Foot protein isoforms are expressed at different times during embryonic chick skeletal muscle development.

We have investigated the time course of expression of the alpha and beta triad junctional foot proteins in embryonic chick pectoral muscle. The level of [3H]ryanodine binding in muscle homogenates is low until day E20 of embryonic development, then increases dramatically at the time of hatching reaching adult levels by day N7 posthatch. The alpha and beta foot protein isoforms increase in abundance concomitantly with [3H]ryanodine binding. Using foot protein isoform-specific antibodies, the alpha foot protein is detected in a majority of fibers in day E10 muscle, while the beta isoform is first observed at low levels in a few fibers in day E15 muscle. A high molecular weight polypeptide, distinct from the alpha and beta proteins, is recognized by antifoot protein antibodies. This polypeptide is observed in day E8 muscle and declines in abundance with continued development. It appears to exist as a monomer and does not bind [3H]ryanodine. In contrast, the alpha isoform present in day E10 muscle and the beta isoform in day E20 muscle are oligomeric and bind [3H]ryanodine suggesting that they may exist as functional calcium channels in differentiating muscle. Comparison of the intracellular distributions of the alpha foot protein, f-actin, the heavy chain of myosin and titin in day E10 muscle indicates that the alpha foot protein is expressed during myofibril assembly and Z line formation. The differential expression of the foot protein isoforms in developing muscle, and their continued expression in mature muscle, is consistent with these proteins making different functional contributions. In addition, the expression of the alpha isoform during the time of organization of a differentiated muscle morphology suggests that foot proteins may participate in events involved in muscle differentiation.

Ryanodine and inositol trisphosphate receptors coexist in avian cerebellar Purkinje neurons.

Two intracellular calcium-release channel proteins, the inositol trisphosphate (InsP3), and ryanodine receptors, have been identified in mammalian and avian cerebellar Purkinje neurons. In the present study, biochemical and immunological techniques were used to demonstrate that these proteins coexist in the same avian Purkinje neurons, where they have different intracellular distributions. Western analyses demonstrate that antibodies produced against the InsP3 and the ryanodine receptors do not cross-react. Based on their relative rates of sedimentation in continuous sucrose gradients and SDS-PAGE, the avian cerebellar InsP3 receptor has apparent native and subunit molecular weights of approximately 1,000 and 260 kD, while those of the ryanodine receptors are approximately 2,000 and 500 kD. Specific [3H]InsP3- and [3H]ryanodine-binding activities were localized in the sucrose gradient fractions enriched in the 260-kD and the approximately 500-kD polypeptides, respectively. Under equilibrium conditions, cerebellar microsomes bound [3H]InsP3 with a Kd of 16.8 nM and Bmax of 3.8 pmol/mg protein; whereas, [3H]ryanodine was bound with a Kd of 1.5 nM and a capacity of 0.08 pmol/mg protein. Immunolocalization techniques, applied at both the light and electron microscopic levels, revealed that the InsP3 and ryanodine receptors have overlapping, yet distinctive intracellular distributions in avian Purkinje neurons. Most notably the InsP3 receptor is localized in endomembranes of the dendritic tree, in both the shafts and spines. In contrast, the ryanodine receptor is observed in dendritic shafts, but not in the spines. Both receptors appear to be more abundant at main branch points of the dendritic arbor. In Purkinje neuron cell bodies, both the InsP3 and ryanodine receptors are present in smooth and rough ER, subsurface membrane cisternae and to a lesser extent in the nuclear envelope. In some cases the receptors coexist in the same membranes. Neither protein is observed at the plasma membrane, Golgi complex or mitochondrial membranes. Both the InsP3 and ryanodine receptors are associated with intracellular membrane systems in axonal processes, although they are less abundant there than in dendrites. These data demonstrate that InsP3 and ryanodine receptors exist as unique proteins in the same Purkinje neuron. These calcium-release channels appear to coexist in ER membranes in most regions of the Purkinje neurons, but importantly they are differentially distributed in dendritic processes, with the dendritic spines containing only InsP3 receptors.

Identification and localization of ryanodine binding proteins in the avian central nervous system.

