Dyadic Junctions Research Articles

Commentaries on Viewpoint: The cardiac contraction cycle: Is Ca2+ going local?

Commentaries on Viewpoint: The cardiac contraction cycle: Is Ca²⁺ going local?

Multiple levels of the single L-type Ca2+ channel conductance in adult mammalian ventricular myocytes

In cardiac muscle, Ca2+ entry through the L-type Ca2+ channel during excitation triggers Ca2+ release from sarcoplasmic reticulum (SR) via the ryanodine receptor, causing muscle contraction. This Ca2+ induced Ca2+ release (CICR) is locally controlled by openings of the single L-type Ca2+ channel that triggers the opening of ryanodine receptors located within the dyadic junction. The unitary current through the single Ca2+ channel determines the open probability of the ryanodine receptors, and hence the efficacy of CICR. Here, we report findings of multiple conductance levels of L-type Ca2+ channels in freshly isolated rat ventricular myocytes. When 10mM Ca2+ was used in the pipette solution as the charge carrier in the cell-attached configuration, the most frequently occurring conductance levels are 6.9pS and 2.9pS. Three distinct conductance levels were also observed, although infrequently, in the same cell, corresponding to unitary currents of 0.33pA, 0.24pA, and 0.17pA upon depolarization to −10mV. In conclusion, our data demonstrate the existence of multiple single L-type Ca2+ channel conductance levels in the adult mammalian ventricular myocytes with Ca2+ as the charge carrier. The multiple conductance levels present heterogeneity in the Ca2+ trigger signal strength at local dyadic junctions. The physiological significance of this heterogeneity on affecting the efficacy of CICR in the cardiac muscle is discussed.

AB31-3: Factors required for graded calcium release in a simplified computational model of the dyadic junction: Importance of junctional variation

AB31-3: Factors required for graded calcium release in a simplified computational model of the dyadic junction: Importance of junctional variation

Triadin

See related article, pages 651–658 The Ca2+ movements that control many cellular functions, including contraction of striated and smooth muscle cells and vesicular secretion from endocrine cells and nerve terminals, are increasingly recognized to involve macromolecular Ca2+-signaling complexes that, in addition to the key Ca2+-transporting proteins, include large numbers of associated proteins that provide a variety of regulatory, structural and Ca2+-sensing/buffering functions. The importance of working out the subtle interactions within these macromolecular complexes is underscored by the genetic diseases that have been associated with mutations of their constituent proteins. In this issue, Terentyev et al1 use a spectrum of molecular and electrophysiological techniques to demonstrate that triadin (TRD) plays an unexpectedly important role in regulating the ryanodine receptors (RyRs), found primarily in dyadic junctions of cardiac sarcoplasmic reticulum (SR; Figure). It has been previously suggested that TRD and junctin are integral membrane proteins of the junctional SR, and serve as linker proteins from the SR Ca release channel (RyR) to calsequestrin (CSQ) complexes, the major Ca2+-buffer in the lumen of the SR (Figure).2 The large (≈4500 aa) cytoplasmic domain of RyR appears to have multiple binding sites for an ever-growing list of proteins that includes: calmodulin, PKA, FKBP 12.6.3 The major findings of the present communication are that overexpression of TRD leads to 3-fold increase in open probability of RyRs in bilayers, a 60% increase in spontaneous spark frequency with only minor decreases in spark amplitude (≈10%) and SR Ca2+ content (≈30%), as well as a marked alteration in the voltage dependence of Ca2+ release. The authors propose the activity of RyRs to be directly modulated by the level of expression of TRD, most likely mediated by amino acid residues 200 to 224 …

Calcium signaling between sarcolemmal calcium channels and ryanodine receptors in heart cells.

Cardiac excitation-Ca2+ release coupling is, in essence, a tale of two molecules, sarcolemmal voltage-gated L-type Ca2+ channels (LCCs) and intracellular ryanodine receptors (RyRs), communicating via the Ca2+-induced Ca2+ release mechanism. Recent advances have provided a microscopic view of the intermolecular Ca2+ signaling between LCCs and RyRs. In a dyadic junction or a "couplon", LCCs open and close stochastically upon depolarization, delivering a train of high local Ca2+ pulses ("Ca2+ sparklets") to the RyRs in the abutting SR terminal cisternae. Stochastic activation of RyRs discharges "Ca2+ sparks" from different couplons, which summate into global Ca2+ transients. Hence, ignition of Ca2+ sparks by Ca2+ sparklets constitute elementary events of EC coupling. While the sparklet-spark coupling is of low fidelity (at 0 mV, about one out of 50 sparklets triggers a spark under physiological conditions), the high-gain amplification of CICR (approximately 15 at 0 mV) is achieved because of the greater single-channel flux and open time of RyRs and multi-RyR origin of Ca2+ spark. The global stability of CICR is safeguarded by many factors acting in synergy, including physical separation of RyR clusters, sheer Ca2+ gradients around the channel pores, low intrinsic Ca2+ sensitivity of RyRs in vivo, and high cooperativity for the Ca2+-dependent spark activation. The local stability of CICR is insured because of strong, use-dependent inactivation of RyRs, that terminates Ca2+ sparks and confers persistent local SR refractoriness.

