cs+ solutions and 47.5 ± 1.1 pS in asymmetric Ca2+ solutions. These channels were sensitive to stimulation by Ca2+, ATP, and caffeine, but not inositol 1,4,5-trisphosphate, and were inhibited by ruthenium red. The channels were also activated by cADP-ribose in a Ca2+-dependent fashion. Calmodulin and Mg2+, but not heparin, modulated channel activity in the presence of cADP-ribose. We propose that these Ca2+ channels constitute the intracellular Ca2+-induced Ca2+ release pathway that is activated by cADP-ribose in sea urchin eggs.
- calcium release channels
- ryanodine receptors
- intracellular calcium
- endoplasmic reticulum
- intracellular channels
release of Ca2+from intracellular stores has a central role in egg development after fertilization (19, 30). Consequently, the characterization of the physiological agonists that elicit Ca2+ release in oocytes is a highly relevant topic that is the subject of active investigation. In the case of sea urchin eggs, three independent Ca2+ release pathways, sensitive to inositol 1,4,5-trisphosphate (IP3), ryanodine, or nicotinic acid adenine dinucleotide phosphate, have been described (5, 7, 12,21). In addition, adenosine 5′-cyclic diphosphoribose (cADPR) promotes Ca2+ release in sea urchin egg homogenates and microsomes (14, 23, 25). Agents that inhibit Ca2+ release through the ryanodine receptors-Ca2+ release channels (RyR-channels) of sarcoplasmic reticulum (SR) block cADPR-induced Ca2+ release in oocytes, and agonists of Ca2+ release from SR, such as Ca2+, caffeine, and ryanodine, also release Ca2+ from sea urchin egg homogenates (12, 14). High concentrations of caffeine or ryanodine, by depleting the same stores, desensitize the releasing activity of cADPR in intact eggs and in egg homogenates. Subthreshold concentrations of caffeine or ryanodine, which enhance the releasing effects of subsequent doses of these agonists, also enhance the releasing effect of subthreshold doses of cADPR and vice versa (3, 12,20).
These similarities between the properties of the cADPR- and ryanodine-sensitive Ca2+ release pathways indicate that sea urchin eggs have a common Ca2+-induced Ca2+ release route sensitive to caffeine, cADPR, and ryanodine. Accordingly, cADPR has been proposed to act as an endogenous agonist in sea urchin eggs (9, 14, 25), and it may act as an agonist of Ca2+ release in a variety of other cells as well (26). However, stimulation of Ca2+ release by cADPR in sea urchin eggs may involve at least two other proteins, besides the putative ryanodine receptors, that change their affinity toward cADPR in the presence of caffeine (29), suggesting that cADPR probably acts indirectly on the sea urchin egg putative RyR-channels. Furthermore, there is no information, to our knowledge, of the properties at the single channel level of the Ca2+release channels through which cADPR-activated release takes place in sea urchin eggs.
We have isolated a microsomal fraction from sea urchin eggs, and using planar lipid bilayers, we have investigated the presence of cADPR-sensitive Ca2+ channels in the isolated microsomes. Single channel experiments revealed channels in the microsomes that were activated by Ca2+, ATP, caffeine, and cADPR. The activation of the channels by cADPR was modulated by Ca2+, calmodulin, and Mg2+ and was blocked by ruthenium red.
Isolation of sea urchin egg microsomes.
