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Am J Physiol Regul Integr Comp Physiol 274: R494-R502, 1998;
0363-6119/98 $5.00
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Vol. 274, Issue 2, R494-R502, February 1998

Characterization of the ryanodine receptor/channel of invertebrate muscle

Kerry E. Quinn1, Loriana Castellani2,3, Karol Ondrias1, and Barbara E. Ehrlich1

1 Department of Physiology, University of Connecticut, Farmington, Connecticut 06030; 2 Rosenstiel Center, Brandeis University, Waltham 02254; and 3 Marine Biological Laboratory, Woods Hole, Massachusetts 02543

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Electron-microscopic analysis was used to show that invertebrate muscle has feetlike structures on the sarcoplasmic reticulum (SR) displaying the typical four-subunit appearance of the calcium (Ca2+) release channel/ryanodine receptor (RyR) observed in vertebrate skeletal muscle (K. E. Loesser, L. Castellani, and C. Franzini-Armstrong. J. Muscle Res. Cell Motil. 13: 161-173, 1992). SR vesicles from invertebrate muscle exhibited specific ryanodine binding and single channel currents that were activated by Ca2+, caffeine, and ATP and inhibited by ruthenium red. The single channel conductance of this invertebrate RyR was lower than that of the vertebrate RyR (49 and 102 pS, respectively). Activation of lobster and scallop SR Ca2+ release channel, in response to cytoplasmic Ca2+ (1 nM-10 mM), reflected a bell-shaped curve, as is found with the mammalian RyR. In contrast to a previous report (J.-H. Seok, L. Xu, N. R. Kramarcy, R. Sealock, and G. Meissner. J. Biol. Chem. 267: 15893-15901, 1992), our results show that regulation of the invertebrate and vertebrate RyRs is quite similar and suggest remarkably similar paths in these diverse organisms.

intracellular calcium release; skeletal muscle; lobster; scallop

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

JUNCTIONAL FEET SPANNING the gap between the terminal cisternae of the sarcoplasmic reticulum (SR) and the transverse tubule (t tubule)/cell surface membrane have been observed in striated muscles of a variety of animal species (31). Events occurring at the level of the t tubule-SR junction have long been recognized as responsible for linking depolarization of the cell's surface membrane with the release of intracellular Ca2+ (8). Although the mechanism of coupling between the two membranes forming the gap is not fully understood, considerable progress has been made recently through the identification of some of the molecular components of the junction (14, 28, 46, 53).

In mammalian skeletal muscle, a Ca2+ release channel has been found in the terminal cisternae of the SR (50). By use of the plant alkaloid ryanodine, the protein responsible for the channel activity has been isolated and identified morphologically with the feet spanning the junction (22, 23). When reconstituted into lipid bilayers, the isolated ryanodine receptor (RyR)/foot protein forms a Ca2+ release channel with pharmacological properties similar to those of the Ca2+ release channel of heavy SR (21, 22, 26). At the single channel level, activation of the RyR by Ca2+ results in a bell-shaped response curve. The concentration of free cytosolic Ca2+ required for peak activation of the mammalian RyR in skeletal muscle is 5 µM (9).

The junctional region between the SR and t tubules (or cell surface membrane) in invertebrates is morphologically very similar to that observed in vertebrates (16, 17). Despite similarities in structure, voltage dependence, and response to caffeine, invertebrate muscle fibers differ from skeletal muscle in their requirement for extracellular Ca2+ in excitation-contraction coupling (19, 20). Recent structural studies of junctional SR from scallop and other invertebrates have shown overall morphology and size of the feet very similar to skeletal muscle, and they are recognized as the Ca2+ release channel/RyR of vertebrate SR. However, the organization of the feet was different between vertebrate and invertebrate species (31).

In vertebrates, several isoforms of the RyR have been identified when different tissues are compared structurally (37, 43, 51, 52) and functionally (13, 47, 50). RyR isoforms also have been identified within the same tissue (2, 4, 36, 51), but the functional consequences of multiple RyR isoforms within one cell type are not known.