Calcium signaling between sarcolemmal calcium channels and ryanodine receptors in heart cells

Cardiac excitation-Ca2+ release coupling is, in essence, a tale of two molecules, sarcolemmal voltage-gated L-type Ca2+ channels (LCCs) and intracellular ryanodine receptors (RyRs), communicating via the Ca2+-induced Ca2+ release mechanism. Recent advances have provided a microscopic view of the intermolecular Ca2+ signaling between LCCs and RyRs. In a dyadic junction or a "couplon", LCCs open and close stochastically upon depolarization, delivering a train of high local Ca2+ pulses ("Ca2+ sparklets") to the RyRs in the abutting SR terminal cisternae. Stochastic activation of RyRs discharges "Ca2+ sparks" from different couplons, which summate into global Ca2+ transients. Hence, ignition of Ca2+ sparks by Ca2+ sparklets constitute elementary events of EC coupling. While the sparklet-spark coupling is of low fidelity (at 0 mV, about one out of 50 sparklets triggers a spark under physiological conditions), the high-gain amplification of CICR (approximately 15 at 0 mV) is achieved because of the greater single-channel flux and open time of RyRs and multi-RyR origin of Ca2+ spark. The global stability of CICR is safeguarded by many factors acting in synergy, including physical separation of RyR clusters, sheer Ca2+ gradients around the channel pores, low intrinsic Ca2+ sensitivity of RyRs in vivo, and high cooperativity for the Ca2+-dependent spark activation. The local stability of CICR is insured because of strong, use-dependent inactivation of RyRs, that terminates Ca2+ sparks and confers persistent local SR refractoriness.

Two-dimensional confocal images of organization, density, and gating of focal Ca2+ release sites in rat cardiac myocytes.

In cardiac myocytes Ca2+ cross-signaling between Ca2+ channels and ryanodine receptors takes place by exchange of Ca2+ signals in microdomains surrounding dyadic junctions, allowing first the activation and then the inactivation of the two Ca2+-transporting proteins. To explore the details of Ca2+ signaling between the two sets of receptors we measured the two-dimensional cellular distribution of Ca2+ at 240 Hz by using a novel confocal imaging technique. Ca2+ channel-triggered Ca2+ transients could be resolved into dynamic "Ca2+ stripes" composed of hundreds of discrete focal Ca2+ releases, appearing as bright fluorescence spots (radius congruent with 0.5 micrometer) at reproducible sites, which often coincided with t-tubules as visualized with fluorescent staining of the cell membrane. Focal Ca2+ releases triggered stochastically by Ca2+ current (ICa) changed little in duration ( congruent with7 ms) and size (congruent with100,000 Ca ions) between -40 and +60 mV, but their frequency of activation and first latency mirrored the kinetics and voltage dependence of ICa. The resolution of 0.95 +/- 0. 13 reproducible focal Ca2+ release sites per micrometer3 in highly Ca2+-buffered cells, where diffusion of Ca2+ is limited to 50 nm, suggests the presence of about one independent, functional Ca2+ release site per half sarcomere. The density and distribution of Ca2+ release sites suggest they correspond to dyadic junctions. The abrupt onset and termination of focal Ca2+ releases indicate that the cluster of ryanodine receptors in individual dyadic junctions may operate in a coordinated fashion.

Modulation of Cardiac Ryanodine Receptors by Sorcin

Sorcin is a widely expressed, 22-kDa Ca2+-binding protein initially identified in multidrug-resistant cells. In the heart, sorcin localizes to the dyadic junctions of transverse tubules and sarcoplasmic reticulum and coimmunoprecipitates with the Ca2+ release channel/ryanodine receptor (RyR) (Meyers, M. B., Pickel, V. M., Sheu, S.-S., Sharma, V. K., Scotto, K. W., and Fishman, G. I. (1995) J. Biol. Chem. 270, 26411-26418). We have investigated a possible functional interaction between sorcin and cardiac RyR using purified recombinant sorcin in [3H]ryanodine binding experiments and single channel recordings of RyR. The open probability of single RyR was decreased significantly by the addition of sorcin to the cytoplasmic side of the channel (IC50 approximately 480 nM). In addition, sorcin completely inhibited [3H]ryanodine binding with an IC50 approximately 700 nM. Inhibition occurred over a wide range of [Ca2+], and sorcin-modulated RyR remained Ca2+-dependent. Furthermore, caffeine-activated RyRs were also inhibited by sorcin at low [Ca2+] (pCa 7), suggesting that Ca2+ is not an obligatory factor for sorcin inhibition of RyR. Comparisons of these inhibitory effects with those of calmodulin and calpain, proteins structurally related to sorcin, suggested that the interaction of sorcin with cardiac RyR was distinct from and independent of either of these modulatory proteins. Phosphorylation of sorcin with the catalytic subunit of protein kinase A significantly decreased the ability of sorcin to modulate RyR. These results suggest that sorcin may modulate RyR function in a normal cell environment and that the level of modulation is in turn influenced by signaling pathways that increase protein kinase A activity.