Eggs were collected from seawater after electrostimulation of sea urchins (Tetrapigus nyger) into their coelomic cavity at room temperature. All subsequent steps were done at 4°C. After removal of jelly coats (10), eggs were washed once in Ca2+-free artificial seawater. This first wash was followed by two brief low-speed sedimentations in sucrose buffer (0.3 M sucrose, 0.1 M KCl, 20 mM 3-(N-morpholino)propanesulfonic acid, pH 7.0, plus protease inhibitors: 10 μg/ml leupeptin, 2 μg/ml pepstatin A, 5 mM benzamidine, 100 μg/ml soybean trypsin inhibitor). Eggs were homogenized in a Dounce homogenizer and sedimented at 2,000g for 20 min, and the resulting supernatant was sedimented at 12,000 gfor 20 min. The pellet was discarded, and the supernatant was sedimented at 100,000 g for 60 min. To remove the egg pigment echinochrome, the 100,000-g pellet was resuspended in sucrose buffer to a protein concentration of ∼10 mg/ml and fractionated in an agarose column (Bio-Gel A-0.5 m, Bio-Rad) equilibrated with sucrose buffer. The column exclusion fraction, containing the microsomes, was sedimented at 100,000g for 60 min, and the pellet was resuspended in sucrose buffer, quickly frozen in liquid nitrogen, and stored at −80°C.
Ca2+ release studies.
Ca2+ release was measured in microsomes actively loaded with Ca2+. For this purpose the isolated sea urchin egg microsomes were added at a protein concentration of 0.5 mg/ml to a fluorometer cuvette containing 1 ml of a solution of 1 mM Mg-ATP, 8 mM phosphocreatine, 4 U/ml of creatine kinase, 0.1 M KCl, and 20 mM 3-(N-morpholino)propanesulfonic acid-tris(hydroxymethyl)aminomethane (Tris), pH 7.2. To measure the concentration of free Ca2+([Ca2+]), 0.5 μM fluo 3 was added to this solution, and fluo 3 fluorescence was determined using a Shimadzu RF-540 spectrofluorometer with excitation and emission wavelengths of 506 and 526 nm, respectively. Because after vesicle addition, external [Ca2+] reached 5–10 μM, no extra Ca2+ was added to the incubation solution. After 30 min of incubation with Mg-ATP at 22°C, external [Ca2+] decreased to 0.3–0.4 μM. At this point, different agonists of Ca2+ release channels were added, and the changes in fluo 3 fluorescence were followed as a function of time. Calibration curves of fluo 3 fluorescence vs. [Ca2+] were done using solutions of known [Ca2+], which was determined with a Ca2+ electrode.
Planar phospholipid bilayers were formed from 5:3:2 palmitoyloleoyl phosphatidylethanolamine-phosphatidylserine-phosphatidylcholine. Fusion of vesicles to negatively charged Muller-Rudin membranes was performed as described previously (4), with slight modifications. Sea urchin egg microsomes were added to the ciscompartment solution, containing 5 ml of 200 mM CsCl, 0.1 mM CaCl2, and 25 mMN-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES)-Tris, pH 7.4. The other (trans) compartment contained 25 mM HEPES-Tris, pH 7.4. After channel fusion, evidenced by the emergence of current fluctuations a few minutes after the addition of the microsomes, the cis compartment was perfused with five times the compartment volume (a total volume of 25 ml) of a solution containing 25 mM HEPES-Tris, pH 7.4. After perfusion, 200 mM Cs-methanesulfonate, 0.5 mM Ca-HEPES, and sufficientN-hydroxyethylethylenediaminotriacetic acid or ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid to give the desired free [Ca2+], which was always checked with a Ca2+electrode, were added to the ciscompartment. The trans compartment was supplemented with 50 mM Cs-methanesulfonate after fusion. Voltage was applied to the cis compartment, and the trans compartment was held at virtual ground through an operational amplifier in a current-to-voltage configuration. Unless indicated otherwise, most single channel records illustrated here were obtained in asymmetric Cs-methanesulfonate solutions (200 mM cis/50 mMtrans) at room temperature (22–24°C), with membranes held at 0 mV. For analysis, data were filtered at 0.5 kHz using an eight-pole low-pass Bessel filter (model 902 LPF, Frequency Devices, Haverhill, MA) and subsequently digitized at 2 kHz with an acquisition system (Axotape, Axon Instruments, Foster City, CA).