A Ca2+ release channel that exhibits properties distinct from those of the vertebrate release channels has recently been characterized in Homarus americanus (lobster) skeletal muscle (48). The lobster Ca2+ release channel was shown to bind ryanodine but exhibited a unique Ca2+ dependence, lacked regulation by ATP and caffeine, and was immunologically distinct from the vertebrate RyR (48). In contrast, other reports of the properties of the Ca2+ release channel from lobster were comparable to mammalian RyR channel properties (29, 41). Similarly, a high-molecular-weight protein from Caenorhabditis elegans was found to bind ryanodine and form a channel biophysically and pharmacologically comparable to those obtained from mammalian muscle (25).

We have characterized the structural and functional properties of a channel involved in Ca2+ release from scallop muscle (Placopecten magellanicus) and lobster tail muscle (H. americanus). These muscles were chosen because they are excellent model systems for studying the physiology of the contractile proteins of muscle. Results shown here and previously (31) demonstrate that SR from invertebrate muscle contains a high-molecular-weight molecule that is morphologically similar to the cardiac and skeletal RyR. We also found that this molecule binds ryanodine and exhibits single channel currents activated by Ca2+, caffeine, and ATP and inhibited by ruthenium red. Although the invertebrate form of the RyR appeared to be structurally similar to the mammalian form of the channel, the conductance was lower in the invertebrate RyR. In addition, the channel was modulated by Ca2+, with a bell-shaped dependence, as demonstrated for the mammalian RyR channel (3, 9, 35). From these properties, it appears that the invertebrate RyR is functionally similar to the vertebrate RyR and represents the Ca2+ release pathway, as in mammalian muscle.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Preparation of SR vesicles. Live scallops (P. magellanicus) and lobsters (H. americanus) were obtained from the Marine Biological Laboratory. For electron microscopy, SR vesicles were isolated from striated adductor muscle of scallop, as described by Castellani and Hardwicke (7), except N-tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid (TES) was substituted for NaPi. TES, leupeptin, and pepstatin A (1 µg/ml) were included in all solutions.

For ryanodine binding and single channel measurements, scallop SR vesicles were prepared as follows. About 10 g of fresh striated muscle were homogenized in 150 ml of 20 mM imidazole and 5 mM sodium azide (pH 7.0), with 0.2 µM aprotinin, 1 µM pepstatin, 1 µM leupeptin, and 0.2 mM phenylmethylsulfonyl fluoride (PMSF), pH 7.4. The homogenate was centrifuged at 2,600 g for 20 min to sediment cell debris. Cellular membranes were pelleted after centrifugation at 70,000 g for 30 min. The pellet was resuspended in a wash buffer containing 150 mM KCl, 20 mM 3-(N-morpholino)propanesulfonic acid, and the protease inhibitors listed above. The suspension was layered over a sucrose step gradient. The 20-40% interface was collected, and the pellet was suspended in wash buffer and centrifuged at 70,000 g for 30 min. The final pellet was resuspended in wash buffer containing 10% sucrose, frozen in liquid nitrogen, and stored at -80°C.

Lobster SR vesicles were prepared according to previously published methods (48). Briefly, the tail was homogenized in a 20 mM tris(hydroxymethyl)aminomethane (Tris) buffer containing 0.1 mM ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 0.2 µM aprotinin, 1 µM pepstatin, 1 µM leupeptin, and 0.2 mM PMSF, pH 7.4. The homogenate was strained through cheesecloth and centrifuged at 2,000 g. The supernatant was centrifuged at 45,000 g for 30 min. The pellet was pipetted over a sucrose step gradient and centrifuged at 89,000 g for 2 h. The 25-35% interface was collected, resuspended in a wash buffer (150 mM KCl, 20 mM 3-(N-morpholino)propanesulfonic acid, 0.1 mM EGTA, 0.2 µM aprotinin, 1 µM pepstatin, 1 µM leupeptin, and 0.2 mM PMSF, pH 7.4). The suspension was centrifuged at 35,000 g for 30 min. The pellet was homogenized in wash buffer, frozen in liquid nitrogen, and stored at -80°C.

