Molecular architecture of membranes involved in excitation-contraction coupling of cardiac muscle

Peripheral couplings are junctions between the sarcoplasmic reticulum (SR) and the surface membrane (SM). Feet occupy the SR/SM junctional gap and are identified as the SR calcium release channels, or ryanodine receptors (RyRs). In cardiac muscle, the activation of RyRs during excitation-contraction (e-c) coupling is initiated by surface membrane depolarization, followed by the opening of surface membrane calcium channels, the dihydropyridine receptors (DHPRs). We have studied the disposition of DHPRs and RyRs, and the structure of peripheral couplings in chick myocardium, a muscle that has no transverse tubules. Immunolabeling shows colocalization of RyRs and DHPRs in clusters at the fiber's periphery. The positions of DHPR and RyR clusters change coincidentally during development. Freeze-fracture of the surface membrane reveals the presence of domains (junctional domains) occupied by clusters of large particles. Junctional domains in the surface membrane and arrays of feet in the junctional gap have similar sizes and corresponding positions during development, suggesting that both are components of peripheral couplings. As opposed to skeletal muscle, membrane particles in junctional domains of cardiac muscle do not form tetrads. Thus, despite their proximity to the feet, they do not appear to be specifically associated with them. Two observations establish the identify of the structurally identified feet arrays/junctional domain complexes with the immunocytochemically defined RyRs/DHPRs coclusters: the concomitant changes during development and the identification of feet as the cytoplasmic domains of RyRs. We suggest that the large particles in junctional domains of the surface membrane represent DHPRs. These observations have two important functional consequences. First, the apposition of DHPRs and RyRs indicates that most of the inward calcium current flows into the restricted space where feet are located. Secondly, contrary to skeletal muscle, presumptive DHPRs do not show a specific association with the feet, which is consistent with a less direct role of charge movement in cardiac than in skeletal e-c coupling.     

Organization of Ca2+ release units in excitable smooth muscle of the guinea-pig urinary bladder

Ca(2+) release from internal stores (sarcoplasmic reticulum or SR) in smooth muscles is initiated either via pharmaco-mechanical coupling due to the action of an agonist and involving IP3 receptors, or via excitation-contraction coupling, mostly involving L-type calcium channels in the plasmalemma (DHPRs), and ryanodine receptors (RyRs), or Ca(2+) release channels of the SR. This work focuses attention on the structural basis for the coupling between DHPRs and RyRs in phasic smooth muscle cells of the guinea-pig urinary bladder. Immunolabeling shows that two proteins of the SR: calsequestrin and the RyR, and one protein the plasmalemma, the L-type channel or DHPR, are colocalized with each other within numerous, peripherally located sites located within the caveolar domains. Electron microscopy images from thin sections and freeze-fracture replicas identify feet in small peripherally located SR vesicles containing calsequestrin and distinctive large particles clustered within small membrane areas. Both feet and particle clusters are located within caveolar domains. Correspondence between the location of feet and particle clusters and of RyR- and DHPR-positive foci allows the conclusion that calsequestrin, RyRs, and L-type Ca(2+) channels are associated with peripheral couplings, or Ca(2+) release units, constituting the key machinery involved in excitation-contraction coupling. Structural analogies between smooth and cardiac muscle excitation-contraction coupling complexes suggest a common basic mechanism of action.     

Numerical analysis of ryanodine receptor activation by L-type channel activity in the cardiac muscle diad.

Computer simulations were used to examine the response of ryanodine receptors (RyRs) to the sarcolemmal calcium influx via L-type calcium channels (DHPRs). The effects of ryanodine receptor organization, diad geometry, DHPR single-channel current, and DHPR gating were examined. In agreement with experimental findings, the simulations showed that RyRs can respond rapidly (approximately 0.4 ms) to calcium influx via DHPRs. The responsiveness of the RyR depends on the geometrical arrangement between the RyRs and the DHPR in the diad, with wider diads being generally less responsive. When the DHPR single-channel current is small (approximately 25 fA), the organization of RyRs into small clusters results in an improved responsiveness. With experimentally observed DHPR mean open and closed times (0.17 ms and 4 ms, respectively) it is the first opening of the DHPR that is most likely to activate the RyR. A measure of the efficiency (Q) by which DHPR gating evokes sarcoplasmic reticulum release is defined. Q is at maximum for tau approximately 0.3 ms, and we interpret this finding in terms of the "tuning" of DHPR gating to RyR response. If certain cardiac myopathies are associated with a mismatch in the "tuning," then modification of DHPR gating with drugs to "retune" calcium-induced calcium release should be possible.     