Because the channels studied displayed four conductance levels, plus rapid and complex kinetics, the normalized mean current ( ) was used as an index of channel activity and was calculated as follows Equation 1whereI max is the channel maximal current amplitude andI mean is the mean current amplitude. Mean channel current and fractional times spent in each subconductance state (Pi ) were always calculated from steady-state records lasting ≥150 s. pClamp 6.0 (Axon Instruments) was used for the former calculations, and Transit software (Baylor College of Medicine, Houston, TX) was used for the latter. Furthermore, to establish that the four equal current levels observed corresponded to true subconductance states and not to four separate single channels, the following analysis was performed. For a binomial system made of four independent units, the frequency of occurrence of each conductance level is given by the following set of equations Equation 2 Equation 3 Equation 4 Equation 5 Equation 6In Eqs. 2-6 ,P o corresponds to the open probability of each unit andP c to the probability of the closed state. To calculateP o,Eq. 7 was used Equation 7From the calculatedP o the binomial frequencies for the open conductance states were estimated, and the resulting theoretical frequency values were compared with the experimental distribution of frequencies using a χ2 test.
IP3- and cADPR-induced Ca2+ release from sea urchin egg microsomes.
The sea urchin egg microsomes isolated according to the procedures described above actively accumulated Ca2+ at 22°C after addition of 1 mM Mg-ATP (not shown). The uptake of Ca2+ ceased after 20–30 min of incubation, when the [Ca2+] of the extravesicular solution reached 0.3–0.4 μM. At this point, addition of 5 μM IP3 to the actively loaded vesicles produced transient Ca2+ release, and subsequent addition of 1 μM cADPR induced a new transient Ca2+ release of smaller magnitude (Fig.1 A). The inverse sequence of agonist addition, first cADPR and then IP3, produced a transient Ca2+ release after addition of cADPR and a second release response to 5 μM IP3 smaller (Fig.1 B) than the release response obtained when 5 μM IP3 was added before cADPR (Fig. 1 A). These results indicate that the isolated microsomes have release pathways responsive to IP3 and cADPR. Addition of the Ca2+ ionophore A-23187 at the end of the experiment produced in all cases a marked increase in extravesicular [Ca2+] (Fig. 1). This response to the ionophore indicated that the vesicles had actively accumulated significant amounts of Ca2+ and that the increase in external [Ca2+] observed after addition of cADPR or IP3 corresponded in effect to Ca2+ release through pathways in the microsomal membranes.
Sea urchin egg microsomes displayed caffeine-sensitive Ca2+ channels.
Using planar lipid bilayers, we investigated the presence of Ca2+ channels in the isolated microsomes. For this purpose, we used experimental conditions similar to those routinely employed to detect ryanodine-sensitive Ca2+ channels in SR vesicles, such as pH 7.4 and high concentrations (up to 50 mM) of cations, Cs+ or Ca2+, in thetrans compartment (seeexperimental procedures). In the standard recording conditions (200 mM Cs+ cis/50 mM Cs+ trans) and in the presence of 12 μM Ca2+ in thecis solution, only single channels that exhibitedI max of 4–4.2 pA, with a low of 0.007, were observed (Fig. 2 A, top trace). Channel activity increased to a of 0.041 after addition of 2 mM caffeine to the cissolution (Fig. 2 A, bottom trace). Consistent channel activation by caffeine was observed in all single channels tested.
In contrast to the consistent activation of channel activity by caffeine, not all channels were consistently activated by ryanodine. After addition of 50–100 μM ryanodine over 12 μM Ca2+ in thecis solution, a distinct increase in channel P o was observed only in three of seven single channel experiments. Also, all our attempts to measure specific [3H]ryanodine binding to sea urchin egg microsomes were unsuccessful. For these reasons, we have not included the effects of ryanodine on single channel activity in this report.
Effect of cADPR on single Ca2+ channel activity.
