Assembly of specialized membrane domains, both of the plasma membrane and of the ER, is necessary for the physiological activity of striated muscle cells. The mechanisms that mediate the structural organization of the sarcoplasmic reticulum with respect to the myofibrils are, however, not known. We report here that ank1.5, a small splice variant of the ank1 gene localized on the sarcoplasmic reticulum membrane, is capable of interacting with a sequence of 25 aa located at the COOH terminus of obscurin. Obscurin is a giant sarcomeric protein of ∼800 kD that binds to titin and has been proposed to mediate interactions between myofibrils and other cellular structures. The binding sites and the critical aa required in the interaction between ank1.5 and obscurin were characterized using the yeast two-hybrid system, in in vitro pull-down assays and in experiments in heterologous cells. In differentiated skeletal muscle cells, a transfected myc-tagged ank1.5 was found to be selectively restricted near the M line region where it colocalized with endogenous obscurin. The M line localization of ank1.5 required a functional obscurin-binding site, because mutations of this domain resulted in a diffused distribution of the mutant ank1.5 protein in skeletal muscle cells. The interaction between ank1.5 and obscurin represents the first direct evidence of two proteins that may provide a direct link between the sarcoplasmic reticulum and myofibrils.In keeping with the proposed role of obscurin in mediating an interaction with ankyrins and sarcoplasmic reticulum, we have also found that a sequence with homology to the obscurin-binding site of ank1.5 is present in the ank2.2 isoform, which in striated muscles has been also shown to associate with the sarcoplasmic reticulum. Accordingly, a peptide containing the COOH terminus of ank2.2 fused with GST was found to bind to obscurin. Based on reported evidence showing that the COOH terminus of ank2.2 is necessary for the localization of ryanodine receptors and InsP3 receptors in the sarcoplasmic reticulum, we propose that obscurin, through multiple interactions with ank1.5 and ank2.2 isoforms, may assemble a large protein complex that, in addition to a structural function, may play a role in the organization of specific subdomains in the sarcoplasmic reticulum.

ABSTRACT2؉ spikes that were enhanced by 3,9-dimethylxanthine, an activator of RYs. Analysis of RNA from islets and insulin-secreting ␤TC-3-cells by RNase protection assay, using type-specific RY probes, revealed low-level expression of mRNA for the type 2 isoform of the receptor (RY 2 ). We conclude that in situ activation of RY 2 in beta cells requires cAMPdependent phosphorylation, a process that recruits the receptor in a functionally operative form.Ryanodine receptors (RY) are Ca 2ϩ channels in the endoplasmic reticulum (ER) composed of four Ϸ550-kDa RY protomers and four molecules of FKBP12 or FKBP12.6. The latter are isoforms of the 12-kDa binding protein for the immunosuppressant drug FK506. cDNAs for three RYs have been cloned. Ryanodine RY 1 , RY 2 , and RY 3 receptors are products of three genes (1). Truncated and splice variants of RYs have been described, all of which might associate in the form of homo-or heterotetramers to produce large diversity of the channels. FKBP12 and FKBP12.6 associate with RY 1 and RY 2 , respectively, and stabilize the channel (2). Studies in cell-free systems have demonstrated that activity of RYs can be modulated by numerous factors including phosphorylationdephosphorylation, Ca 2ϩ