Ryanodine binding proteins of the CNS have been identified using monoclonal antibodies against avian skeletal muscle ryanodine binding proteins. These proteins were localized to intracellular membranes of the dendrites, perikarya, and axons of cerebellar Purkinje neurons using laser confocal microscopy and immunoelectron microscopy. Ryanodine binding proteins were not found in dendritic spines. Immunoprecipitation and [3H]epiryanodine binding experiments revealed that the cerebellar ryanodine binding proteins have a native molecular weight of approximately 2000 kd and are composed of two high molecular weight (approximately 500 kd) polypeptide subunits. A comparable protein having a single high molecular weight polypeptide subunit was observed in the remainder of the brain. If the ryanodine binding proteins in muscle and nerve are similar in function, then the neuronal proteins may participate in the release of calcium from intracellular stores that are mechanistically and spatially distinct from those gated by inositol trisphosphate receptors.

Concentrations of caffeine greater than 20 mM increase the indo-1 fluorescence ratio in a Ca(2+)-independent manner.

The methylxanthine, caffeine, quenches the fluorescence of the ratiometric Ca2+ indicator indo-1, but does not affect the ratio (R) of indo-1 fluorescence at 400 and 500 nm in the presence of caffeine concentrations up to 10 mM [1]. We have found that when caffeine is at concentrations of 20 mM or greater in vitro, or in saponinpermeabilized skeletal muscle fibers, a Ca(2+)-independent increase in R occurs, which leads to an overestimation of the free Ca2+ concentration. Depending on experimental conditions, two factors contribute to the alteration in R in vitro. First, when indo-1 fluorescence is low, fluorescence by caffeine, at 400 nm, can be significant. A second, and more dramatic effect, is that quenching of indo-1 fluorescence by 20-50 mM caffeine is dissimilar at 400 and 500 nm. Quenching at 500 nm is not linear, with respect to the concentration of caffeine, and causes a Ca(2+)-independent increase in R, that occurs even when the fluorescence of caffeine is a small portion of total fluorescence. However, unlike R, the Ca2+ calibration constant of indo-1, KD beta, is unchanged in 50 mM caffeine. Therefore, an accurate quantitation of Ca2+ in the presence of even high concentrations of caffeine can be made in vitro by determining the Ca2+ calibration factors of indo-1 (RMIN and RMAX) for each caffeine concentration. These effects of concentrations of caffeine greater than 20 mM are not observed in intact cells loaded with the cell permeant form of indo-1 when caffeine is applied extracellularly. This suggests either that the concentration of caffeine within the cell does not reach that necessary to produce the effect, or that the effects of caffeine on the dye are modified by the environment within the cell.

Immunohistochemical localization of ryanodine binding proteins in the central nervous system of gymnotiform fish.

The ryanodine receptor, an integral membrane protein of the sarcoplasmic reticulum in muscle, embodies a high conductance channel permeable to calcium ions. Recent studies have identified ryanodine-binding proteins in avian and mammalian central nervous systems. These neuronal ryanodine receptors appear to function as Ca2+ channels which may gate the release of Ca2+ from caffeine-sensitive intracellular pools in neurons. In the present investigation, we employed monoclonal antibodies against ryanodine-binding proteins of avian muscle cells to the brain of weakly electric gymnotiform fish. Immunoprecipitation and Western blot analysis revealed two isoforms in the fish brain, with molecular weights comparable to those of avian and fish muscle ryanodine-binding proteins. By employing immunohistochemical techniques, we mapped these proteins in fish brain. Ryanodine receptor-like immunoreactivity was found in nerve cell bodies as well as dendrites and axonal processes. The ryanodine-binding protein is distributed throughout the neuraxis in specific cell types of the gymnotiform brain. In the telencephalon, immunoreactive cells were found in the glomerular layer of the olfactory bulb, in the supracommissural subdivision of the ventral telencephalon, and in the intermediate rostral subdivision of the ventral telencephalon. In the diencephalon, immunoreactive cells or fibers were observed in the nucleus prethalamicus and the habenula, within the nucleus at the base of the optic tract and the adjacent dorsal tegmental nucleus, the pretectal nuclei A and B, and the nucleus electrosensorius. In addition, immunopositive cells were seen in several nuclei of the hypothalamus, with the inferior and lateral subdivision of the nucleus recessus lateralis displaying the highest concentration of neurons. In the mesencephalon, the optic tectum contained the greatest number of immunopositive cells. In the rhombencephalon, labelling was seen in the nucleus of the lateral valvula, central gray, lateral tegmental nucleus, in boundary cells of the nucleus praeminentialis, efferent octavolateral nucleus, an area adjacent to the medial edge of the lateral reticular nucleus, nucleus medialis, and electrosensory lateral line lobe. As in avian brain, cerebellar Purkinje cells were positive for ryanodine-binding protein, although only subsets of Purkinje cells were labelled.