Addition of cADPR to the cis solution containing 12 μM Ca2+ produced more stimulation of channel activity than 2 mM caffeine. Aftercis addition of 2 and 5 μM cADPR, channel activity increased from a basal of 0.006 to a of 0.039 and 0.125, respectively (Fig. 2 B). At this level of activity it became evident that cADPR-activated channels exhibited a complex behavior. Channel activity presented bursting kinetics, and fluctuations among a single closed state and four near-equal subconductance open states were clearly discernible with an amplified time scale (Fig. 2 B, bottom traces). Subconductance states, observed as well in native ryanodine-sensitive Ca2+channels from SR (1), were consistently observed in all channels studied. Several channel experiments (n = 4) revealed that, in the presence of 12 μM Ca2+ in thecis solution, increased as a function of cADPR concentration, reaching 0.095 ± 0.011 at 5 μM cADPR, the highest concentration tested. Significant channel activation over the control condition became noticeable at ≥0.5 μM cADPR (Fig.3 A, filled symbols).
To analyze in more detail the effects of cADPR on the distribution of subconductance open states, an experiment was performed using a higher Cs-methanesulfonate gradient (300 mMcis/50 mMtrans) to increase channel current and thus increase the signal-to-noise ratio of the experimental records. An increase in by cADPR as low as 0.2 μM was observed when 300 mM Cs+was used in the cis solution (Fig.3 A, open circles). This observation suggests that increasing Cs+ in the cis solution makes the channels more sensitive to stimulation by cADPR and is consistent with previous reports showing that the effect of agonists on mammalian ryanodine-sensitive Ca2+ channels is modulated by ionic strength (8, 31). In these conditions, a comparison of the fractional time spent in each subconductance state (Pi ) for the control and in the presence of two different concentrations of cADPR, 0.2 or 0.5 μM, revealed that cADPR increased channel activity by increasing all individualPi values (Fig.3 B). However, as reflected in the percent distribution of the states (Fig. 3 B, inset), the highest conductance state seemed to be somewhat more prevalent in the presence of cADPR. Further analysis of these experimental records in terms of the frequencies for the open conductance states (see experimental procedures) showed that they did not follow the pattern expected for a binomial distribution. In fact, as shown in Table 1, a comparison of the experimentally determined open substate frequencies (data of Fig.3 B) with those predicted byEqs. 2-6 revealed that in all these cases the χ2 test detected a significant difference between both frequencies. These findings indicate that the four current levels recorded in cADPR-activated channels are not due to a complex formed by four independent single channels.
Channels recorded in asymmetric Ca2+ gradients (12 μM Ca2+ cis/43 mM Ca2+ trans) were likewise activated bycis addition of 1 μM cADPR (Fig.4 A). Because 43 mM Ca2+, the concentration present in the transcompartment in these experiments, effectively blocks SR ryanodine-sensitive Ca2+ channels at the cytoplasmic surface (8, 31), the observed channel activity most likely corresponds to channels fused with their cytosolic side oriented toward the cis compartment.
A plot of maximal current values vs. voltage, obtained in channels activated by cis addition of micromolar cADPR in 12 μM Ca2+, revealed oocyte channel conductances of 47.5 ± 1.1 pS in asymmetric Ca2+ solutions and 155.0 ± 8.0 pS in asymmetric Cs+ solutions (Fig. 4 B). The displacement of theI max-voltage curves toward the right after transaddition of Ca2+ to channels recorded in Cs+ (data not shown) implies that the channels were more selective for Ca2+ than for Cs+. The higher selectivity for Ca2+ than for monovalent cations such as Cs+, K+, or Na+ is a well-known feature of mammalian ryanodine-sensitive Ca2+channels (8). For practical reasons, because higher currents were obtained in Cs+, making readily visible the different subconductance states and increasing the differences between the lowest subconductance state and baseline noise (as shown in Fig. 4 A), all subsequent channel experiments were done in asymmetric Cs+ solutions (200 mMcis/50 mMtrans).
Lack of effect of IP3 on channel activity.