Electron microscopy. Freshly prepared SR vesicles were diluted (~1:30) in 100 mM sodium acetate, 1 mM magnesium acetate, 0.2 mM EGTA, 1 mM Mg-ATP, and 10 mM TES, pH 7.0. The suspension (~5-10 µl) was placed on a freshly cleaved sheet of mica, fixed with 1% uranyl acetate, and rinsed extensively with 10% glycerol, and excess solution was drawn to form a thin film. The samples were visualized by rotary shadowing with platinum at an angle of 20° in an Edwards High Vacuum (Grand Island, NY) evaporator. Replicas, floated off in distilled water, were applied on 400-mesh copper grids. Electron micrographs were recorded in a Philips 301 electron microscope operated at 60 kV.

Ligand-binding assay. SR vesicles (0.4 mg/ml) were incubated with Bodipy-ryanodine (3-50 nM; Molecular Probes, Eugene, OR). Blank samples were incubated with 1,000× unlabeled ryanodine. Vesicles were incubated in a buffer containing 350 mM KCl and 50 mM Tris · HCl, pH 7.4, for 2 h at room temperature. Channels were activated by addition of 1 mM ATP and adjustment of the free Ca2+ to maximize channel openings (i.e., 5 µM for rabbit and scallop and 0.5 µM for lobster). After a 2-h incubation period, samples were spun and pellets were resuspended. Fluorescence was read (480 nm excitation, 510 nm emission), and the concentration of Bodipy-ryanodine was determined. Specific ryanodine binding is defined as the difference between total Bodipy-ryanodine binding and nonspecific binding measured in the presence of 1,000× unlabeled ryanodine.

Single channel measurements. The RyR was incorporated into planar lipid bilayers, as described previously (13). Bilayers were formed by painting a lipid mixture of phosphatidylethanolamine-phosphatidylcholine (Avanti Polar Lipids, Alabaster, AL) in decane, across a 100-µm hole in a Teflon membrane, which separated two halves of a Lucite chamber. The cis solution was a N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-Tris buffer without monovalent alkali metal and halide ions (250 mM HEPES and 125 mM Tris, pH 7.3). Ca2+ on the trans side [50 mM Ca(OH)2 and 250 mM HEPES] served as the only permeant ion in the system. SR vesicles were added to the cis side, and fusion with the lipid bilayer was induced by making the cis side hyperosmotic by the addition of 400-500 mM KCl. After the appearance of potassium and chloride channels, the cis side was perfused with the HEPES-Tris buffer. The free Ca2+ concentration on the cis side was adjusted to 5 µM using a Ca2+-EGTA-buffered solution. Channel currents were amplified using a bilayer clamp amplifier (Warner Instruments, New Haven, CT) and recorded on VHS tapes. Data were filtered to 1 kHz, digitized at 5 kHz, then transferred to a personal computer and analyzed with pClamp 6.0 (Axon Instruments, Foster City, CA). Mean open times are defined as the total time the channel was open divided by the number of openings.

    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

The Ca2+ release proteins were characterized by morphological analysis, ligand-binding properties, and electrophysiological measurements of SR vesicles incorporated into planar lipid bilayers.

Morphology. Preparations of scallop SR vesicles, visualized in the electron microscope by rotary shadowing, showed a mixture of cisternae and tubules (Fig. 1). The region of the cisternae responsible for the coupling with the surface membrane lacked the Ca2+-adenosinetriphosphatase and displayed arrays of feet, characterized by a fourfold symmetrical appearance. The overall morphology and dimensions of these structures are remarkably similar to those observed in skeletal muscle fibers of vertebrates, where they have been identified as the ryanodine-binding Ca2+ release channels (22, 26). These junctional regions are sometimes observed to extend into longitudinal SR tubules, which are characterized by the crystalline arrangement of the Ca2+-adenosinetriphosphatase molecules in ribbons of dimers.


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  		Fig. 1.   Electron micrographs of scallop sarcoplasmic reticulum (SR) vesicles rotary shadowed with platinum. Junctional SR regions display arrays of feet, characterized by a 4-lobed appearance (×180,000). A and B: junctional regions (arrowheads) extending into longitudinal SR tubes, characterized by pronounced striations due to regular arrangement of Ca2+-ATPase molecules in ribbons of dimers (arrows). C and D: junctional regions laying flat on mica; these regions have retained only a few feet. E: selected images of individual feet displaying typical 4-subunit appearance, resembling those observed in skeletal muscle (×348,000).