The Advantages of Cryotechniques: Application To Bioluminescent Cells

The use of fast-freeze fixation (FFF) followed by freeze-substitution (FS) brings substantial advantages which are due to the extreme rapidity of this fixation compared to the conventional one. The initial step, FFF, physically immobilizes most molecules and therefore arrests the biological reactions in a matter of milliseconds. The second step, FS, slowly removes the water content still in solid state and, at the same time, chemically fixes the other cell components in absence of external water. This procedure results in an excellent preservation of the ultrastructure, avoids osmotic artifacts,maintains in situ most soluble substances and keeps up a number of cell activities including antigenicities. Another point of interest is that the rapidity of the initial immobilization enables the capture of unstable structures which, otherwise, would slip towards a more stable state. When combined with electrophysiology, this technique arrests the ultrastructural modifications at a well defined state, allowing a precise timing of the events.We studied the epithelium of the elytra of the scale-worm, Harmothoe lunulata which has excitable, conductible and bioluminescent properties. The intracellular sites of the light emission are paracrystals of endoplasmic reticulum (PER), named photosomes (Fig.1). They are able to flash only when they are coupled with plasma membrane infoldings by dyadic or triadic junctions (Fig.2) basically similar to those of the striated muscle fibers. We have studied them before, during and after stimulation. FFF-FS showed that these complexes are labile structures able to diffentiate and dedifferentiate within milliseconds. Moreover, a transient network of endoplasmic reticulum was captured which we have named intermediate endoplasmic reticulum (IER) surrounding the PER (Fig.1). Numerous gap junctions are found in the membranous infoldings of the junctional complexes (Fig.3). When cryofractured, they cleave unusually (Fig.4-5). It is tempting to suggest that they play an important role in the conduction of the excitation.

Multiple SR-T tubule junctions in a single insect flight muscle fiber

The membranes of mesothoracic dorsoventral flight muscle of Libellua needhami and Erythemis simplicicolis (Odonata: Libellulidae) were studied by transmission electron microscopy using uranyl acetate en bloc staining. Intact fibers were studied by conventional scanning electron microscopy procedures. Dyadic, triadic, and tetradic transverse tubule (TT)—sarcoplasmic reticulum (SR) junctions as well as plasma membrane—SR junctions are described for the first time in the same insect muscle fiber. Triadic (SR—TT—SR) and, rarely tetradic (TT—SR—TT—SR) junctions were limited to regions between adjacent myofibrils and near the ends of fibers. Dyadic (TT—SR) junctions are found between myofibrils or within indentations of slablike mitochondria. Interspecific variation in SR—TT junctional assemblies from previous similar studies is reviewed and summarized and a three-dimensional model based on SEM and TEM observations is presented.

The membrane systems of the cardiac muscle cell of Tmetonyx cicada O. Fabricius (Crustacea, Amphipoda).

The membrane systems of the cardiac muscle cell of the amphipod Tmetonyx cicada (O. Fabricus) are described. The sarcolemma invaginates and forms a transverse network of tubules at the level of the Z band. Narrow longitudinal tubules branch from the network and connect to another transverse network of tubules at the H band level, where dyadic and triadic junctions are formed with the sarcoplasmic reticulum. Adjacent myofibrils are normally separated by a well developed double layer of the sarcoplasmic reticulum. In areas where the myofibrils closely approach the outer sarcolemma, peripheral couplings have been found at the level of the H band.

The membrane systems of the cardiac muscle cell of Cirolana borealis Lilljeborg (Crustacea, Isopoda).

The membrane systems of the cardiac muscle cell of the isopod Cirolana borealis Lilljeborg are described. The sarcolemma invaginates at the level of the Z band, forming transverse tubules. Narrow tubules branch off in a longitudinal direction from these transverse and radially arranged Tz-tubules forming a transverse collar at each A-I level, where dyadic and triadic junctions are formed with the sarcoplasmic reticulum. Two different orientations of the coupling discs have been detected in the supercontracted sarcomere, and this observation has been discussed. Adjacent myofibrils are separated by a double layer of sarcoplasmic reticulum.