Addition of 2 μM IP3 to thecis solution containing 12 μM Ca2+ had no effect on channel activity and did not interfere with subsequent channel activation by 5 μM cADPR (Fig. 5 A, middle traces). In contrast, decreasing [Ca2+] in thecis solution from 12 to 0.72 μM in the continuous presence of 2 μM IP3 and 5 μM cADPR produced a significant reduction in channel activity, decreasing from 0.115 to 0.005 (Fig. 5 A, bottom trace). This decrease in channel activity, which took place when [Ca2+] in the cis solution was lowered to levels that optimize the Ca2+ release response to IP3 in sea urchin egg homogenates (5), indicates that these cADPR-activated channels are not responsive to IP3. In addition, this reduction implies that stimulation by cADPR requires >0.72 μM Ca2+ in thecis solution, as reported in further detail below.
Effects of calmodulin, ruthenium red, and heparin on channel activity.
In a separate experiment, addition of 4 μg/ml (0.24 μM) calmodulin to a channel already activated by 2 μM cADPR in 12 μMcisCa2+ did not modify channel activity (Fig. 5 B, 3rd trace). Yet, further addition of ruthenium red produced a marked inhibition, lowering from 0.063 to 0.005 (Fig. 5 B, bottom trace). Likewise, no effect of cis addition of calmodulin on the activity of cADPR-activated channels was observed when the [Ca2+] of thecis compartment was lowered to 0.72 μM (records not shown). Because cADPR-activated Ca2+ release in sea urchin egg microsomes has an absolute requirement for calmodulin (22, 23, 27), endogenous calmodulin may be associated with the channels fused in the bilayers. That this is most likely the case is shown by the experiments described below.
Addition of heparin to the cissolution containing 12 μM Ca2+plus 5 μM cADPR had a marginal stimulatory effect on channel activity and did not interfere with subsequent channel inhibition by ruthenium red. Thus channels (n = 2) that, after addition of 5 μM cADPR to the cissolution containing 12 μM Ca2+, increased their activity from a of 0.010 ± 0.001 to 0.083 ± 0.026, on addition of 300 μg/ml heparin increased their only to 0.125 ± 0.019. This small increase in channel activity brought about by 300 μg/ml heparin was not statistically significant. Further increasing heparin to 600 μg/ml had a negligible effect on channel activity, as reflected by a of 0.127 ± 0.028. Subsequent addition of 30 μM ruthenium red decreased channel activity to a of 0.008 ± 0.004.
Ca2+dependence of channel activity.
In the absence of cADPR, varying cis[Ca2+] from 0.7 to 270 μM had a modest effect on single channel activity (Fig.6, open circles); increased from <0.003 in 0.7 μM Ca2+ to ∼0.01 in 12 μM Ca2+. Channel remained constant at 12–30 μM cisCa2+. Further increasing [Ca2+] to 270 μM produced some decrease in , to an average of 0.006.
In contrast, in the presence of 1 μM cADPR, increasingcis[Ca2+] to 12 μM had a marked stimulatory effect on channel activity (Fig. 6, filled circles), as reflected by an increase in to ∼0.035. As observed in the absence of cADPR, channel activity remained constant at 12–30 μM cisCa2+. Further increasingcis[Ca2+] to 60 μM reduced to ∼0.020.
In the experimental conditions used to record channel activity, 1 μM cADPR did not activate the channels at ≤0.7 μM Ca2+ (Fig. 6). Yet, as shown in Fig. 1, 1 μM cADPR was effective in eliciting Ca2+ release from sea urchin egg microsomes actively loaded with Ca2+ and bathed in solutions containing ∼0.4 μM Ca2+. Furthermore, concentrations of cADPR as low as 10 nM have been reported to activate Ca2+ release from sea urchin egg homogenates actively loaded with Ca2+ (13). Because vesicular Ca2+ release and bilayer experiments were done in different conditions, such as the presence of millimolar Mg2+ and ATP in the external solution when vesicular release was measured, we investigated whether cADPR stimulation of channel activity was modified by addition of ATP or Mg2+ to thecis solution.