Ryanodine binding to vesicles. Specific ryanodine binding has been used to identify the protein responsible for Ca2+ release from the SR of vertebrate muscle. Specific ryanodine binding to scallop and lobster SR vesicles was found and is compared with binding to rabbit SR vesicles (Fig. 2). Lobster muscle SR vesicles bound ryanodine with a maximum value (Bmax) of 13 pmol/mg protein and a K1/2 of 8 nM (n = 2; Fig. 2A). Scallop bound ryanodine with a Bmax of 18 pmol/mg protein and a K1/2 of 12 nM (n = 3; Fig. 2B). Rabbit muscle SR vesicles bound ryanodine with a Bmax of 70 pmol/mg protein and a K1/2 of 8 nM (n = 3; Fig. 2C).


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  		Fig. 2.   Ryanodine binding to lobster (n = 2; A), scallop (n = 3; B), and rabbit (n = 3; C) SR vesicles. Ryanodine binding to SR vesicles was determined as described in EXPERIMENTAL PROCEDURES. Insets: Scatchard analysis for each respective binding curve.

Ca2+ channels incorporated into bilayers. Compared with current recordings of SR channels from lobster, scallop, and rabbit, the main difference in channel properties was the amplitude of the current, which was smaller in the invertebrate muscle (Fig. 3A). All-points histograms reveal that the maximal current flow through the open channel for lobster and scallop channels is approximately one-half that for rabbit (Fig. 3B). Specifically, the current, at a holding potential of 0 mV, with Ca2+ as the current carrier, was 1.8 ± 0.3 pA for lobster (n = 3), 2.1 ± 0.1 pA for scallop (n = 7), and 3.9 ± 0.1 pA for rabbit (n = 5). With use of the maximum opening observed as the unit current, the single channel slope conductances are shown for lobster, scallop, and rabbit (Fig. 4). The slope conductance was 49 pS for lobster (n = 3), 54 pS for scallop (n = 5), and 102 pS for rabbit (n = 5).


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  		Fig. 3.   Current recordings of a Ca2+-gated channel from lobster, scallop, and rabbit SR. A: current recordings at 0 mV from lobster (1 of 3 similar experiments), scallop (1 of 7 similar experiments), and rabbit SR (1 of 5 similar experiments); all were recorded with 2 µM free Ca2+ on cytoplasmic side. Channel openings are downward. Solid line indicates zero current; dashed line is drawn at 4 pA from zero-current baseline. B: analysis of channel current records; current amplitudes are shown at a holding potential of 0 mV for lobster channels (1 of 3 similar experiments), scallop channels (1 of 7 similar experiments), and rabbit ryanodine receptor (RyR) channels (1 of 5 similar experiments).


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  		Fig. 4.   Slope conductances for lobster and scallop Ca2+ release channels compared with rabbit RyR channel. Current-voltage relationship was measured on Ca2+ release channels from lobster and scallop SR, incorporated into lipid bilayers. Slope conductances for lobster (bullet , n = 3) and scallop (open circle , n = 5) were smaller than those for rabbit skeletal muscle RyR (black-square, n = 5).

The distribution of open and closed times for channels was similar among lobster, scallop, and rabbit (Fig. 5). Mean open times were 5 ± 3 ms for lobster (n = 3), 6 ± 2 ms for scallop (n = 7), and 12 ± 2 ms for rabbit (n = 5). The invertebrate channels responded to ligands that normally activate the mammalian RyR. Caffeine (Fig. 6) and Ca2+ (Fig. 7) activated the invertebrate RyR as well as ATP (not shown). Ryanodine (25 nM) modified the invertebrate channel, and the hexavalent cation ruthenium red (20 µM) blocked the caffeine-activated channel (Fig. 6). The channels from invertebrate muscle are less sensitive to ruthenium red than the Ca2+ release channel from vertebrate skeletal muscle: 1 µM ruthenium red completely inhibits the channel from vertebrate skeletal muscle, whereas 15-20 µM ruthenium red is needed to completely block the channel from invertebrate muscle in our experiments. A similar concentration of ruthenium red was required to inhibit ryanodine binding to SR vesicles of lobster muscle (41). In contrast, 1 mM ruthenium red was needed for partial inhibition of the channel from lobster muscle in a previous report (48).


