Ankyrin-B Is Required for Intracellular Sorting of Structurally Diverse Ca 2 1 Homeostasis Proteins (original) (raw)
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Calcium Homeostasis Is Modified in Skeletal Muscle Fibers of Small Ankyrin1 Knockout Mice
International Journal of Molecular Sciences, 2019
Small Ankyrins (sAnk1) are muscle-specific isoforms generated by the Ank1 gene that participate in the organization of the sarcoplasmic reticulum (SR) of striated muscles. Accordingly, the volume of SR tubules localized around the myofibrils is strongly reduced in skeletal muscle fibers of 4- and 10-month-old sAnk1 knockout (KO) mice, while additional structural alterations only develop with aging. To verify whether the lack of sAnk1 also alters intracellular Ca2+ handling, cytosolic Ca2+ levels were analyzed in stimulated skeletal muscle fibers from 4- and 10-month-old sAnk1 KO mice. The SR Ca2+ content was reduced in sAnk1 KO mice regardless of age. The amplitude of the Ca2+ transients induced by depolarizing pulses was decreased in myofibers of sAnk1 KO with respect to wild type (WT) fibers, while their voltage dependence was not affected. Furthermore, analysis of spontaneous Ca2+ release events (sparks) on saponin-permeabilized muscle fibers indicated that the frequency of spark...
Ankyrin-B Regulates Cav2.1 and Cav2.2 Channel Expression and Targeting
Journal of Biological Chemistry, 2014
Background: Calcium channels control membrane excitability. The mechanisms underlying Ca v 2.1/Ca v 2.2 targeting are not well understood. Results: Ankyrin-B associates with Ca v 2.1/Ca v 2.2. Loss of ankyrin-B results in reduced expression of Ca v 2.1/Ca v 2.2 in select brain regions. Conclusion: Ankyrin-B plays a role in the expression of Ca v 2.1/Ca v 2.2. Significance: Results identify pathway for membrane targeting of calcium channels and regulation of membrane excitability. N-type and P/Q-type calcium channels are documented players in the regulation of synaptic function; however, the mechanisms underlying their expression and cellular targeting are poorly understood. Ankyrin polypeptides are essential for normal integral membrane protein expression in a number of cell types, including neurons, cardiomyocytes, epithelia, secretory cells, and erythrocytes. Ankyrin dysfunction has been linked to defects in integral protein expression, abnormal cellular function, and disease. Here, we demonstrate that ankyrin-B associates with Ca v 2.1 and Ca v 2.2 in cortex, cerebellum, and brain stem. Additionally, using in vitro and in vivo techniques, we demonstrate that ankyrin-B, via its membrane-binding domain, associates with a highly conserved motif in the DII/III loop domain of Ca v 2.1 and Ca v 2.2. Further, we demonstrate that this domain is necessary for proper targeting of Ca v 2.1 and Ca v 2.2 in a heterologous system. Finally, we demonstrate that mutation of a single conserved tyrosine residue in the ankyrin-binding motif of both Ca v 2.1 (Y797E) and Ca v 2.2 (Y788E) results in loss of association with ankyrin-B in vitro and in vivo. Collectively, our findings identify an interaction between ankyrin-B and both Ca v 2.1 and Ca v 2.2 at the amino acid level that is necessary for proper Ca v 2.1 and Ca v 2.2 targeting in vivo.
Calcium homeostasis alterations in a mouse model of the Dynamin 2-related centronuclear myopathy
Biology open, 2016
Autosomal dominant centronuclear myopathy (CNM) is a rare congenital myopathy characterized by centrally located nuclei in muscle fibers. CNM results from mutations in the gene encoding dynamin 2 (DNM2), a large GTPase involved in endocytosis, intracellular membrane trafficking, and cytoskeleton regulation. We developed a knock-in mouse model expressing the most frequent DNM2-CNM mutation; i.e. the KI-Dnm2(R465W) model. Heterozygous (HTZ) KI-Dnm2 mice progressively develop muscle atrophy, impairment of contractile properties, histopathological abnormalities, and elevated cytosolic calcium concentration. Here, we aim at better characterizing the calcium homeostasis impairment in extensor digitorum longus (EDL) and soleus muscles from adult HTZ KI-Dnm2 mice. We demonstrate abnormal contractile properties and cytosolic Ca(2+) concentration in EDL but not soleus muscles showing that calcium impairment is correlated with muscle weakness and might be a determinant factor of the spatial mu...
