Cardiac calsequestrin (Casq2) is thought to be the key sarcoplasmic reticulum (SR) Ca 2+ storage protein essential for SR Ca 2+ release in mammalian heart. Human CASQ2 mutations are associated with catecholaminergic ventricular tachycardia. However, homozygous mutation carriers presumably lacking functional Casq2 display surprisingly normal cardiac contractility. Here we show that Casq2-null mice are viable and display normal SR Ca 2+ release and contractile function under basal conditions. The mice exhibited striking increases in SR volume and near absence of the Casq2-binding proteins triadin-1 and junctin; upregulation of other Ca 2+ -binding proteins was not apparent. Exposure to catecholamines in Casq2-null myocytes caused increased diastolic SR Ca 2+ leak, resulting in premature spontaneous SR Ca 2+ releases and triggered beats. In vivo, Casq2-null mice phenocopied the human arrhythmias. Thus, while the unique molecular and anatomic adaptive response to Casq2 deletion maintains functional SR Ca 2+ storage, lack of Casq2 also causes increased diastolic SR Ca 2+ leak, rendering Casq2-null mice susceptible to catecholaminergic ventricular arrhythmias.

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").

The ryanodine receptor (RyR) is a high-conductance Ca2+ channel of the sarcoplasmic reticulum in muscle and of the endoplasmic reticulum in other cells. In striated muscle fibers, RyRs are responsible for the rapid release of Ca2+ that activates contraction. Ryanodine receptors are complex molecules, with unusually large cytoplasmic domains containing numerous binding sites for agents that control the state of activity of the channel-forming domain of the molecule. Structural considerations indicate that long-range interactions between cytoplasmic and intramembrane domains control channel function. Ryanodine receptors are located in specialized regions of the SR, where they are structurally and functionally associated with other intrinsic proteins and, indirectly, also with the luminal Ca2(+)-binding protein calsequestrin. Activation of RyRs during the early part of the excitation-contraction coupling cascade is initiated by the activity of surface-membrane Ca2+ channels, the dihydropyridine receptors (DHPRs). Skeletal and cardiac muscles contain different RyR and DHPR isoforms and both contribute to the diversity in cardiac and skeletal excitation-contraction coupling mechanisms. The architecture of the sarcoplasmic reticulum-surface junctions determines the types of RyR-DHPR interactions in the two muscle types.

Excitation-contraction coupling, the process that regulates contractions by skeletal muscles, transduces changes in membrane voltage by activating release of Ca2+ from internal stores to initiate muscle contraction. Defects in EC coupling are associated with muscle diseases. Here we identify Stac3 as a novel component of the EC coupling machinery. Using a zebrafish genetic screen, we generate a locomotor mutation that is mapped to stac3. We provide electrophysiological, Ca2+ imaging, immunocytochemical and biochemical evidence that Stac3 participates in excitation-contraction coupling in muscles. Furthermore, we reveal that a mutation in human STAC3 as the genetic basis of the debilitating Native American myopathy (NAM). Analysis of NAM stac3 in zebrafish shows that the NAM mutation decreases excitation-contraction coupling. These findings enhance our understanding of both excitation-contraction coupling and the pathology of myopathies.