RYR1 and RYR3 have different roles in the assembly of calcium release units of skeletal muscle

Calcium release units (CRUs) are junctions between the sarcoplasmic reticulum (SR) and exterior membranes that mediates excitation contraction (e-c) coupling in muscle cells. In skeletal muscle CRUs contain two isoforms of the sarcoplasmic reticulum Ca(2+)release channel: ryanodine receptors type 1 and type 3 (RyR1 and RyR3). 1B5s are a mouse skeletal muscle cell line that carries a null mutation for RyR1 and does not express either RyR1 or RyR3. These cells develop dyspedic SR/exterior membrane junctions (i.e., dyspedic calcium release units, dCRUs) that contain dihydropyridine receptors (DHPRs) and triadin, two essential components of CRUs, but no RyRs (or feet). Lack of RyRs in turn affects the disposition of DHPRs, which is normally dictated by a linkage to RyR subunits. In the dCRUs of 1B5 cells, DHPRs are neither grouped into tetrads nor aligned in two orthogonal directions. We have explored the structural role of RyR3 in the assembly of CRUs in 1B5 cells independently expressing either RyR1 or RyR3. Either isoform colocalizes with DHPRs and triadin at the cell periphery. Electron microscopy shows that expression of either isoform results in CRUs containing arrays of feet, indicating the ability of both isoforms to be targeted to dCRUs and to assemble in ordered arrays in the absence of the other. However, a significant difference between RyR1- and RyR3-rescued junctions is revealed by freeze fracture. While cells transfected with RyR1 show restoration of DHPR tetrads and DHPR orthogonal alignment indicative of a link to RyRs, those transfected with RyR3 do not. This indicates that RyR3 fails to link to DHPRs in a specific manner. This morphological evidence supports the hypothesis that activation of RyR3 in skeletal muscle cells must be indirect and provides the basis for failure of e-c coupling in muscle cells containing RyR3 but lacking RyR1 (see the accompanying report, ).     

Functional implications of RyR-dHPR relationships in skeletal and cardiac muscles

Dihydropyridine receptors (DHPRs) and ryanodine receptors (RyRs) interact during EC coupling within calcium release units, CRUs. The location of the two channels and their positioning are related to their role in EC coupling. alphals DHPR and RyR1 of skeletal muscle form interlocked arrays. Groups of four DHPRs (forming a tetrad) are located on alternate RyR1s. This association provides the structural framework for reciprocal signaling between the two channels. RyR3 are present in some skeletal muscles in association with RyR1 and in ratios up to 1:1. RyR3 neither induce formation of tetrads by DHPRs nor sustain EC coupling. RyR3 are located in a parajunctional position, in proximity of the RyR1-DHPR complexes, and they may be indirectly activated by calcium liberated via the RyR1 channels. RyR2 have two locations in cardiac muscle. One is at CRUs that contain DHPRs and RyRs. In these cardiac CRUs, RyR2 and alpha1c DHPR are in proximity of each other, but not closely linked, so that they may not have a direct molecular interaction. A second location of RyR2 is on SR cisternae that are not attached to surface membrane/T tubules. The RyR2 in these cisternae, which are often several microns away from any DHPRs, must necessarily be activated indirectly.     