Distribution of inositol-1,4,5-trisphosphate and ryanodine receptors in rat neostriatum.

The distribution of the inositol-1,4,5-trisphosphate (IP3) and the cardiac form of the ryanodine receptor, two intracellular calcium channels, was examined in the rat neostriatum. Both IP3 and ryanodine receptor labeling occurred within striatal medium spiny cells but only ryanodine receptor labeling was present in choline acetyltransferase- and parvalbumin-positive interneurons. IP3 receptor labeling was observed within cell bodies, dendrites and spines of spiny striatal neurons, as seen at both the light and electron microscopic levels. Subcellular labeling for the ryanodine receptor was restricted to cell bodies and proximal dendrites when a polyclonal antibody raised against a peptide sequence from the dog cardiac ryanodine receptor was employed. More extensive dendritic labeling was seen using monoclonal antibody MA3-916, also raised against the canine cardiac ryanodine receptor. At the ultrastructural level, labeled dendritic spines were observed frequently with the monoclonal but not the polyclonal antibody. Ryanodine receptor labeling also was present within astrocytic processes surrounding blood vessels and within the neuropil, regardless of the antibody used. The results of these studies suggest that the ryanodine receptor plays a general role in intracellular calcium regulation within striatal cells while the IP3 receptor plays a specialized role within spiny neurons.

Distribution of ryanodine receptors in the chicken central nervous system.

The ryanodine receptor (RR), an intracellular calcium release channel, has been identified in the nervous system but its contributions to neuronal function are unknown. We have utilized immunohistochemical techniques to establish the distribution of RRs in the central nervous system (CNS) of the chick as a step toward elucidating the function of RRs in this system. RR immunoreactivity is observed throughout the brain, most prominently in large neurons. The strongest immunoreactivity is found in cerebellar Purkinje neurons, but nuclei in the motor, visual and vestibular systems are also intensely labeled, and immunoreactive neurons are observed the olfactory bulb and the hippocampus. In these neurons, labeling is prominent in cell bodies, dendrites and axons, but is not observed in the dendritic spines or in plasma membranes. The neuronal RRs bind [3H]ryanodine with high affinity and this activity is regulated by calcium, caffeine, MgCl2/ATP and ionic strength. Multiple forms of the RRs are found in the chicken CNS. Immunoprecipitation and localization studies using RR isoform specific monoclonal antibodies reveal major differences in their distribution. The predominant species in the cerebellum is similar to the skeletal muscle isoform while there is a lower level of expression of either the cardiac or beta skeletal isoforms. In the remainder of the brain, the predominant isoform is similar to the cardiac or beta skeletal muscle isoforms. The broad distribution of RRs in the CNS suggests that calcium release events mediated by these proteins may have a functional role in a diverse array of neurons. Moreover within the populations of neurons expressing RR's, the presence of specific RR isoforms may correlate with specialization in the calcium release events mediated by these proteins.

Differential distribution and subcellular localization of ryanodine receptor isoforms in the chicken cerebellum during development.

The distribution of ryanodine receptor (RyR) isoforms was examined using isoform-specific monoclonal antibodies in the developing chicken brain, from E18 through adulthood, using light and electron microscopic immunocytochemistry. Monoclonal antibody 110F is specific for the alpha-skeletal muscle form of RyR, while monoclonal antibody 110E recognizes both the beta-skeletal muscle and cardiac isoforms, but does not distinguish between the two. Significant differences in the distribution of the alpha- and beta/cardiac forms were observed. Labeling for the alpha-form was restricted to cerebellar Purkinje neurons while the beta/cardiac form was observed in neurons throughout the brain. A major finding was the presence of labeling for the beta/cardiac in presynaptic terminals of the parallel fibers in the molecular layer and the mossy fiber terminals in the granular layer glomeruli in late development and during adulthood. Labeling for the beta/cardiac, but not the alpha-form, underwent a major redistribution in the cerebellum during the course of development. At 1 day of age, beta/cardiac labeling was present mainly in Purkinje neurons. From 1 day to 4 weeks, immunolabeling for the beta/cardiac form gradually disappeared from Purkinje neurons, but increased in granule cells. Within the molecular layer, the labeling pattern changed from being primarily within Purkinje dendrites to a more diffuse pattern. Electron microscopic examination of the cerebellar molecular layer of 2-week-old chicks revealed that beta/cardiac-labeling was mainly present in the axons and presynaptic processes of the parallel fibers. No developmental changes were observed in other brain regions. This study represents the first demonstration of ryanodine receptor immunoreactivity in presynaptic boutons and suggests that the ryanodine receptor may modulate neurotransmitter release through local regulation of intracellular calcium in the parallel fiber synapse.

