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.

Diffusion of single cardiac ryanodine receptors in lipid bilayers is decreased by annexin 12.

Diffusion of cardiac ryanodine receptors (RyR2) in lipid bilayers was characterized. RyR2 location was monitored by imaging fluo-3 fluorescence due to Ca2+ flux through RyR2 channels or fluorescence from RyR2 conjugated with Alexa 488 or containing green fluorescent protein. Single channel currents were recorded to ensure that functional channels were studied. RyR2 exhibited an apparent diffusion coefficient (DRyR) of 1.2 x 10(-8) cm2 s(-1) and a mean path length of 5.0 microm. Optimal use of optical methods for analysis of RyR2 channel function requires that RyR2 diffusion be limited. Therefore, we tested the effect of annexin 12, which interacts with anionic phospholipids in a Ca2+-dependent manner. Addition of annexin 12 (0.25-4.0 microM) to the trans side of bilayers containing an 80:20 ratio of phosphatidylethanolamine/phosphatidylserine decreased RyR2 diffusion in a concentration-dependent manner. Annexin 12 (2 microM) decreased the apparent DRyR 683-fold from 1.2-10(-8) to 1.8 x 10(-11) cm2 s(-1) and the mean path length 10-fold from 5.0 to 0.5 micro m without obvious changes in the conductance of the native bilayer or in activation of RyR2 channels by Ca2+ or suramin. Thus, annexin 12 may provide a useful tool for optimizing optical analysis of RyR2 channels in lipid bilayers.

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.

Localization of ryanodine receptors in smooth muscle.

The ryanodine receptor (RyR) in aortic and vas deferens smooth muscle was localized using immunofluorescence confocal microscopy and immunoelectron microscopy. Indirect immunofluorescent labeling of aortic smooth muscle with anti-RyR antibodies showed a patchy network-like staining pattern throughout the cell cytoplasm, excluding nuclei, in aortic smooth muscle and localized predominantly to the cell periphery in the vas deferens. This distribution is consistent with that of the sarcoplasmic reticulum (SR) network, as demonstrated by electron micrographs of osmium ferrocyanide-stained SR in the two smooth muscles. Immunoelectron microscopy of vas deferens smooth muscle showed anti-RyR antibodies localized to both the sparse central and predominant peripheral SR elements. We conclude that RyR-Ca2+-release channels are present in both the peripheral and central SR in aortic and vas deferens smooth muscle. This distribution is consistent with the possibility that both regions are release sites, as indicated by results of electron probe analysis, which show a decrease in the Ca2+ content of both peripheral and internal SR in stimulated smooth muscles. The complex distribution of inositol 1,4,5-trisphosphate and ryanodine receptors (present study) is compatible with their proposed roles as agonist-induced Ca2+-release channels and origins of Ca2+ sparks, Ca2+ oscillations, and Ca2+ waves.

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.

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.

Structural components of ryanodine responsible for modulation of sarcoplasmic reticulum calcium channel function.

Comparative molecular field analysis (CoMFA) was used to analyze the relationship between the structure of a group of ryanoids and the modulation of the calcium channel function of the ryanodine receptor. The conductance properties of ryanodine receptors purified from sheep heart were measured using the planar, lipid bilayer technique. The magnitude of the ryanoid-induced fractional conductance was strongly correlated to specific structural loci on the ligand. Briefly, electrostatic effects were more prominent than steric effects. The 10-position of the ryanoid had the greatest influence on fractional conductance. Different regions of the ligand have opposing effects on fractional conductance. For example, steric bulk at the 10-position is correlated with decreased fractional conductance, whereas steric bulk at the 2-position (isopropyl position) is correlated with increased fractional conductance. In contrast to fractional conductance, the 3-position (the pyrrole locus) had the greatest influence on ligand binding, whereas the 10-position had comparatively little influence on binding. Two possible models of ryanodine action, a direct (or channel plug) mechanism and an allosteric mechanism, were examined in light of the CoMFA. Taken together, the data do not appear to be consistent with direct interaction between ryanodine and the translocating ion. The data appear to be more consistent with an allosteric mechanism. It is suggested the ryanoids act by inducing or stabilizing a conformational change in the ryanodine receptor that results in the observed alterations in cation conductance.

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.

Remodeling of cytoskeleton and triads following activation of v-Src tyrosine kinase in quail myotubes.

To study the cellular signals underlying the regulatory mechanisms involved in maintenance of sarcomeric integrity, we have used quail skeletal muscle cells that reach a high degree of structural maturation in vitro, and also express a temperature-sensitive mutant of the v-Src tyrosine kinase that allows the control of differentiation in a reversible manner. By immunofluorescence and electron microscopy we show that v-Src activity in myotubes leads to an extensive cellular remodeling which affects components of the sarcomeres, the cytoskeleton network and the triad junctions. We have previously shown that activation of v-Src causes a selective dismantling of the I-Z-I segments coupled to the formation of aggregates of sarcomeric actin, alpha-actinin and vinculin, called actin bodies. We now show that intermediate filaments do not participate in the formation of actin bodies, while talin, a component of costameres, does. The I-Z-I segments are completely dismantled within 24 hours of v-Src activity, but the A-bands persist for a longer time, implying distinct pathways for the turnover of sarcomeric subdomains. Immunofluorescence labeling of markers of the triad junctions demonstrates that the localization of the alpha 1 subunit of the dihydropyridine receptor is disrupted earlier than that of the ryanodine receptor after tyrosine kinase activation. Furthermore, the location of junctional sarcoplasmic reticulum and transverse tubule membranes is maintained in myotubes in which the I-Z-I have been removed and the regular disposition of the intermediate filaments is disrupted, supporting a role for sarcoplasmic reticulum in the proper positioning of triad junctions. Altogether these results point to a tyrosine kinase signaling cascade as a mechanism for selectively destabilizing sarcomere subdomains and their tethering to the cytoskeleton and the sarcolemma.