The relative position of RyR feet and DHPR tetrads in skeletal muscle

In skeletal muscle, L-type calcium channels (or dihydropyridine receptors, DHPRs) are coupled functionally to the calcium release channels of the sarcoplasmic reticulum (or ryanodine receptors, RyRs) within specialized structures called calcium release units (CRUs). The functional linkage requires a specific positioning of four DHPRs in correspondence of the four identical subunits of a single RyR type 1. Four DHPRs linked to the four binding sites of the RyR1 cytoplasmic domain (or foot), define the corners of a square, constituting a tetrad. RyRs self-assemble into ordered arrays and by associating with them, DHPRs also assemble into ordered arrays. The approximate location of the four DHPRs relative to the four identical subunits of a RyR-foot can be predicted on the basis of the relative position of tetrads and feet within the arrays. However, until recently one vital piece of information has been lacking: the orientation of the two arrays relative to one another. In this work we have defined the relative orientation of the RyR and DHPR arrays by directly superimposing replicas of rotary shadowed images of rows of feet, obtained from isolated SR vesicles, and replicas of tetrad arrays obtained by freeze-fracture. If the orientation for the two sets of images is carefully maintained, the superimposition provides specific constraints on the DHPR-RyR relative position.     

Ryanodine and dihydropyridine receptor binding in ventricular cardiac muscle of fish with different temperature preferences

Ca(2+)-induced Ca2+ release (CICR) mechanism of cardiac excitation-contraction (e-c) coupling is dependent on the close apposition between the sarcolemmal dihydropyridine receptors (DHPR) and the sarcoplasmic reticulum (SR) ryanodine receptors (RyR). In particular, high RyR/DHPR ratio is considered to reflect strong dependence on SR Ca2+ stores for the intracellular Ca2+ transient. To indirectly evaluate the significance of CICR in fish hearts, densities of cardiac DHPRs and RyRs were compared in ventricular homogenates of three fish species (burbot, rainbow trout, and crucian carp) and adult rat by [3H] PN200-110 and [3H] ryanodine binding. The density of RyRs was significantly (P<0.05) higher in the adult rat (124+/-10 channels/microm3 myocyte volume) than in any of the fish species. Among the fish species, cold-acclimated (4 degrees C) trout had more RyRs than burbot, and crucian carp. The density of DHPRs was highest in the trout heart. RyR/DHPR ratio was significantly (P<0.05) higher in rat (4.1+/-0.5) than in the fish hearts (varying from 0.97+/-0.16 to 1.91+/-0.49) suggesting that "mammalian type" CICR is less important during e-c coupling in fish ventricular myocytes. In rainbow trout, acclimation to cold did not affect the RyR/DHPR ratio, while in crucian carp it was depressed in cold-acclimated animals (4 degrees C; 0.97+/-0.16) when compared to warm-acclimated fish (23 degrees C; 1.91+/-0.49). Although RyR/DHPR ratios were relatively low in fish hearts, there was a close correlation (r2=0.78) between the RyR/DHPR ratio and the magnitude of the Ry-sensitive component of contraction in ventricular muscle among the fish species examined in this study.     

Colocalization of dihydropyridine and ryanodine receptors in neonate rabbit heart using confocal microscopy

Because of undeveloped T tubules and sparse sarcoplasmic reticulum, Ca(2+)-induced Ca(2+) release (CICR) may not be the major mechanism providing contractile Ca(2+) in the neonatal heart. Spatial association of dihydropyridine receptors (DHPRs) and ryanodine receptors (RyRs), a key factor for CICR, was examined in isolated neonatal rabbit ventricular myocytes aged 3-20 days by double-labeling immunofluorescence and confocal microscopy. We found a significant increase (P < 0.0005) in the degree of colocalization of DHPR and RyR during development. The number of voxels containing DHPR that also contained RyR in the 3-day-old group (62 +/- 1.8%) was significantly lower than in the other age groups (76 +/- 1.3 in 6-day old, 75 +/- 1.2 in 10-day old, and 79 +/- 0.9% in 20-day old). The number of voxels containing RyR that also contained DHPR was significantly higher in the 20-day-old group (17 +/- 0.5%) compared with the other age groups (10 +/- 0.7 in 3-day old, 11 +/- 0.6 in 6-day old, and 11 +/- 0.5% in 10-day old). During this period, the pattern of colocalization changed from mostly peripheral to mostly internal couplings. Our results provide a structural basis for the diminished prominence of CICR in neonatal heart.     