Luminal Ca 2؉ in the endoplasmic and sarcoplasmic reticulum (ER͞ SR) plays an important role in regulating vital biological processes, including store-operated capacitative Ca 2؉ entry, Ca 2؉ -induced Ca 2؉ release, and ER͞SR stress-mediated cell death. We report rapid and substantial decreases in luminal [Ca 2؉ ], called ''Ca 2؉ blinks,'' within nanometer-sized stores (the junctional cisternae of the SR) during elementary Ca 2؉ release events in heart cells. Blinks mirror small local increases in cytoplasmic Ca 2؉ , or Ca 2؉ sparks, but changes of [Ca 2؉ ] in the connected free SR network were below detection. Store microanatomy suggests that diffusional strictures may account for this paradox. Surprisingly, the nadir of the store depletion trails the peak of the spark by about 10 ms, and the refilling of local store occurs with a rate constant of 35 s ؊1 , which is Ϸ6-fold faster than the recovery of local Ca 2؉ release after a spark. These data suggest that both local store depletion and some time-dependent inhibitory mechanism contribute to spark termination and refractoriness. Visualization of local store Ca 2؉ signaling thus broadens our understanding of cardiac store Ca 2؉ regulation and function and opens the possibility for local regulation of diverse store-dependent functions.calcium-induced calcium release ͉ calcium spark ͉ cardiac myocytes ͉ endoplasmic reticulum ͉ sarcoplasmic reticulum L ocal Ca 2ϩ releases from the endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) in muscle have been shown to underlie neurosecretion, memory encoding, neurite growth, muscle contraction, and apoptosis (1-4). Whereas the ER͞SR serves primarily as the intracellular Ca 2ϩ store, luminal Ca 2ϩ plays an active role in many regulatory systems, including store-operated capacitative Ca 2ϩ entry (5, 6), Ca 2ϩ -induced Ca 2ϩ release (7-9), and ER͞SR stress-mediated cell death (10, 11). Over the last decade, the elementary Ca 2ϩ release events have been directly visualized as Ca 2ϩ ''sparks'' (12-15), ''puffs'' (16), ''syntillas'' (17), or the equivalent (18) in the cytoplasm of both excitable and nonexcitable cells. However, the reciprocal store depletion signals, which were speculated in various models of spark termination (refs. 7 and 19; see ref. 20 for a review), have not been seen experimentally. In theory, a rapid refilling of local store Ca 2ϩ from the bulk of ER͞SR might occur and prevent significant local Ca 2ϩ depletion (21,22). Alternatively, this failing could be due to lack of a means to probe Ca 2ϩ inside this delicate membrane-bound intracellular structure with the required sensitivity, resolution, and speed, given the extremely small release flux involved (Ϸ2⅐10 Ϫ19 mol of Ca 2ϩ ) (12, 17). Using confocal imaging, electron microscopy, and electrophysiological approaches, we investigated dynamic Ca 2ϩ regulation inside nanometer-sized SR structures during elementary Ca 2ϩ release events in intact heart muscle cells. Our results afforded insights into mechanisms underlying spark termination and refract...

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Cytoplasmic dynein is the major minus-end-directed microtubule-based motor in cells. Dynein processivity and cargo selectivity depend on cargo-specific effectors that, while generally unrelated, share the ability to interact with dynein and dynactin to form processive dynein–dynactin-effector complexes. How this is achieved is poorly understood. Here, we identify a conserved region of the dynein Light Intermediate Chain 1 (LIC1) that mediates interactions with unrelated dynein–dynactin effectors. Quantitative binding studies map these interactions to a conserved helix within LIC1 and to N-terminal fragments of Hook1, Hook3, BICD2, and Spindly. A structure of the LIC1 helix bound to the N-terminal Hook domain reveals a conformational change that creates a hydrophobic cleft for binding of the LIC1 helix. The LIC1 helix competitively inhibits processive dynein–dynactin-effector motility in vitro, whereas structure-inspired mutations in this helix impair lysosomal positioning in cells. The results reveal a conserved mechanism of effector interaction with dynein–dynactin necessary for processive motility.

Motor actions of myosin were directly visualized by electron tomography of insect flight muscle quick-frozen during contraction. In 3D images, active cross-bridges are usually single myosin heads, bound preferentially to actin target zones sited midway between troponins. Active attached bridges (approximately 30% of all heads) depart markedly in axial and azimuthal angles from Rayment's rigor acto-S1 model, one-third requiring motor domain (MD) tilting on actin, and two-thirds keeping rigor contact with actin while the light chain domain (LCD) tilts axially from approximately 105 degrees to approximately 70 degrees. The results suggest the MD tilts and slews on actin from weak to strong binding, followed by swinging of the LCD through an approximately 35 degrees axial angle, giving an approximately 13 nm interaction distance and an approximately 4-6 nm working stroke.