In adult skeletal muscles, 2 junctophilin isoforms (JPH1 and JPH2) tether the sarcoplasmic reticulum (SR) to transverse tubule (T-tubule) membranes, generating stable membrane contact sites known as triads. JPHs are anchored to the membrane of the SR by a C-terminal transmembrane domain (TMD) and bind the T-tubule membrane through their cytosolic N-terminal region, which contains 8 lipid-binding (MORN) motifs. By combining expression of GFP-JPH1 deletion mutants in skeletal muscle fibers with in vitro biochemical experiments, we investigated the molecular determinants of JPH1 recruitment at triads in adult skeletal muscle fibers. We found that MORN motifs bind PI(4,5)P2 in the sarcolemma, but do not mediate the selective localization of JPH1 at the T-tubule compartment of triads. On the contrary, fusion proteins containing only the TMD of JPH1 were able to localize at the junctional SR compartment of the triad. Bimolecular fluorescence complementation experiments indicated that the TMD of JPH1 can form dimers, suggesting that the observed localization at triads may result from dimerization with the TMDs of resident JPH1. A second domain, capable of mediating homo- and heterodimeric interactions between JPH1 and JPH2 was identified in the cytosolic region. FRAP experiments revealed that removal of either one of these 2 domains in JPH1 decreases the association of the resulting mutant proteins with triads. Altogether, these results suggest that the ability to establish homo- and heterodimeric interactions with resident JPHs may support the recruitment and stability of newly synthesized JPHs at triads in adult skeletal muscle fibers.

Ca2+ release, which is necessary for muscle contraction, occurs at the j-SR (junctional domain of the sarcoplasmic reticulum). It requires the assembly of a large multiprotein complex containing the RyR (ryanodine receptor) and additional proteins, including triadin and calsequestrin. The signals which drive these proteins to the j-SR and how they assemble to form this multiprotein complex are poorly understood. To address aspects of these questions we studied the localization, dynamic properties and molecular interactions of triadin. We identified three regions, named TR1 (targeting region 1), TR2 and TR3, that contribute to the localization of triadin at the j-SR. FRAP experiments showed that triadin is stably associated with the j-SR and that this association is mediated by TR3. Protein pull-down experiments indicated that TR3 contains binding sites for calsequestrin-1 and that triadin clustering can be enhanced by binding to calsequestrin-1. These findings were confirmed by FRET experiments. Interestingly, the stable association of triadin to the j-SR was significantly decreased in myotubes from calsequestrin-1 knockout mice. Taken together, these results identify three regions in triadin that mediate targeting to the j-SR and reveal a role for calsequestrin-1 in promoting the stable association of triadin to the multiprotein complex associated with RyR.

confocal microscope. In situ calibration determined the half signal of fluo-5N and rhod-5N to be 335 and 872 mM, respectively. Rhod-5N was selected for ongoing experiments. Chronic depletion of [Ca 2þ ]SR with caffeine reduced [Ca 2þ ]t-sys to 0.1 mM via chronic activation of storeoperated Ca 2þ entry (Launikonis et al 2003, PNAS). We then exposed Ca 2þdepleted preparations to 0-800 nM [Ca 2þ ]cyto in 50 mM EGTA. At [Ca 2þ ] cyto > 100 nM the [Ca 2þ ]t-sys reached a plateau at 1.8-1.9 mM after 3-5 s. At [Ca 2þ ]cyto < 100 nM the [Ca 2þ ]t-sys did not always reach this plateau and showed a biphasic uptake of Ca 2þ . At the plateau [Ca 2þ ]t-sys lowering [Ca 2þ ]cyto to < 1 nM did not cause a significant loss of [Ca 2þ ]t-sys. There was an apparent absence of effect of removing [Na þ ]cyto on these results. Mathematical modeling of these results suggests that the plasma membrane CaATPase (PMCA) with its low Km for Ca 2þ is the major protein responsible for t-system Ca 2þ uptake in the resting muscle, despite the higher transport capacity of the Na-Ca exchanger.

The sarcoplasmic reticulum (SR) of striated muscle cells is mainly dedicated to Ca 2þ homeostasis and regulation of muscle contraction. The SR is organized in longitudinal and junctional SR (j-SR). In skeletal muscle, this latter domain together with the T-tubules form specific junctions called triads, where proteins regulating the excitation-contraction coupling mechanism assemble. Junctophilins (JPs) are directly involved in the formation and maintenance of triads. Basically, they are anchored to the SR via their C-terminal transmembrane domain (TMD), while their N-terminus contains eight MORN motifs, which associate with the phospholipids of the T-tubules. Nevertheless, how JPs are targeted to triads is not known. The roles of the N-terminal and the C-terminal regions of JP1 in this process were investigated. Expression in primary myotubes and/or muscle fibers of JP1 deletion mutants lacking the TMD resulted in protein distribution at both the surface sarcolemma and the T-tubules, confirming that MORN motifs are involved in JP1 interaction with the sarcolemma, but are not sufficient to restrict its localization at the T-tubules. On the other hand, progressive deletion of MORN motifs I-VI, MORN motifs I-VIII or of the entire region 1-635, did no affect JP1 localization at triads, indicating that the presence of the TMD is sufficient for JP1 localization at the j-SR. These results indicate that the localization of JP1 at the triads appears to be mainly directed by mechanisms acting at the level of the SR, rather than at T-tubules. FRAP analysis performed on a GFP-TMD fusion protein expressed in myotubes indicated that this protein has a high mobility, suggesting the absence of strong protein-protein interactions occurring at the j-SR. Further work is needed to better understand the molecular mechanisms driving TMD-mediated JP1 localization at triads.

Gd þ3 and GsMTx-4 are more effective than BTP2 or over-expression of a dominant negative Orai1 (E190Q) in decreasing [Ca 2þ ] i in MH-RyR1 R163C myotubes suggesting that it is mediated by an alternative non-Stim1/Orai1 entry pathway. Exposure to a DAG analog induced a larger increase in Ca 2þ in MH-RyR1 R163C myotubes than in WT cells and was completely blocked by Gd 3þ and GsMTx-4 suggesting that TRPC3/6 could be involved. Further support is that these non-selective cationic channels are over-expressed and that [Na] i is elevated in MHRyR1 R163C cells. In vivo in MH-RyR1 R163C/Wt muscle intracellular Ca 2þ and Na þ overload can be blocked by dantrolene, and attenuated by Gd 3þ and GsMTx-4. Exposure of MH-RyR1 R163C/Wt mice to halothane markedly increased both [Ca 2þ ] i and [Na þ ] i in their muscle fibers. These effects of halothane were markedly attenuated by Gd þ3 or GsMTx-4 and completely suppressed by dantrolene. Taken together these results strongly suggest that sarcolemmal Ca 2þ and Na þ entry via TRPC3/6 are important contributors to the altered Ca 2þ and Na þ homeostasis observed in MH muscle both at rest and during an MH crisis.