Ca2+ entry-independent effects of L-type Ca2+ channel modulators on Ca2+ sparks in ventricular myocytes

During the cardiac action potential, Ca²⁺ entry through dyhidropyridine receptor L-type Ca²⁺ channels (DHPRs) activates ryanodine receptors (RyRs) Ca²⁺-release channels, resulting in massive Ca²⁺ mobilization from the sarcoplasmic reticulum (SR). This global Ca²⁺ release arises from spatiotemporal summation of many localized elementary Ca²⁺-release events, Ca²⁺ sparks. We tested whether DHPRs modulate Ca²⁺sparks in a Ca²⁺ entry-independent manner. Negative modulation by DHPR of RyRs via physical interactions is accepted in resting skeletal muscle but remains controversial in the heart. Ca²⁺ sparks were studied in cat cardiac myocytes permeabilized with saponin or internally perfused via a patch pipette. Bathing and pipette solutions contained low Ca²⁺ (100 nM). Under these conditions, Ca²⁺ sparks were detected with a stable frequency of 3–5 sparks·s^–1·100 µm^–1. The DHPR blockers nifedipine, nimodipine, FS-2, and calciseptine decreased spark frequency, whereas the DHPR agonists Bay-K8644 and FPL-64176 increased it. None of these agents altered the spatiotemporal characteristics of Ca²⁺ sparks. The DHPR modulators were also without effect on SR Ca²⁺ load (caffeine-induced Ca²⁺ transients) or sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA) activity (Ca²⁺ loading rates of isolated SR microsomes) and did not change cardiac RyR channel gating (planar lipid bilayer experiments). In summary, DHPR modulators affected spark frequency in the absence of DHPR-mediated Ca²⁺ entry. This action could not be attributed to a direct action of DHPR modulators on SERCA or RyRs. Our results suggest that the activity of RyR Ca²⁺-release units in ventricular myocytes is modulated by Ca²⁺ entry-independent conformational changes in neighboring DHPRs. exitation-contraction coupling; ryanodine receptor; sarco(endo)plasmic reticulum Ca²⁺-ATPase; dihydropyridine receptor; sarcoplasmic reticulum     

Multiple regions of RyR1 mediate functional and structural interactions with alpha(1S)-dihydropyridine receptors in skeletal muscle

Excitation-contraction (e-c) coupling in muscle relies on the interaction between dihydropyridine receptors (DHPRs) and RyRs within Ca(2+) release units (CRUs). In skeletal muscle this interaction is bidirectional: alpha(1S)DHPRs trigger RyR1 (the skeletal form of the ryanodine receptor) to release Ca(2+) in the absence of Ca(2+) permeation through the DHPR, and RyR1s, in turn, affect the open probability of alpha(1S)DHPRs. alpha(1S)DHPR and RyR1 are linked to each other, organizing alpha(1S)-DHPRs into groups of four, or tetrads. In cardiac muscle, however, alpha(1C)DHPR Ca(2+) current is important for activation of RyR2 (the cardiac isoform of the ryanodine receptor) and alpha(1C)-DHPRs are not organized into tetrads. We expressed RyR1, RyR2, and four different RyR1/RyR2 chimeras (R4: Sk1635-3720, R9: Sk2659-3720, R10: Sk1635-2559, R16: Sk1837-2154) in 1B5 dyspedic myotubes to test their ability to restore skeletal-type e-c coupling and DHPR tetrads. The rank-order for restoring skeletal e-c coupling, indicated by Ca(2+) transients in the absence of extracellular Ca(2+), is RyR1 > R4 > R10 > R16 > R9 > RyR2. The rank-order for restoration of DHPR tetrads is RyR1 > R4 = R9 > R10 = R16 > RyR2. Because the skeletal segment in R9 does not overlap with that in either R10 or R16, our results indicate that multiple regions of RyR1 may interact with alpha(1S)DHPRs and that the regions responsible for tetrad formation do not correspond exactly to the ones required for functional coupling.     