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  		Fig. 5.   Mean open time of lobster (1 of 3 similar experiments, A), scallop (1 of 7 similar experiments, B), and rabbit (1 of 5 similar experiments, C) SR RyR channel.


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  		Fig. 6.   Effect of caffeine, ryanodine, and ruthenium red (R red) on invertebrate (scallop, 1 of 3 similar experiments) and vertebrate (rabbit, 1 of 7 similar experiments) SR Ca2+ release channels. Control conditions were recorded with 2 µM free Ca2+ on cytoplasmic side. Caffeine (10 mM) activated both channels. Ryanodine (25 nM) induced a modified substrate in both channels for scallop (n = 1). Inactivation of both channels is observed after treatment with 20 µM ruthenium red. Channel openings are downward. Solid line indicates zero current; dashed line is drawn at 4 pA from zero-current baseline.


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  		Fig. 7.   Ca2+ dependence of Ca2+ release channel from lobster (n = 3; A), scallop (n = 3; B), and rabbit (n = 4; C). Current traces of Ca2+-gated channels were examined, and open probability was determined at 10 nM-10 mM Ca2+. Open probabilities were plotted as a function of Ca2+ concentration, and resulting curves were calculated. For each species, data from at least 3 experiments were averaged and normalized to maximum Ca2+-activated open probability determined for rabbit.

The Ca2+ dependence of lobster and scallop SR Ca2+ release channels was compared with that of vertebrate SR channels. Channel activity followed a bell-shaped curve in response to increasing Ca2+ concentrations, with a peak activity at 0.5-1 µM for lobster SR (Fig. 7) and 5 µM for scallop SR. Peak activation of rabbit RyR occurred at 5 µM free Ca2+. Open probability values were fitted to functions similar to those used to describe the bell-shaped Ca2+ response curve of the Ca2+ release channels of cerebellum (3). A fitted curve was generated with an activation binding constant in lobster of 0.05 µM Ca2+ and an inhibitory binding constant of 5 µM Ca2+ (n = 3). In one of the three experiments, lobster channel activity on either side of the peak did not drop below an open probability of 0.2. For scallop the curve was fit with an activation binding constant of 0.15 µM and an inhibitory binding constant of 50 µM (n = 3). Rabbit RyR had an activating binding constant of 0.8 µM and an inhibitory binding constant of 50 µM (n = 4).

At 10 nM free Ca2+ the basal activity for channels was similar for lobster (5 ± 1%, n = 3), scallop (1 ± 1%, n = 3), and rabbit (4 ± 1%, n = 4). Absolute values for open probabilities at maximum Ca2+ activation were less for lobster (15 ± 6%, n = 3) and scallop (10 ± 2.4%, n = 3) than for rabbit (39 ± 5%, n = 4).

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

We have shown that SR membranes from invertebrate muscle contain a Ca2+ release channel/RyR that is structurally similar to the vertebrate striated muscle RyR (13, 47, 50). Several techniques have been used to identify the receptor/channels in these membranes: electron-microscopic analysis, ligand binding, and single channel recordings of channels in planar lipid bilayers.

The SR from scallop has arrays of bumps in rotary shadowed replicas that have the symmetrical four-lobed appearance of the feet structures from vertebrate muscle. The dimensions of the structures from scallop SR are identical to those observed in skeletal muscle fibers of vertebrates (270 × 270 Å) (56). These structures have been identified as the ryanodine-binding Ca2+ release channels in vertebrate muscles (4).

Other properties of the invertebrate RyR are qualitatively similar to those of vertebrate muscle RyR. We found little or no difference in ryanodine-binding characteristics between mammalian and invertebrate species. Previously reported values for ryanodine binding are K1/2 of 6-12 nM and Bmax of 5-30 pmol/mg in mammalian muscle (1, 11, 57) and K1/2 of 6.5 nM and Bmax of 10-17 pmol/mg in lobster (41, 48). Crude homogenates from C. elegans generated values for K1/2 similar to those obtained with lobster and scallop muscle SR vesicles (K1/2 = 26 nM) (25). However, Bmax was much lower in C. elegans (Bmax = 110 fmol/mg protein) (25), because ryanodine binding was measured in the whole organism, rather than in the isolated SR. Our experiments in scallop and lobster generated values for ryanodine binding similar to those previously reported.