Subcellular analysis of Ca2+ homeostasis in primary cultures of skeletal muscle myotubes
Molecular Biology of the Cell, 1997
Specifically targeted aequorin chimeras were used for studying the dynamic changes of Ca2+ concentration in different subcellular compartments of differentiated skeletal muscle myotubes. For the cytosol, mitochondria, and nucleus, the previously described chimeric aequorins were utilized; for the sarcoplasmic reticulum (SR), a new chimera (srAEQ) was developed by fusing an aequorin mutant with low Ca2+ affinity to the resident protein calsequestrin. By using an appropriate transfection procedure, the expression of the recombinant proteins was restricted, within the culture, to the differentiated myotubes, and the correct sorting of the various chimeras was verified with immunocytochemical techniques. Single-cell analysis of cytosolic Ca2+ concentration ([Ca2+]c) with fura-2 showed that the myotubes responded, as predicted, to stimuli known to be characteristic of skeletal muscle fibers, i.e., KCl-induced depolarization, caffeine, and carbamylcholine. Using these stimuli in cultures transfected with the various aequorin chimeras, we show that: 1) the nucleoplasmic Ca2+ concentration ([Ca2+]n) closely mimics the [Ca2+Ic, at rest and after stimulation, indicating a rapid equilibration of the two compartments also in this cell type; 2) on the contrary, mitochondria amplify 4-6-fold the [Ca2+]c increases; and 3) the lumenal concentration of Ca2+ within the SR ([Ca2+Isr) is much higher than in the other compartments (>100 JIM), too high to be accurately measured also with the aequorin mutant with low Ca2+ affinity. An indirect estimate of the resting value (-1-2 mM) was obtained using Sr2+, a surrogate of Ca2+ which, because of the lower affinity of the photoprotein for this cation, elicits a lower rate of aequorin consumption. With Sr2 , the kinetics and amplitudes of the changes in [cationii]sr evoked by the various stimuli could also be directly analyzed.
Altered Ca2+ homeostasis and endoplasmic reticulum stress in Myotonic Dystrophy type 1 muscle cells
Genes, 2013
The pathogenesis of Myotonic Dystrophy type 1 (DM1) is linked to unstable CTG repeats in the DMPK gene which induce the mis-splicing to fetal/neonatal isoforms of many transcripts, including those involved in cellular Ca2+ homeostasis. Here we monitored the splicing of three genes encoding for Ca2+ transporters and channels (RyR1, SERCA1 and CACN1S) during maturation of primary DM1 muscle cells in parallel with the functionality of the Excitation-Contraction (EC) coupling machinery. At 15 days of differentiation, fetal isoforms of SERCA1 and CACN1S mRNA were significantly higher in DM1 myotubes compared to controls. Parallel functional studies showed that the cytosolic Ca2+ response to depolarization in DM1 myotubes did not increase during the progression of differentiation, in contrast to control myotubes. While we observed no differences in the size of intracellular Ca2+ stores, DM1 myotubes showed significantly reduced RyR1 protein levels, uncoupling between the segregated ER/SR Ca2+ store and the voltage-induced Ca2+ release machinery, parallel with induction of endoplasmic reticulum (ER) stress markers. In conclusion, our data suggest that perturbed Ca2+ homeostasis, via activation of ER stress, contributes to muscle degeneration in DM1 muscle cells likely representing a premature senescence phenotype.
Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2002
Calcium is the most ubiquitous second messenger. Its concentration inside the cell is tightly regulated by a series of mechanisms, among which some have been extensively studied in nonmuscle cells. This is the case of the "store-operated entry of Ca(2+)", the uptake of Ca(2+) by mitochondria and the inositol 1,4,5-trisphosphate (IP(3)) cascade. These processes were recently found to be also present in skeletal muscle and are reviewed here. The "store-operated entry of Ca(2+)" allows the refilling of the stores after muscle fiber depolarization and is activated even after a partial depletion of the sarcoplasmic reticulum (SR). The uptake of Ca(2+) by mitochondria accelerates muscle relaxation and allows the adaptation of ATP supply to the increased energy demand. IP(3) receptors are found in the nuclear envelope and are involved in Ca(2+) waves propagating from one nucleus to another. This pathway is possibly involved in gene expression regulation. Finally, cytosolic Ca(2+) buffers like parvalbumins modify [Ca(2+)](i) transients and, therefore, muscle mechanics.The importance of these regulation mechanisms is also evaluated in Duchenne muscular dystrophy (DMD), a disease in which impairment of [Ca(2+)](i) homeostasis has been postulated but remains, however, controversial. This genetic disease is indeed characterized by the absence of a cytoskeletal protein called dystrophin, a situation leading to a disorganization of the cytoskeleton and to an abnormal influx of Ca(2+). How this increased entry of Ca(2+) affects the local concentration of Ca(2+) in subcellular compartmen...
The Biochemical journal, 1992
The effect of thyroid hormone (L-tri-iodothyronine; T3) on the cytosolic free Ca2+ concentration ([Ca2+]i) in L6 myotubes was studied at rest and during activation to explore the possible mediating role of [Ca2+]i in the T3-induced net synthesis of fast-type sarcoplasmic reticulum (SR) Ca(2+)-ATPase. The mean [Ca2+]i at rest was approx. 115 nM in myoblasts, control myotubes and T3-treated myotubes. Therefore it is unlikely that the T3-induced elevation of Ca(2+)-ATPase levels is mediated by [Ca2+]i changes. To investigate the influence of the 4-fold higher Ca(2+)-ATPase levels in T3-treated myotubes (compared with controls) on [Ca2+]i, interventions with caffeine (10 mM) and a high extracellular K+ concentration ([K+]o) (30 mM) were applied which initially mobilize Ca2+ predominantly from the SR. The results showed a lower (caffeine) or not significantly different (high [K+]o) increase in [Ca2+]i in T3-treated myotubes compared with controls. No rise in [Ca2+]i was found in myoblast...
The Journal of Cell Biology, 1995
Calsequestrin (CSQ) is the low affinity, high capacity Ca2+-binding protein concentrated within specialized areas of the muscle fiber sarcoplasmic reticulum (a part of the ER) where it is believed to buffer large amounts of Ca 2÷. Upon activation of intracellular channels this Ca 2+ pool is released, giving rise to the [Ca2+]i increases that sustain contraction. In order to investigate the ER retention and the functional role of the protein, L6 rat myoblasts were infected with a viral vector with or without the cDNA of chicken CSQ, and stable clones were investigated before and after differentiation to myotubes. In the undifferentiated L6 cells, expression of considerable amounts of heterologous CSQ occurred with no major changes of other ER components. Ca 2+ release from the ER, induced by the peptide hormone vasopressin, remained however unchanged, and the same occurred when other treatments were given in sequence to deplete the ER and other intraceUular stores: with the Ca 2+ pump blocker, thapsigargin; and with the Ca 2+ ionophore, ionomycin, followed by the Na÷/H + ionophore, monensin. The lack of effect of CSQ expression on the vasopressin-induced [Ca2+]i responses was explained by immunocytochemistry showing the heter-ologous protein to be localized not in the ER but in large vacuoles of acidic content, positive also for the lysosomal enzyme, cathepsin D, corresponding to a lysosomal subpopulation. After differentiation, all L6 cells expressed small amounts of homologous CSQ. In the infected cells the heterologous protein progressively decreased, yet the [Ca2+]i responses to vasopressin were now larger with respect to both control and undifferentiated cells. This change correlated with the drop of the vacuoles and with the accumulation of CSQ within the ER lumen, where a clustered distribution was observed as recently shown in developing muscle fibers. These results provide direct evidence for the contribution of CSQ, when appropriately retained, to the Ca 2÷ capacity of the rapidly exchanging, ER-located Ca 2+ stores; and for the existence of specific mechanism(s) (that in L6 cells develop in the course of differentiation) for the ER retention of the protein. In the growing L6 myoblasts the Ca2+-binding protein appears in contrast to travel along the exocytic pathway, down to post-Golgi, lysosome-related vacuoles which, based on the lack of [Ca2+]~ response to ionomycin-monensin, appear to be incompetent for Ca 2+ accumulation.