Triad formation: organization and function of the sarcoplasmic reticulum calcium release channel and triadin in normal and dysgenic muscle in vitro

Excitation-contraction (E-C) coupling is thought to involve close interactions between the calcium release channel (ryanodine receptor; RyR) of the sarcoplasmic reticulum (SR) and the dihydropyridine receptor (DHPR) alpha 1 subunit in the T-tubule membrane. Triadin, a 95- kD protein isolated from heavy SR, binds both the RyR and DHPR and may thus participate in E-C coupling or in interactions responsible for the formation of SR/T-tubule junctions. Immunofluorescence labeling of normal mouse myotubes shows that the RyR and triadin co-aggregate with the DHPR in punctate clusters upon formation of functional junctions. Dysgenic myotubes with a deficiency in the alpha 1 subunit of the DHPR show reduced expression and clustering of RyR and triadin; however, both proteins are still capable of forming clusters and attaining mature cross-striated distributions. Thus, the molecular organization of the RyR and triadin in the terminal cisternae of SR as well as its association with the T-tubules are independent of interactions with the DHPR alpha 1 subunit. Analysis of calcium transients in dysgenic myotubes with fluorescent calcium indicators reveals spontaneous and caffeine-induced calcium release from intracellular stores similar to those of normal muscle; however, depolarization-induced calcium release is absent. Thus, characteristic calcium release properties of the RyR do not require interactions with the DHPR; neither do they require the normal organization of the RyR in the terminal SR cisternae. In hybrids of dysgenic myotubes fused with normal cells, both action potential- induced calcium transients and the normal clustered organization of the RyR are restored in regions expressing the DHPR alpha 1 subunit.     

Rapid activation of the cardiac ryanodine receptor by submillisecond calcium stimuli

The local control concept of excitation-contraction coupling in the heart postulates that the activity of the sarcoplasmic reticulum ryanodine receptor channels (RyR) is controlled by Ca(2+) entry through adjoining sarcolemmal single dihydropyridine receptor channels (DHPRs). One unverified premise of this hypothesis is that the RyR must be fast enough to track the brief (<0.5 ms) Ca(2+) elevations accompanying single DHPR channel openings. To define the kinetic limits of effective trigger Ca(2+) signals, we recorded activity of single cardiac RyRs in lipid bilayers during rapid and transient increases in Ca(2+) generated by flash photolysis of DM-nitrophen. Application of such Ca(2+) spikes (amplitude approximately 10-30 microM, duration approximately 0.1-0.4 ms) resulted in activation of the RyRs with a probability that increased steeply (apparent Hill slope approximately 2.5) with spike amplitude. The time constants of RyR activation were 0.07-0.27 ms, decreasing with spike amplitude. To fit the rising portion of the open probability, a single exponential function had to be raised to a power n approximately 3. We show that these data could be adequately described with a gating scheme incorporating four sequential Ca(2+)-sensitive closed states between the resting and the first open states. These results provide evidence that brief Ca(2+) triggers are adequate to activate the RyR, and support the possibility that RyR channels are governed by single DHPR openings. They also provide evidence for the assumption that RyR activation requires binding of multiple Ca(2+) ions in accordance with the tetrameric organization of the channel protein.     

Differential contribution of skeletal and cardiac II-III loop sequences to the assembly of dihydropyridine-receptor arrays in skeletal muscle