Ryanodine receptor Ca2+ release channels: does diversity in form equal diversity in function?

Complexities in calcium signaling in eukaryotic cells require diversity in the proteins involved in generating these signals. In this review, we consider the ryanodine receptor (RyR) family of intracellular calcium release channels. This includes species, tissue, and cellular distributions of the RyRs and mechanisms of activation, deactivation, and inactivation of RyR calcium release events. In addition, as first observed in nonmammalian vertebrate skeletal muscles, it is now clear that more than one RyR isoform is frequently coexpressed within many cell types. How multiple ryanodine receptor release channels are used to generate intracellular calcium transients is unknown. Therefore, a primary focus of this review is why more than one RyR is required for this purpose, particularly in a tissue, such as vertebrate fast-twitch skeletal muscles, where a relatively simple and straightforward change in calcium would appear to be required to elicit contraction. Finally, the roles of the RyR isoforms and the calcium release events they mediate in the development of embryonic skeletal muscle are considered.

Ryanodine receptor protein is expressed during differentiation in the muscle cell lines BC3H1 and C2C12.

BC3H1 and C2C12, murine cell lines, were assessed as model systems for the expression of ryanodine receptor protein during myogenesis. The ryanodine receptor is a calcium release channel of the sarcoplasmic reticulum and a component of the triad junction, a structure which is essential to excitation-contraction coupling in mature striated muscle. BC3H1 and C2C12 cells do not express the ryanodine receptor at detectable levels in a proliferative, nondifferentiated state. The ryanodine receptor protein is expressed during differentiation in BC3H1 and C2C12 cells, becoming detectable within 24 hr of the onset of differentiation. In both cell lines the ryanodine receptor is assembled in oligomeric form and binds [3H]ryanodine with high affinity. Fusion is not required for expression of the ryanodine receptor in either BC3H1 or nonfusing C2C12 cells. The level of expression of the ryanodine receptor protein is modulated by incubation with the growth factors TGF-beta and bFGF in a manner similar to that of other muscle-specific proteins. These initial observations suggest that the BC3H1 and C2C12 cell lines provide a model system for further investigations of the expression and function of the ryanodine receptor during myogenic differentiation.

Positioning of major tryptic fragments in the Ca2+ release channel (ryanodine receptor) resulting from partial digestion of rabbit skeletal muscle sarcoplasmic reticulum.

Site-specific antibodies against different regions of the Ca2+ release channel of skeletal muscle sarcoplasmic reticulum (ryanodine receptor) were developed and used as probes for immunoblotting of the major tryptic fragments resulting from partial digestion of the ryanodine receptor in sarcoplasmic reticulum membranes. Five major tryptic fragments, some of which migrated as doublets, with apparent masses of 150/140, 110/100, 55, 170/160, and 76 kDa were ordered so that they covered the bulk of the protein from the NH2 to the COOH terminus. Tryptic subfragments of 53, 63, and 115/95 kDa were also derived from the 150/140-, 110/100-, and 170/160-kDa fragments, respectively. All of these fragments and subfragments were detected only in the insoluble membrane fraction of the trypsinized sarcoplasmic reticulum. Upon Na2CO3 extraction, the 150/140-, 110/100-, and 55-kDa fragments could be solubilized, suggesting their origin in the cytoplasmic domain of the ryanodine receptor. The 170/160- and 76-kDa fragments and the 115/95-kDa subfragment remained insoluble, suggesting their origin in the transmembrane region of the ryanodine receptor. The 150/140-, 110/100-, 170/160-, and 76-kDa fragments and the 115/95 subfragment co-migrated near the bottom of a sucrose density gradient after CHAPS solubilization, suggesting that they were associated in an oligomeric complex. By contrast, the 53- and 63-kDa subfragments and the 55-kDa fragment were detected near the top of the sucrose gradient after CHAPS solubilization, suggesting that they were not involved in the formation of the core of the oligomeric complex. These studies identify 7 sites that are exposed to trypsin in the ryanodine receptor in sarcoplasmic reticulum, 3 of which are novel and 4 of which are in the same location as proteolytic cleavage sites identified previously (Marks, A. R., Fleischer, S., and Tempst, P. (1990) J. Biol. Chem. 265, 13143-13149).