Stimulation of channel activity by ATP.
Addition of 2 mM ATP to the cissolution containing 12 μM Ca2+produced a significant stimulation of , from 0.007 to 0.022 (Fig. 7). Subsequent addition of 0.5 μM cADPR did not stimulate further channel activity but produced a small reduction in channel to 0.015 (not shown). Even increasing cADPR to 2 μM in the presence of ATP did not stimulate channel activity, because remained at 0.015 (Fig. 7, top, 3rd trace). After extensive washing of the ciscompartment (see experimental procedures) and addition of 12 μM Ca2+ to thecis solution, the channel regained its low activity, with a of 0.005. At this point, addition of 1 μM cADPR increased to 0.032 (Fig. 7,bottom). These results indicate that the stimulatory effect of ATP on channel activity did not add to that of cADPR and was wholly reversible. In concordance with these results, several experiments revealed that cisaddition of 1–2 mM ATP to cADPR-activated channels did not stimulate further channel activity (data not shown).
Stimulation of channel activity by cADPR plus Mg2+.
To analyze the effects of Mg2+ on cADPR-activated Ca2+ channels,cis[Ca2+] was held at 24 μM, a concentration that was within the range of [Ca2+] that produced optimal channel activation. To avoid changes in [Ca2+] after Mg2+ addition, ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid was used as a buffer. Addition of 1 μM cADPR to a single Ca2+ channel increased from 0.003 to 0.020 (Fig.8 A). Subsequent addition of 1 mM Mg2+to the cis solution markedly enhanced channel activity, increasing 12.8-fold, from 0.020 to 0.256 (Fig. 8 A). Addition of Mg2+ in the absence of cADPR had no effect on channel activity (not shown).
Yet, not all channels studied displayed the same marked activation by Mg2+. In the example of the single channel illustrated in Fig. 8 B, after addition of 1 μM cADPR in 24 μMcisCa2+, the channel increased its activity from a of 0.009 to 0.012 (Fig. 8 B,top and middle traces). Subsequent addition of 1 mM Mg2+ produced only a 4.4-fold stimulation of channel activity, to a of 0.053 (Fig.8 B, bottom trace).
The difference between these two types of responses is further illustrated in Fig. 9. All channels that presented a more marked activation by Mg2+ (Fig. 9, filled squares) were clearly activated by a cADPR concentration as low as 0.1 μM, with an apparent saturation at 1–2 μM cADPR. In contrast, the channels less activated by Mg2+ required >0.5 μM cADPR for discernible activation and did not seem to saturate at a cADPR concentration as high as 2 μM (Fig. 9, open circles). These results indicate that, in the combined presence of 24 μM cisCa2+ and 1 mM Mg2+, some of the channels markedly increased their sensitivity to activation by cADPR. This finding suggests that one of the additional components present in Ca2+ release experiments makes the channels more responsive to cADPR. However, the differences in the requirements for higher cis[Ca2+] to observe channel activation by cADPR in bilayers, as opposed to release experiments, cannot be attributed to Mg2+. Whencis[Ca2+] was decreased to the submicromolar range, channels were not consistently activated by addition of cADPR, even in the presence of 1 mM Mg2+ (data not shown).
A study of the response of the channels less sensitive to activation by Mg2+ showed that concentrations of Mg2+ >1 mM were less effective in enhancing channel activation by cADPR, and channel activity in 9 mM Mg2+ was lower than in its absence (Fig. 9, inset).I max was reduced as well by >7 mM Mg2+ (data not shown).
Effects of calmodulin antagonists plus Mg2+ on channel activity.