The plasmalemmal dihydropyridine receptor (DHPR) is the voltage sensor in skeletal muscle excitation-contraction (e-c) coupling. It activates calcium release from the sarcoplasmic reticulum via protein-protein interactions with the ryanodine receptor (RyR). To enable this interaction, DHPRs are arranged in arrays of tetrads opposite RyRs. In the DHPR alpha(1S) subunit, the cytoplasmic loop connecting repeats II and III is a major determinant of skeletal-type e-c coupling. Whether the essential II-III loop sequence (L720-L764) also determines the skeletal-specific arrangement of DHPRs was examined in dysgenic (alpha(1S)-null) myotubes reconstituted with distinct alpha(1) subunit isoforms and II-III loop chimeras. Parallel immunofluorescence and freeze-fracture analysis showed that alpha(1S) and chimeras containing L720-L764, all of which restored skeletal-type e-c coupling, displayed the skeletal arrangement of DHPRs in arrays of tetrads. Conversely, alpha(1C) and those chimeras with a cardiac II-III loop and cardiac e-c coupling properties were targeted into junctional membranes but failed to form tetrads. However, an alpha(1S)-based chimera with the heterologous Musca II-III loop produced tetrads but did not reconstitute skeletal muscle e-c coupling. These findings suggest an inhibitory role in tetrad formation of the cardiac II-III loop and that the organization of DHPRs in tetrads vis-a-vis the RyR is necessary but not sufficient for skeletal-type e-c coupling.     

Ryanoids and imperatoxin affect the modulation of cardiac ryanodine receptors by dihydropyridine receptor Peptide A

Ca²⁺-entry via L-type Ca²⁺ channels (DHPR) is known to trigger ryanodine receptor (RyR)-mediated Ca²⁺-release from sarcoplasmic reticulum (SR). The mechanism that terminates SR Ca²⁺ release is still unknown. Previous reports showed evidence of Ca²⁺-entry independent inhibition of Ca²⁺ sparks by DHPR in cardiomyocytes. A peptide from the DHPR loop II-III (PepA) was reported to modulate isolated RyRs. We found that PepA induced voltage-dependent “flicker block” and transition to substates of fully-activated cardiac RyRs in planar bilayers. Substates had less voltage-dependence than block and did not represent occupancy of a ryanoid site. However, ryanoids stabilized PepA-induced events while PepA increased RyR2 affinity for ryanodol, which suggests cooperative interactions. Ryanodol stabilized Imperatoxin A (IpTx_A) binding but when IpTx_A bound first, it prevented ryanodol binding. Moreover, IpTx_A and PepA excluded each other from their sites. This suggests that IpTx_A generates a vestibular gate (either sterically or allosterically) that prevents access to the peptides and ryanodol binding sites. Inactivating gate moieties (“ball peptides”) from K⁺ and Na⁺ channels (ShakerB and KIFMK, respectively) induced well resolved slow block and substates, which were sensitive to ryanoids and IpTx_A and allowed, by comparison, better understanding of PepA action. The RyR2 appears to interact with PepA or ball peptides through a two-step mechanism, reminiscent of the inactivation of voltage-gated channels, which includes binding to outer (substates) and inner (block) vestibular regions in the channel conduction pathway. Our results open the possibility that “ball peptide-like” moieties in RyR2-interacting proteins could modulate SR Ca²⁺ release in cells.     

Role of Ryanodine Receptors in the Assembly of Calcium Release Units in Skeletal Muscle

Abstract. In muscle cells, excitation–contraction (e–c) coupling is mediated by “calcium release units,” junctions between the sarcoplasmic reticulum (SR) and exterior membranes. Two proteins, which face each other, are known to functionally interact in those structures: the ryanodine receptors (RyRs), or SR calcium release channels, and the dihydropyridine receptors (DHPRs), or L-type calcium channels of exterior membranes. In skeletal muscle, DHPRs form tetrads, groups of four receptors, and tetrads are organized in arrays that face arrays of feet (or RyRs). Triadin is a protein of the SR located at the SR–exterior membrane junctions, whose role is not known. We have structurally characterized calcium release units in a skeletal muscle cell line (1B5) lacking Ry₁R. Using immunohistochemistry and freeze-fracture electron microscopy, we find that DHPR and triadin are clustered in foci in differentiating 1B5 cells. Thin section electron microscopy reveals numerous SR–exterior membrane junctions lacking foot structures (dyspedic). These results suggest that components other than Ry₁Rs are responsible for targeting DHPRs and triadin to junctional regions. However, DHPRs in 1B5 cells are not grouped into tetrads as in normal skeletal muscle cells suggesting that anchoring to Ry₁Rs is necessary for positioning DHPRs into ordered arrays of tetrads. This hypothesis is confirmed by finding a “restoration of tetrads” in junctional domains of surface membranes after transfection of 1B5 cells with cDNA encoding for Ry₁R.     