Embryonic chicken skeletal muscle cells fail to develop normal excitation-contraction coupling in the absence of the alpha ryanodine receptor. Implications for a two-ryanodine receptor system.

Two ryanodine receptor (RyR), sarcoplasmic reticulum Ca2+ release channels, alpha and beta, co-exist in chicken skeletal muscles. To investigate a two-RyR Ca2+ release system, we compared electrically evoked Ca2+ transients in Crooked Neck Dwarf (cn/cn) cultured muscle cells, which do not make alpha RyR, and normal (+/?) cells. At day 3 in culture, Ca2+ release in +/? cells required extracellular Ca2+ (Ca2+o), and Ca2+ transients had slow kinetics. At day 5, Ca2+ release was Ca2+o-independent in 40% of the cells, and transients were more rapid. By day 7, all +/? cells had Ca2+o-independent Ca2+ release. Contractions were observed in +/? cells on all days. Ca2+ transients were observed in cn/cn cells on days 3, 5, and 7, but in each case they were Ca2+o-dependent and exhibited slow kinetics. Localized vesiculations, not contractions, occurred in cn/cn cells. By day 10, Ca2+ transients were no longer observed in cn/cn cells even in Ca2+o. Sarcoplasmic reticulum Ca2+ was not depleted, as caffeine induced Ca2+ transients. Thus, in the absence of alpha RyR there is a failure to develop Ca2+o-independent Ca2+ release and contractions and to sustain Ca2+o-dependent release. Moreover, contributions by the alpha RyR cannot be duplicated by the beta RyR alone.

A caffeine- and ryanodine-sensitive Ca2+ store in avian sensory neurons.

1. We identified and studied the function of ryanodine receptors in neurons isolated from dorsal root ganglia (DRG) of 10-day-old chick embryos. 2. A monoclonal antibody (mAb 34C) that recognizes all known ryanodine receptor isoforms in skeletal and cardiac muscle and CNS identified ryanodine receptor-like immunoreactivity in cultured DRG neurons. 3. Using the permeabilized patch technique to record membrane currents, we found that calcium currents were followed by a current with characteristics of a Ca(2+)-activated Cl- current (ICl(Ca)) in approximately two-thirds of the neurons. In these cells, acute application of 10 mM caffeine activated a similar ICl(Ca) and this effect was inhibited by 10 microM ryanodine. The activation of ICl(Ca) by caffeine was not dependent on extracellular Ca2+. These data suggest that caffeine raises intracellular free Ca2+ (Cai2+) by activating the release of Ca2+ from an intracellular store and that this Ca2+ activates the membrane conductance responsible for ICl(Ca). 4. The magnitude of ICl(Ca) activated by depolarization was not affected by ryanodine, implying that the Ca2+ that activates ICl(Ca) in this protocol is supplied by the Ca2+ current without amplification by a ryanodine-sensitive mechanism such as Ca(2+)-induced Ca2+ release. 5. We also used indo-1 to measure Cai2+ in DRG neurons. Ten millimolar caffeine caused a transient increase in Cai2+ that was inhibited by 10 microM ryanodine. 6. The ability of caffeine to elevate Cai2+ and activate ICl(Ca) was reduced at higher temperatures, suggesting increased Ca2+ sequestration. 7. These data demonstrate the existence of an intracellular store of Ca2+ that can be mobilized by a caffeine- and ryanodine-sensitive mechanism. The release of Ca2+ from this store can elevate Cai2+ and modulate membrane conductances.

Functional expression of cDNA encoding the Ca2+ release channel (ryanodine receptor) of rabbit skeletal muscle sarcoplasmic reticulum in COS-1 cells.