Channels activated by cADPR were not affected by calmodulin addition (Fig. 5), even at a concentration (4 μg/ml) that induces near full activation of Ca2+ release in sea urchin egg microsomes (22, 23, 27). Because calmodulin seems to be essential for activation of Ca2+release by cADPR in these microsomes (23), the lack of effect of calmodulin on single channel activity may reflect the presence of endogenous calmodulin tightly associated with the channel protein. To test this hypothesis, we examined the effects of the calmodulin antagonist W-7 (17) on channel activity. A single channel that displayed a of 0.274 in the presence of 24 μM Ca2+, 1 mM Mg2+, and 2 μM cADPR in thecis solution (Fig.10, top trace) decreased its activity to a of 0.047 aftercis addition of 10 μM W-7 (Fig. 10,middle trace). To test for the specificity of this effect, 10 μM calmodulin was subsequently added to the cis solution. After addition of this large excess of calmodulin, the channel increased its to 0.223, which is similar to that measured before W-7 addition (Fig. 10,bottom trace). Inhibitory effects comparable to those obtained with W-7 were observed with use of compound R-24571 (calmidazolium), a different calmodulin antagonist (data not shown). These results support the above hypothesis that channels fused in the bilayer contain sufficient endogenous calmodulin to support channel activity.
The results described here demonstrate that microsomes isolated fromT. nyger eggs contain Ca2+ channels that were activated by cis addition of caffeine, micromolar Ca2+, or millimolar ATP. Channels were also activated bycis addition of micromolar cADPR, and stimulation by cADPR was modulated bycis[Ca2+] and by Mg2+.
Comparison with mammalian RyR-channels.
Activation by caffeine, ATP, and cis[Ca2+] in the micromolar range (8, 11, 12) is characteristic of vertebrate RyR-channels. Furthermore, as described for RyR-channels isolated from mammalian skeletal muscle (11), the egg channels displayed a bell-shaped Ca2+ dependence of in the presence of cADPR. In addition, the egg channels were inhibited by ruthenium red and displayed multiple subconductance states, behaviors displayed as well by the mammalian RyR-channels (1, 8). On the basis of these similarities, it is tempting to assign the cADPR-activated channels from sea urchin eggs to the ryanodine receptor family. Although the conductance of the cADPR-activated egg channels (50 pS) was lower than the conductances described for RyR-channels from mammalian skeletal and cardiac muscle (100–120 pS) (8), this difference may not be very significant, because variations in the conductance of the mammalian channels have been reported (2). Yet, in contrast to the well-reported effects of ryanodine on RyR-channels (8, 31), we did not observe consistent modulation of the sea urchin channels by ryanodine. We have no explanation for this lack of consistent effects of ryanodine on channel activity.
Channels activated by cADPR were not activated by IP3.
The egg channels activated by cADPR were not IP3-gated channels, since addition of IP3 (2 μM) to thecis chamber had no effect on channel activity. However, IP3 addition to sea urchin microsomes actively loaded with Ca2+ caused significant Ca2+ release, indicating that the microsomes described in this study have release pathways responsive to IP3. Accordingly, a straightforward interpretation of the lack of effect of IP3 on channel activity is that the IP3-gated channels of sea urchin eggs (not described so far in bilayers) are different from the Ca2+ channels activated by cADPR studied in this work. It is pertinent to mention in this regard that there is significant evidence indicating that the cADPR- and IP3-sensitive Ca2+ release pathways of sea urchin eggs are different. 1) Ruthenium red and procaine, two antagonists of Ca2+-induced Ca2+ release in SR, inhibit cADPR-sensitive Ca2+ release in egg homogenates without affecting IP3-sensitive release (12, 13).2) Even high concentrations of IP3 do not alter the Ca2+-releasing activity induced by cADPR (12). 3) cADPR-induced Ca2+ release is insensitive to heparin (13, 24), a competitive inhibitor of IP3 binding to its receptor. Furthermore, the lack of effect of heparin on Ca2+ release is in agreement with the negligible effect of heparin on channel activity found in this work, even at heparin concentrations that completely inhibit IP3-sensitive Ca2+ release.
Ca2+dependence of channel activity in the presence of cADPR.