Shape, size, and distribution of Ca(2+) release units and couplons in skeletal and cardiac muscles.

Excitation contraction (e-c) coupling in skeletal and cardiac muscles involves an interaction between specialized junctional domains of the sarcoplasmic reticulum (SR) and of exterior membranes (either surface membrane or transverse (T) tubules). This interaction occurs at special structures named calcium release units (CRUs). CRUs contain two proteins essential to e-c coupling: dihydropyridine receptors (DHPRs), L-type Ca(2+) channels of exterior membranes; and ryanodine receptors (RyRs), the Ca(2+) release channels of the SR. Special CRUs in cardiac muscle are constituted by SR domains bearing RyRs that are not associated with exterior membranes (the corbular and extended junctional SR or EjSR). Functional groupings of RyRs and DHPRs within calcium release units have been named couplons, and the term is also loosely applied to the EjSR of cardiac muscle. Knowledge of the structure, geometry, and disposition of couplons is essential to understand the mechanism of Ca(2+) release during muscle activation. This paper presents a compilation of quantitative data on couplons in a variety of skeletal and cardiac muscles, which is useful in modeling calcium release events, both macroscopic and microscopic ("sparks").     

Arg(615)Cys substitution in pig skeletal ryanodine receptors increases activation of single channels by a segment of the skeletal DHPR II-III loop

The effect of peptides, corresponding to sequences in the skeletal muscle dihydropyridine receptor II-III loop, on Ca(2+) release from sarcoplasmic reticulum (SR) and on ryanodine receptor (RyR) calcium release channels have been compared in preparations from normal and malignant hyperthermia (MH)-susceptible pigs. Peptide A (Thr(671)-Leu(690); 36 microM) enhanced the rate of Ca(2+) release from normal SR (SR(N)) and from SR of MH-susceptible muscle (SR(MH)) by 10 +/- 3.2 nmole/mg/min and 76 +/- 9.7 nmole/mg/min, respectively. Ca (2+) release from SR(N) or SR(MH) was not increased by control peptide NB (Gly(689)-Lys(708)). AS (scrambled A sequence; 36 microM) did not alter Ca (2+) release from SR(N), but increased release from SR(MH) by 29 +/- 4.9 nmoles/mg/min. RyR channels from MH-susceptible muscle (RyR(MH)) were up to about fourfold more strongly activated by peptide A (> or =1 nM) than normal RyR channels (RyR(N)) at -40 mV. Neither NB or AS activated RyR(N). RyR(MH) showed an approximately 1.8-fold increase in mean current with 30 microM AS. Inhibition at +40 mV was stronger in RyR(MH) and seen with peptide A (> or = 0.6 microM) and AS (> or = 0.6 microM), but not NB. These results show that the Arg(615)Cys substitution in RyR(MH) has multiple effects on RyRs. We speculate that enhanced DHPR activation of RyRs may contribute to increased Ca(2+) release from SR in MH-susceptible muscle.     

Changes in the organization of excitation-contraction coupling structures in failing human heart

Background

The cardiac myocyte t-tubular system ensures rapid, uniform cell activation and several experimental lines of evidence suggest changes in the t-tubular system and associated excitation-contraction coupling proteins may occur in heart failure.

Methods and Results

The organization of t-tubules, L-type calcium channels (DHPRs), ryanodine receptors (RyRs) and contractile machinery were examined in fixed ventricular tissue samples from both normal and failing hearts (idiopathic (non-ischemic) dilated cardiomyopathy) using high resolution fluorescent imaging. Wheat germ agglutinin (WGA), Na-Ca exchanger, DHPR and caveolin-3 labels revealed a shift from a predominantly transverse orientation to oblique and axial directions in failing myocytes. In failure, dilation of peripheral t-tubules occurred and a change in the extent of protein glycosylation was evident. There was no change in the fractional area occupied by myofilaments (labeled with phalloidin) but there was a small reduction in the number of RyR clusters per unit area. The general relationship between DHPRs and RyR was not changed and RyR labeling overlapped with 51±3% of DHPR labeling in normal hearts. In longitudinal (but not transverse) sections there was an ∼30% reduction in the degree of colocalization between DHPRs and RyRs as measured by Pearson''s correlation coefficient in failing hearts.