The arrangement and ultrastructure of the musculature, nerves and epidermis, in the tail of the cercaria of Cryptocotyle lingua (Creplin) from Littorina littorea (L.)

The ultrastructure of the caudal muscles of the cercaria of Cryptocotyle lingua is related to the rapid swimming movements. Dorsal and ventral striated longitudinal muscle cells extend in a straight line along the tail length each containing a single myofibril, U-shaped in transverse section and divided into sarcomeres. A-, I- and H-bands are recognizable and the fragmented Z-band consists of a row of dense bars to which the thin myofilaments are attached. A pair of proximal ventro-lateral striated muscles may be responsible for the figure of eight pattern of movement. Large mitochondria are abundant in the non-fibrillar region of the muscle cells. Tubular sarcoplasmic reticulum envelops the myofibril giving off branches at the Z-bands which extend into and across the myofibril alternating with the dense bars. T-tubules are absent but cisternae of the sarcoplasmic reticulum form numerous dyadic junctions with the sarcolemma. Dorsal and ventral motor caudal nerves arise from a nerve plexus behind the tail root; axo-axonal and neuromuscular synapses are frequent. Clear and dense synaptic vesicles occur in the presynaptic terminals.

Electrical properties of obliquely striated muscle fibre membrane ofAnodonta glochidium

(1) Electrical properties of the obliquely striated fibers of the adductor muscle of glochidium larvae ofAnodonta implicata were studied by means of intracellular recording and polarization techniques. (2) The muscle fiber lacks transverse tubules and a large fraction (60–85%) of the surface membrane is involved in dyadic junctions with cisternae of the sarcoplasmic reticulum. (3) The resting potential and the overshoot of the action potential in the normal saline are −42.4±6.6 mV and 3.8±4.2 mV, respectively. (4) The overshoot is not altered by the total removal of external Na or by the addition of the 3 μM tetrodotoxin. (5) The overshoot is decreased by reducing the external Ca, and the action potential is eliminated by 1 mM LaCl3 in normal saline. (6) The action potential is produced by an increase in Ca permeability. (7) The specific membrane resistance and capacitance are 9.8±3.6 kΩ·cm2 and 2.1±0.4 μF/cm2, respectively. (8) The fiber membrane shows a marked delayed rectification but no significant inward-going or anomalous rectification. (9) After the suppression of action potentials by La, electronmicroscopy reveals an accumulation of La on the surface membrane including at the dyadic junctions.

Ultrastructural features of cardiac muscle cells in a tarantula spider.

AbstractThe ultrastructural features of cardiac muscle cells and their innervation were examined in the tarantula spider Eurypelma marxi Simon. The cells are transversely striated and have an A band length of about three microns. H zones are indistinct and M lines are absent. Thick and thin myofilament diameters are approximately 200 and 70 Å respectively. Eight to 12 thin filaments usually surround each thick one. Accumulations of thick and thin myofilaments occur perpendicular to the bulk of the myofilaments in some cells. The Z line is discontinuous and thick filaments may pass through the spaces in the Z line. Extensive systems of sarcoplasmic reticulum and transverse tubules are present; these form numerous dyadic junctions in both A and I band regions. Sarcolemmal invaginations form Z line tubules; lateral extensions of the plasma membrane portion of these invaginations form dyads. Nerve branches of the cardiac ganglion make multiple neuromuscular synapses with at least some of the cardiac muscle cells. Both large granular and small agranular vesicles are present in the presynaptic terminals. Intercalated discs similar to those present in other arthropod hearts occur between the ends of adjacent cardiac muscle cells.

The sarcoplasmic reticulum and its association with the T system in an insect.

The fine structure of the sarcoplasmic reticulum and the transverse tubular system of the femoral muscle of the cockroach, Leucophaea maderae, was studied after prefixation in glutaraldehyde, postfixation in osmium tetroxide, and embedding in Epon. The sarcoplasmic reticulum in this muscle reveals features not previously reported. The sarcoplasmic reticulum is abundant, consisting mainly of a fenestrated envelope which surrounds each myofibril at all levels in the sarcomere. This sarcoplasmic reticulum envelope is continuous transversally as well as longitudinally along the myofibrils. Dyadic junctions are formed by a single T system element which contacts the unfenestrated sarcoplasmic reticulum of adjacent myofibrils in an alternating manner at the ends of the A band. At the dyads, regularly spaced thickenings of the sarcoplasmic reticulum membranes bordering the dyadic spaces are noted. These thickenings, however, do not contact the T tubule membrane. Typical dyadic contacts also are seen between the cell surface membrane and sarcoplasmic reticulum. Z line-like material is seen in contact with the membranes of the cell surface and longitudinal branches of the T systems.