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.

Molecular mechanisms that regulatein situactivation of ryanodine receptors (RY) in different cells are poorly understood. Here we demonstrate that caffeine (10 mM) released Ca²⁺from the endoplasmic reticulum (ER) in the form of small spikes in only 14% of cultured fura-2 loaded beta cells fromob/obmice. Surprisingly, when forskolin, an activator of adenylyl cyclase was present, caffeine induced larger Ca²⁺spikes in as many as 60% of the cells. Forskolin or the phosphodiesterase-resistant PKA activator Sp-cAMPS alone did not release Ca²⁺from ER. 4-Chloro-3-ethylphenol (4-CEP), an agent that activates RYs in other cell systems, released Ca²⁺from ER, giving rise to a slow and small increase in [Ca²⁺]_iin beta cells. Prior exposure of cells to forskolin or caffeine (5 mM) qualitatively altered Ca²⁺release by 4-CEP, giving rise to Ca²⁺spikes. In glucose-stimulated beta cells forskolin induced Ca²⁺spikes that were enhanced by 3,9-dimethylxanthine, an activator of RYs. Analysis of RNA from islets and insulin-secreting βTC-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 (RY₂). We conclude thatin situactivation of RY₂in beta cells requires cAMP-dependent phosphorylation, a process that recruits the receptor in a functionally operative form.

In vertebrate skeletal muscles, the type 1 isoform of ryanodine receptor (RyR1) is essential in triggering contraction by releasing Ca²⁺ 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-β 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 Ca²⁺ stores remains controversial. It is open to question whether cADPR regulates ryanodine receptors (RyRs) directly, as originally proposed, or indirectly by promoting Ca²⁺ uptake into the sarco/endoplasmic reticulum by sarco/endoplasmic reticulum Ca²⁺-ATPases. Conversely, although we have proposed that NAADP mobilizes endolysosomal Ca²⁺ stores by activating two-pore domain channels (TPCs), others suggest that NAADP directly activates RyRs. We therefore assessed Ca²⁺ signals evoked by intracellular dialysis from a patch pipette of cADPR and NAADP into HEK293 cells that stably overexpress either TPC1, TPC2, RyR1, or RyR3. No change in intracellular Ca²⁺ concentration was triggered by cADPR in either wild-type HEK293 cells (which are devoid of RyRs) or in cells that stably overexpress TPC1 and TPC2, respectively. By contrast, a marked Ca²⁺ transient was triggered by cADPR in HEK293 cells that stably expressed RyR1 and RyR3. The Ca²⁺ transient was abolished following depletion of endoplasmic reticulum stores by thapsigargin and block of RyRs by dantrolene but not following depletion of acidic Ca²⁺ stores by bafilomycin. By contrast, NAADP failed to evoke a Ca²⁺ transient in HEK293 cells that expressed RyR1 or RyR3, but it induced robust Ca²⁺ transients in cells that stably overexpressed TPC1 or TPC2 and in a manner that was blocked following depletion of acidic stores by bafilomycin. We conclude that cADPR triggers Ca²⁺ release by activating RyRs but not TPCs, whereas NAADP activates TPCs but not RyRs.