Conclusions

The results show that extensive remodelling of the t-tubular network and associated excitation-contraction coupling proteins occurs in failing human heart. These changes may contribute to abnormal calcium handling in heart failure. The general organization of the t-system and changes observed in failure samples have subtle differences to some animal models although the general direction of changes are generally similar.     

Morphology and molecular composition of sarcoplasmic reticulum surface junctions in the absence of DHPR and RyR in mouse skeletal muscle

Calcium release during excitation-contraction coupling of skeletal muscle cells is initiated by the functional interaction of the exterior membrane and the sarcoplasmic reticulum (SR), mediated by the "mechanical" coupling of ryanodine receptors (RyR) and dihydropyridine receptors (DHPR). RyR is the sarcoplasmic reticulum Ca(2+) release channel and DHPR is an L-type calcium channel of exterior membranes (surface membrane and T tubules), which acts as the voltage sensor of excitation-contraction coupling. The two proteins communicate with each other at junctions between SR and exterior membranes called calcium release units and are associated with several proteins of which triadin and calsequestrin are the best characterized. Calcium release units are present in diaphragm muscles and hind limb derived primary cultures of double knock out mice lacking both DHPR and RyR. The junctions show coupling between exterior membranes and SR, and an apparently normal content and disposition of triadin and calsequestrin. Therefore SR-surface docking, targeting of triadin and calsequestrin to the junctional SR domains and the structural organization of the two latter proteins are not affected by lack of DHPR and RyR. Interestingly, simultaneous lack of the two major excitation-contraction coupling proteins results in decrease of calcium release units frequency in the diaphragm, compared with either single knockout mutation.     

The Ca 2+ leak paradox and rogue ryanodine receptors: SR Ca 2+ efflux theory and practice

Ca²⁺ efflux from the sarcoplasmic reticulum (SR) is routed primarily through SR Ca²⁺ release channels (ryanodine receptors, RyRs). When clusters of RyRs are activated by trigger Ca²⁺ influx through L-type Ca²⁺ channels (dihydropyridine receptors, DHPR), Ca²⁺ sparks are observed. Close spatial coupling between DHPRs and RyR clusters and the relative insensitivity of RyRs to be triggered by Ca²⁺ together ensure the stability of this positive-feedback system of Ca²⁺ amplification. Despite evidence from single channel RyR gating experiments that phosphorylation of RyRs by protein kinase A (PKA) or calcium-calmodulin dependent protein kinase II (CAMK II) causes an increase in the sensitivity of the RyR to be triggered by [Ca²⁺]_i there is little clear evidence to date showing an increase in Ca²⁺ spark rate. Indeed, there is some evidence that the SR Ca²⁺ content may be decreased in hyperadrenergic disease states. The question is whether or not these observations are compatible with each other and with the development of arrhythmogenic extrasystoles that can occur under these conditions. Furthermore, the appearance of an increase in the SR Ca²⁺ “leak” under these conditions is perplexing. These and related complexities are analyzed and discussed in this report. Using simple mathematical modeling discussed in the context of recent experimental findings, a possible resolution to this paradox is proposed. The resolution depends upon two features of SR function that have not been confirmed directly but are broadly consistent with several lines of indirect evidence: (1) the existence of unclustered or “rogue” RyRs that may respond differently to local [Ca²⁺]_i in diastole and during the [Ca²⁺]_i transient; and (2) a decrease in cooperative or coupled gating between clustered RyRs in response to physiologic phosphorylation or hyper-phosphorylation of RyRs in disease states such as heart failure. Taken together, these two features may provide a framework that allows for an improved understanding of cardiac Ca²⁺ signaling.