Similar to the vertebrate RyR, the invertebrate RyR can be activated by Ca2+ or caffeine and can be inhibited by ruthenium red or ryanodine. Although there are some differences in the absolute amount of agonist or antagonist needed to activate or inhibit the channel (see below), the differences are within the range observed with vertebrate skeletal and cardiac muscle.

When single channel currents were observed, the recordings were different from those obtained with vertebrate muscle in Ca2+ conductance and Ca2+ dependence. The channel from scallop and lobster muscle opened with a current amplitude approximately one-half of that from the mammalian RyR. The single channel slope conductance of the RyR/channel is 49 pS for lobster and 50 pS for scallop, values that are considerably smaller than those for vertebrate skeletal muscle (120 pS) (13, 50), cardiac muscle (100 pS) (13, 47), and brain (107 pS) (34). The slope conductance for C. elegans, another invertebrate, is also reduced compared with vertebrate RyR (25). In symmetrical 250 mM potassium the slope conductance was 215 pS for C. elegans (25) and 700 pS for mammalian RyR (55).

The difference in the slope conductance between the vertebrate and the invertebrate species studied here may be related to reorientation of the subunits, substitutions in the amino acids lining the conduction pathway, or the presence (or concentration) of proteins associated with the invertebrate RyR, such as FK binding protein (FKBP). The possibility that a small shift in the orientation of the subunits comprising the channel complex may reduce the current amplitude has been demonstrated through the actions of ryanodine (30). Ryanodine appears to reorient the subunits of the channel complex such that the cytoplasmic vestibule has a smaller radius (30). This reorientation is associated with a reduction in conductance. The possibility that substitutions in the amino acids lining the conduction pathway may reduce the current amplitude has been demonstrated by the addition of sulfhydryl-reacting compounds such as methanethiosulfonate ethylammonium (45). These sulfhydryl-reactive compounds bind irreversibly to the channel complex and restrict cation movement through the RyR via a conformation shift in the subunits that comprise the central conduction pathway or by producing a partial block within the conduction pathway itself. The possibility that an associated protein could regulate the amplitude of the current has been demonstrated through the actions of FKBP (5, 12, 24). FKBP is a protein functionally associated with the RyR (5, 12, 24, 33, 54). When FKBP is dissociated from the RyR of mammalian muscle current, conductance is decreased (5, 12, 24) to a value similar to the conductance of native invertebrate RyR. It is not presently possible to test the hypothesis that the FKBP-RyR interaction is altered, because available antibodies to human FKBP do not recognize a specific protein in invertebrate muscle. Other hypotheses could be tested after the receptor has been cloned or with in-depth biochemical analysis.

The invertebrate channels were regulated by cytoplasmic Ca2+, and the relationship of the open probability to Ca2+ concentration could be described by a bell-shaped distribution, as is seen with the RyR of mammalian skeletal muscle. Our results are similar to other studies (29, 41), in which micromolar Ca2+ caused peak activation of the ryanodine binding to SR vesicles from lobster muscle. These results contrast with a previous report which showed that activated release of Ca2+ from lobster SR vesicles required Ca2+ in the millimolar range (48). In one study (41) properties of Ca2+ release channels were inferred from ryanodine binding to SR vesicles, results of which were different from the data presented by Seok et al. (48). The second study (29) described caffeine-induced contractions of muscle fibers. The data by Seok et al. also were not consistent with their findings.

There are several possible explanations for the differences observed in the Ca2+ dependence of channel activation. It is possible that the vesicles isolated by Seok et al. (48) were mostly inside-out vesicles. In this case, activation by high Ca2+ may reflect an intravesicular Ca2+-binding site that is sensitive to Ca2+ in the millimolar range (49). Another, more likely, explanation for the differences between the single channel measurements seen here and those found for Ca2+ release from lobster SR vesicles (48) may reflect two distinct Ca2+-sensitive release systems in lobster muscle: one Ca2+ release channel is similar to the RyR of mammalian skeletal muscle in its responses to the activators Ca2+, ATP, and caffeine; the other is insensitive to submicromolar cytosolic Ca2+ and may have an endogenous activator other than Ca2+. Some of the authors repeated their experiments and now detect, through single channel analysis and ryanodine-binding experiments, a bell-shaped distribution for Ca2+ activation of lobster SR Ca2+ release channel that matches closely lobster and scallop RyR channel activity described here (G. Meissner and L. Xu, personal correspondence).