A full-length cDNA encoding the ryanodine receptor of rabbit skeletal muscle sarcoplasmic reticulum was transiently expressed in COS-1 cells. Immunoblotting studies showed that the expressed ryanodine receptor and the native ryanodine receptor of rabbit skeletal muscle were indistinguishable in molecular size and immunoreactivity. Scatchard analysis of [3H]ryanodine binding to transfected COS-1 cell microsomes resulted in a Bmax of 0.22 pmol/mg of protein and a Kd of 16.2 nM. Expressed ryanodine receptors were solubilized in CHAPS and were shown to cosediment with native ryanodine receptors in a sucrose density gradient. Thus, the expressed receptor, like the native receptor, is assembled as a large oligomeric complex. Single-channel recordings in planar lipid bilayers were used to investigate the functional properties of the sucrose gradient-purified complex. The expressed ryanodine receptor formed a large conductance channel activated by ATP and Ca2+ and inhibited by Mg2+ and ruthenium red. Ryanodine reduced the conductance and increased the mean open time in a manner consistent with that of native channels. These results demonstrated that functional binding sites for the physiological ligands (Ca2+, Mg2+, and ATP) and pharmacological ligands (ruthenium red and ryanodine) controlling gating of the Ca2+ release channel are encoded in the ryanodine receptor cDNA and are faithfully expressed in COS-1 cells. Ryanodine receptors expressed in COS-1 cells displayed several conductance states > or = 1 nS not present in native channels. Such anomalous conductance states of the expressed channel might be referable to lack of muscle-specific posttranslational processing or to the need for components not present in COS-1 cells, which may be required to stabilize the channel structure.

Interaction between ryanodine receptor function and sarcolemmal Ca2+ currents.

We used the whole cell voltage-clamp technique to investigate the effects of disruption of Ca2+ release from the sarcoplasmic reticulum (SR) on sarcolemmal Ca2+ currents of chick myotubes kept in culture for 7 or 8 days. Ca2+ currents were recorded in 145 mM tetraethylammonium chloride and 10 mM Ca2+ with pipettes containing cesium and 10 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. We found two components of Ca2+ current: 1) relatively large T-type currents that were activated near -50 mV and inactivated during 100-ms depolarizations to potentials positive to -60 mV (they were of similar magnitude in Ba2+ or Ca2+ and were insensitive to nifedipine) and 2) L-type currents that were activated near 0 mV and showed little or no inactivation during 100-ms depolarizations (they were larger when Ba2+ was the charge carrier and were blocked by 10 microM nifedipine). Addition of 1 or 100 microM ryanodine to the culture medium for 6-7 days caused a modest but significant increase in the L-type Ca2+ current density (pA/pF). Ryanodine (1 or 100 microM) exposure for 1-7 days reduced the T-type Ca2+ current density to < 10% of control. In contrast, exposure to 1 microM ryanodine for 0.5-3 h had no significant effect on either component of Ca2+ current. These data indicate that ryanodine has no direct action on Ca2+ currents in chick myotubes. However, disruption of SR Ca2+ release for > 24 h changes sarcolemmal Ca2+ channel expression or function.

Three ryanodine receptor isoforms exist in avian striated muscles.

Two isoforms of the ryanodine receptor (termed alpha and beta) are coexpressed in avian fast twitch skeletal muscle, whereas a single isoform is expressed in avian cardiac muscle. We have investigated the relationship between these three proteins, comparing several different properties. First, the three receptor isoform subunits have different mobilities on SDS-polyacrylamide gels. Second, monoclonal antibodies against the chicken skeletal muscle receptor isoforms recognize shared and unique epitopes in each receptor protein, indicating there is not a simple antigenic relationship between the isoforms. Third, the three receptor isoforms exhibit different susceptibilities to proteolysis by trypsin, and limited tryptic digestion yields a different peptide map for each isoform. Fourth, in native sarcoplasmic reticulum membranes, the chicken muscle receptor isoforms are phosphorylated to different extents by the multifunctional calcium/calmodulin-dependent protein kinase II (beta > cardiac > alpha). Fifth, the sites phosphorylated by the calcium/calmodulin-dependent protein kinase in the chicken cardiac and skeletal receptor isoforms are not equivalent. A polyclonal serum, produced against a synthetic peptide containing the site phosphorylated by this kinase in the mammalian cardiac muscle receptor, by immunoprecipitation showed markedly different avidities for the receptor isoforms, and recognized only the cardiac receptor isoform on Western blots. Sixth, the chicken ryanodine receptor isoforms differ in the extent to which they bind azido[125I]calmodulin (alpha > beta > cardiac). These results indicate that three distinct ryanodine receptor proteins are expressed in chicken striated muscles.