Sea urchin egg Ca2+ channels were moderately activated by increasing cis[Ca2+] from 0.7 to 30 μM, but in the presence of micromolar concentrations of cADPR, channel activation by increasing cis[Ca2+] in this range became more prominent. These results agree with a recent report describing significant enhancement of cADPR-gated Ca2+ release rates in sea urchin eggs by increasing [Ca2+] (16).
In the standard experimental conditions used to record channel activity, 1 μM cADPR did not activate the channels at ≤0.7 μM Ca2+. In contrast, we found that 1 μM cADPR was effective in eliciting Ca2+ release from microsomes actively loaded with Ca2+ and bathed in solutions containing 0.3–0.4 μM Ca2+. These results are in agreement with a previous report showing that cADPR induces Ca2+ release from sea urchin egg homogenates at submicromolar [Ca2+] (5). Nonetheless, it is important to note in this regard that Ca2+ release from microsomal vesicles actively loaded with Ca2+is measured under experimental conditions quite different from those prevailing in bilayer experiments. In release experiments,1) the vesicles have actively accumulated significant luminal Ca2+,2) they are bathed in solutions containing millimolar Mg-ATP, and 3) other vesicular proteins are present that may or may not fuse with the channels into the bilayers. Each one of these factors might in principle enhance the effect of cADPR at submicromolar [Ca2+], since all of them regulate the activity of mammalian RyR-channels (8, 18, 31). However, even in the presence of 1 mM Mg2+ channels were not consistently activated by cADPR in submicromolar [Ca2+], making unlikely Mg2+ by itself as the cause of the different Ca2+sensitivity. Whether the other factors mentioned above are responsible for the differences between bilayer and vesicular release experiments should be investigated. In particular, the effects of luminal Ca2+ should be studied, since we have preliminary results (not shown) indicating that cADPR was more effective in stimulating channel activity when thetrans compartment contained 50 μM Ca2+ in addition to 50 mM Cs+. Additionally, other factors that enhance the effects of cADPR on Ca2+ release from sea urchin egg homogenates, such as the oxidation state of critical SH groups (13, 28) or the presence of palmitoyl-CoA (6), may enhance the channel response to cADPR at low [Ca2+].
Effects of Mg2+ on channel activity.
It has been reported that 1 mM MgCl2 is required for optimal activation of Ca2+ release by cADPR (12-15) and that cADPR-activated Ca2+ release from sea urchin egg homogenates is inhibited by millimolar concentrations of Mg2+ (6, 15). In agreement with these reports, our results indicate that Mg2+ presented a dual effect over the activity of cADPR-stimulated channels. Thus 1 mM Mg2+ increased markedly the stimulatory effect of cADPR on channel activity, but at higher concentrations it was less effective and eventually became inhibitory. Furthermore, in the presence of 1 mM Mg2+, the sea urchin egg channels displayed two types of responses toward activation by cADPR. Why channels presented these two different responses is not clear and should be the subject of future investigation.
The results of this work indicate that sea urchin egg microsomes possess Ca2+ channels that share similar properties with vertebrate RyR channels, such as activation by Ca2+, ATP, and caffeine and inhibition by ruthenium red. Because activation of oocyte channels by cADPR was enhanced by Ca2+, we propose that these Ca2+ channels of sea urchin eggs constitute an intracellular Ca2+-induced Ca2+ release pathway in these cells.
We thank Dr. M. T. Nuñez for helpful criticism of the manuscript.
Address for reprint requests: C. Hidalgo, Centro de Estudios Cientı́ficos de Santiago, Casilla 16443, Santiago 9, Chile.
This study was supported by Fondo Nacional de Ciencia y Tecnologı́a Grants 2950037, 1940369, and 1970914, by European Community Grant CI1CT940129, and by institutional support to the Centro de Estudios Cientı́ficos de Santiago from a group of Chilean companies.
- Copyright © 1998 the American Physiological Society