It is still possible that lobster muscle contains two isoforms of the Ca2+ release channel. Several reports describe two immunologically, and sometimes functionally, distinct RyRs in skeletal muscle from fish (15, 39), amphibian (27, 42), and avian species (2). Ryanodine-sensitive Ca2+ release channels from the SR of frog skeletal muscle (6) and rat brain (32) respond to cytosolic Ca2+ in a biphasic manner. The authors fit the Ca2+ response data to two curves: one was a bell-shaped curve, with a peak channel activity at 4.5 µM free Ca2+, and resembled the Ca2+ response curve of the mammalian skeletal muscle RyR; the second curve reflected a sigmoidal Ca2+ response, in which the channel could be turned on by micromolar free Ca2+ but could not be blocked even at millimolar Ca2+. This later Ca2+ response curve is similar to that found in Ca2+ release studies from lobster muscle SR (48). RyR isoforms have been proposed to contribute to the different and specialized functions of a variety of muscle types. For example, RyR1 predominates in skeletal muscle, whereas RyR2 is the primary Ca2+ release channel in heart, and RyR3 exists primarily in brain (for review see Ref. 40), and there are increasing reports that these isoforms coexist within the same cell (10, 18, 38, 39, 44). Our findings add to the suggestion that divergent functional properties of RyRs may exist within the same muscle type to create a multitude of mechanisms for excitation-contraction coupling.

In summary, the morphological appearance of the foot structures and the pharmacology of the channels indicate that invertebrate RyR is structurally similar to the vertebrate striated muscle RyR. Regulation of the invertebrate Ca2+ release channel, studied here, by cytosolic compounds also was similar to the RyR from mammalian skeletal muscle. Although small differences in the response to cytoplasmic Ca2+ and the single channel conductance were observed, these differences may reflect subtle changes of small functional consequence. The possibility that two signaling pathways with distinct regulatory characteristics exist allows diversity in the pattern for Ca2+ signaling in invertebrate and vertebrate species.

Perspectives

The information provided by this study, which demonstrates several functional aspects of the RyR for invertebrate and vertebrate species, can be used as a reference for future studies. For example, the RyR gene sequences for the invertebrate and vertebrate RyR can be compared, and conserved regions could be mapped out as potential regions encoding ligand-binding sites, since the functional responses for the ligands tested are similar between these species. Likewise, regions of the gene sequence for vertebrate and invertebrate channels, purported to be responsible for forming the conduction pathway, can be compared, and differences could be examined for possible alterations in structure and function.

    ACKNOWLEDGEMENTS

We are grateful to Dr. Clara Franzini-Armstrong for suggesting scallop muscle for our study. We thank Dr. G. Meissner for discussions and for providing preprints of unpublished work and Drs. Ed Kaftan, Jim Watras, and Ilya Bezprozvanny for comments on the manuscript.

    FOOTNOTES

This study was supported in part by a grant from Telethon, Italy (L. Castellani) and National Institutes of Health Grants HL-33026 and GM-51480 (B. E. Ehrlich). K. E. Quinn was supported by a grant from the Connecticut Affiliate of the American Heart Association and the Grass Foundation.

Present addresses: K. Ondrias, Institute of Molecular Physiology and Genetics, Slovak Academy of Sciences, Vlarska 5, 83334 Bratislava, Slovak Republic; L. Castellani, Experimental Medicine, University di Roma "Tor Vergata," via de Tor Vergata 135, 1-00137 Rome, Italy.

Address for reprint requests: K. E. Quinn, Laboratory of Molecular Hermeneutics, Dept. of Pharmacology, Yale University School of Medicine, 333 Cedar St., New Haven, CT 06520.

Received 5 June 1997; accepted in final form 15 September 1997.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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