The pharmacology of ryanodine and related compounds.

The goal of this review has been to describe the current state of the pharmacology of ryanodine and related compounds relative to the vertebrate RyRs. Resolution of questions concerning the molecular properties of RyR channel function and the contributions made by the RyR isoforms to cellular signaling in a variety of tissues will require the production of new pharmacological agents directed against these proteins. Novel naturally occurring ryanodine congeners have been identified, and significant advances have been made in developing chemical approaches that permit the structure of ryanodine to be derivatized in selective ways. Moreover, several of these changes have yielded compounds that differ in their binding affinities and in their abilities to modify the properties of the RyR channels. These advances give substance to the possibility of designing the required pharmacological agents based on rational design changes of the structure ryanodine.

Chicken skeletal muscle ryanodine receptor isoforms: ion channel properties.

To define the roles of the alpha- and beta-ryanodine receptor (RyR) (sarcoplasmic reticulum Ca2+ release channel) isoforms expressed in chicken skeletal muscles, we investigated the ion channel properties of these proteins in lipid bilayers. alpha- and beta RyRs embody Ca2+ channels with similar conductances (792, 453, and 118 pS for K+, Cs+ and Ca2+) and selectivities (PCa2+/PK+ = 7.4), but the two channels have different gating properties. alpha RyR channels switch between two gating modes, which differ in the extent they are activated by Ca2+ and ATP, and inactivated by Ca2+. Either mode can be assumed in a spontaneous and stable manner. In a low activity mode, alpha RyR channels exhibit brief openings (tau o = 0.14 ms) and are minimally activated by Ca2+ in the absence of ATP. In a high activity mode, openings are longer (tau o1-3 = 0.17, 0.51, and 1.27 ms), and the channels are activated by Ca2+ in the absence of ATP and are in general less sensitive to the inactivating effects of Ca2+. beta RyR channel openings are longer (tau 01-3 = 0.34, 1.56, and 3.31 ms) than those of alpha RyR channels in either mode. beta RyR channels are activated to a greater relative extent by Ca2+ than ATP and are inactivated by millimolar Ca2+ in the absence, but not the presence, of ATP. Both alpha- and beta RyR channels are activated by caffeine, inhibited by Mg2+ and ruthenium red, inactivated by voltage (cytoplasmic side positive), and modified to a long-lived substate by ryanodine, but only alpha RyR channels are activated by perchlorate anions. The differences in gating and responses to channel modifiers may give the alpha- and beta RyRs distinct roles in muscle activation.

Imaging single cardiac ryanodine receptor Ca2+ fluxes in lipid bilayers.

In this and an accompanying report we describe two steps, single-channel imaging and channel immobilization, necessary for using optical imaging to analyze the function of ryanodine receptor (RyR) channels reconstituted in lipid bilayers. An optical bilayer system capable of laser scanning confocal imaging of fluo-3 fluorescence due to Ca2+ flux through single RyR2 channels and simultaneous recording of single channel currents was developed. A voltage command protocol was devised in which the amplitude, time course, shape, and hence the quantity of Ca2+ flux through a single RyR2 channel is controlled solely by the voltage imposed across the bilayer. Using this system, the voltage command protocol, and concentrations of Ca2+ (25-50 mM) that result in saturating RyR2 Ca2+ currents, proportional fluo-3 fluorescence was recorded simultaneously with Ca2+ currents having amplitudes of 0.25-14 pA. Ca2+ sparks, similar to those obtained with conventional microscope-based laser scanning confocal systems, were imaged in mouse ventricular cardiomyocytes using the optical bilayer system. The utility of the optical bilayer for systematic investigation of how cellular factors extrinsic to the RyR2 channel, such as Ca2+ buffers and diffusion, alter fluo-3 fluorescent responses to RyR2 Ca2+ currents, and for addressing other current research questions is discussed.