In vertebrate skeletal muscles, the type 1 isoform of ryanodine receptor (RyR1) is essential in triggering contraction by releasing Ca 2؉ from the sarcoplasmic reticulum in response to plasma membrane depolarisation. Recently, the presence of another RyR isoform, RyR3, has been detected in mammalian skeletal muscle cells, raising the question of the eventual relevance of RyR3 for muscle cell physiology. The expression of RyR3 was investigated during differentiation of skeletal muscle cells. Using antibodies able to distinguish the different RyR isoforms and Western blot analysis, the RyR3 protein was detected in the microsomal fractions of differentiated skeletal muscle cells but not of undifferentiated cells. Accordingly, blocking muscle differentiation by the addition of either transforming growth factor-␤ or basic fibroblast growth factor prevented the expression of the RyR3 protein. In differentiated skeletal muscle cells, RyR3 was expressed independent of cell fusion and myotube formation. The expression of RyR3 was also investigated during development of the diaphragm muscle. The RyR3 content in the diaphragm muscle increased between the late stage of fetal development and the first postnatal days. However, at variance with RyR1, which reached maximum levels of expression 2-3 weeks after birth, the expression of RyR3 was found to be higher in the neonatal phase of the diaphragm muscle development (2-15 days after birth) than in the same muscle from adult mice. The differential content of RyR3 in adult skeletal muscles was found not to be mediated by neurotrophic factors or electrical activity. These findings indicate that RyR3 is preferentially expressed in differentiated skeletal muscle cells. In addition, during skeletal muscle development, its expression is regulated differently from that of RyR1.

The mechanism by which cyclic adenosine diphosphate ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) mobilize intracellular Ca 2؉ Intracellular Ca 2ϩ signals are initiated by Ca 2ϩ release from intracellular stores, and traditionally, the sarco/endoplasmic reticulum (S/ER) 2 has been considered to be the major releasable store. From this store, Ca 2ϩ may be released through the opening of inositol 1,4,5-trisphosphate receptors (IP 3 Rs) and/or ryanodine receptors (RyRs), the two groups of intracellular Ca 2ϩ release channels located on S/ER membranes. It is generally accepted that of the recognized Ca 2ϩ -mobilizing second messengers, inositol 1,4,5-trisphosphate facilitates this process by activating IP 3 Rs (1).By contrast, the mechanism by which the pyridine nucleotides cyclic adenosine diphosphate ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) (2) mobilize intracellular Ca 2ϩ stores remains controversial. Although a wealth of evidence across a variety of cell types (3-9) supports the original proposal that cADPR activates RyRs (10), studies on reconstituted RyRs in lipid bilayers have failed to conclusively demonstrate direct regulation of these channels by cADPR (11), and it has been suggested that cADPR may initiate Ca 2ϩ signals via RyRs and IP 3 Rs by promoting Ca 2ϩ uptake into the S/ER by S/ER Ca 2ϩ ATPases (SERCA) (12)(13)(14). This proposal has not been effectively countered by studies on ventricular myocytes, which exclusively express RyR2. This is due to the fact that the principal regulatory effect of cADPR with respect to RyR2 is to increase the sensitivity of this RyR subtype to Ca 2ϩ -induced Ca 2ϩ release (8, 9), and in light of the fact that the sensitivity of RyRs to Ca 2ϩ -induced Ca 2ϩ release may be augmented by an increase in Ca 2ϩ concentration within the cytoplasm and/or S/ER lumen (15).The mechanism by which NAADP triggers intracellular Ca 2ϩ release has also been hotly debated. We recently identified a family of two-pore domain channels (TPC1-3, TPCN1-3 for gene name) as endolysosome-targeted, NAADP-gated Ca 2ϩ release channels (16, 17), and our findings have since been confirmed by others (18,19

A missense mutation in the calsequestrin-1 gene (CASQ1) was found in a group of patients with a myopathy characterized by weakness, fatigue and the presence of large vacuoles containing characteristic inclusions resulting from the aggregation of sarcoplasmic reticulum (SR) proteins. The mutation affects a conserved aspartic acid in position 244 (p.Asp244Gly) located in one of the high-affinity Ca2+ binding sites of CASQ1 and alters the kinetics of Ca2+ release in muscle fibers. Expression of the mutated CASQ1 protein in COS-7 cells showed a markedly reduced ability in forming elongated polymers, while both in cultured myotubes and in in-vivo mouse fibers induced the formation of electron-dense SR vacuoles containing aggregates of the mutant CASQ1 protein that resemble those observed in muscle biopsies of patients. Altogether, these results support the view that a single missense mutation in the CASQ1 gene causes the formation of abnormal SR vacuoles containing aggregates of CASQ1 and other SR proteins, results in altered Ca2+ release in skeletal muscle fibers and, hence, is responsible for the clinical phenotype observed in these patients.