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The triad targeting signal of the skeletal muscle calcium channel is localized in the COOH terminus of the alpha(1S) subunit.

Flucher BE, Kasielke N, Grabner M - J. Cell Biol. (2000)

Bottom Line: In contrast, expression of the neuronal alpha(1A) subunit gives rise to robust Ca(2+) currents but not to triad localization.Mapping of the COOH terminus revealed a triad-targeting signal contained in the 55 amino-acid sequence (1607-1661) proximal to the putative clipping site of alpha(1S).Transferring this triad targeting signal to alpha(1A) was sufficient for targeting and clustering the neuronal isoform into skeletal muscle triads and caused a marked restoration of Ca(2+)-dependent EC coupling.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemical Pharmacology, University of Innsbruck, A-6020 Innsbruck, Austria. bernhard.e.flucher@uibk.ac.at

ABSTRACT
The specific localization of L-type Ca(2+) channels in skeletal muscle triads is critical for their normal function in excitation-contraction (EC) coupling. Reconstitution of dysgenic myotubes with the skeletal muscle Ca(2+) channel alpha(1S) subunit restores Ca(2+) currents, EC coupling, and the normal localization of alpha(1S) in the triads. In contrast, expression of the neuronal alpha(1A) subunit gives rise to robust Ca(2+) currents but not to triad localization. To identify regions in the primary structure of alpha(1S) involved in the targeting of the Ca(2+) channel into the triads, chimeras of alpha(1S) and alpha(1A) were constructed, expressed in dysgenic myotubes, and their subcellular distribution was analyzed with double immunofluorescence labeling of the alpha(1S)/alpha(1A) chimeras and the ryanodine receptor. Whereas chimeras containing the COOH terminus of alpha(1A) were not incorporated into triads, chimeras containing the COOH terminus of alpha(1S) were correctly targeted. Mapping of the COOH terminus revealed a triad-targeting signal contained in the 55 amino-acid sequence (1607-1661) proximal to the putative clipping site of alpha(1S). Transferring this triad targeting signal to alpha(1A) was sufficient for targeting and clustering the neuronal isoform into skeletal muscle triads and caused a marked restoration of Ca(2+)-dependent EC coupling.

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Normal incorporation of heterologously expressed GFP-α1S in t tubule/SR junctions of dysgenic myotubes. (a and b) Double immunofluorescence labeling of α1S subunits (a) and RyRs (b) shows that GFP-α1S is colocalized with RyRs in clusters (examples indicated by arrows), presumably representing triad junctions and peripheral couplings. A nontransfected myotube in the same field (#) shows RyR clusters but no α1S labeling. (c and d) Double staining of GFP-α1S with antibodies against α1S and against GFP shows that the fusion protein can be localized using either one of the antibodies. (e and f) As with anti–α1S (a), clusters labeled with anti–GFP (e) are colocalized with RyR clusters (examples indicated by arrows). In poorly differentiated myotubes that lack RyR clusters (*), GFP-α1S accumulates in a reticular membrane system with densities in the perinuclear region, presumably the ER/SR network. N, nuclei. Bar, 10 μm.
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Figure 1: Normal incorporation of heterologously expressed GFP-α1S in t tubule/SR junctions of dysgenic myotubes. (a and b) Double immunofluorescence labeling of α1S subunits (a) and RyRs (b) shows that GFP-α1S is colocalized with RyRs in clusters (examples indicated by arrows), presumably representing triad junctions and peripheral couplings. A nontransfected myotube in the same field (#) shows RyR clusters but no α1S labeling. (c and d) Double staining of GFP-α1S with antibodies against α1S and against GFP shows that the fusion protein can be localized using either one of the antibodies. (e and f) As with anti–α1S (a), clusters labeled with anti–GFP (e) are colocalized with RyR clusters (examples indicated by arrows). In poorly differentiated myotubes that lack RyR clusters (*), GFP-α1S accumulates in a reticular membrane system with densities in the perinuclear region, presumably the ER/SR network. N, nuclei. Bar, 10 μm.

Mentions: The triad localization of GFP-α1S can be detected in double immunofluorescence labeling experiments with antibodies against the α1S subunit and against the RyR (Fig. 1, a and b). Immunolabeling results in a punctate distribution pattern of anti–α1S that is colocalized with similar clusters of anti–RyR. This clustered distribution pattern is characteristic of triad proteins in developing myotubes, and the coexistence of the t-tubule protein α1S with the SR protein, the RyR, is indicative of their localization in junctions between the two membrane systems (Flucher et al. 1994). Myotubes not expressing GFP-α1S form RyR clusters (Fig. 1 b), which have previously been shown to correspond to t tubule/SR junctions by electron microscopy (Powell et al. 1996).


The triad targeting signal of the skeletal muscle calcium channel is localized in the COOH terminus of the alpha(1S) subunit.

Flucher BE, Kasielke N, Grabner M - J. Cell Biol. (2000)

Normal incorporation of heterologously expressed GFP-α1S in t tubule/SR junctions of dysgenic myotubes. (a and b) Double immunofluorescence labeling of α1S subunits (a) and RyRs (b) shows that GFP-α1S is colocalized with RyRs in clusters (examples indicated by arrows), presumably representing triad junctions and peripheral couplings. A nontransfected myotube in the same field (#) shows RyR clusters but no α1S labeling. (c and d) Double staining of GFP-α1S with antibodies against α1S and against GFP shows that the fusion protein can be localized using either one of the antibodies. (e and f) As with anti–α1S (a), clusters labeled with anti–GFP (e) are colocalized with RyR clusters (examples indicated by arrows). In poorly differentiated myotubes that lack RyR clusters (*), GFP-α1S accumulates in a reticular membrane system with densities in the perinuclear region, presumably the ER/SR network. N, nuclei. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2192640&req=5

Figure 1: Normal incorporation of heterologously expressed GFP-α1S in t tubule/SR junctions of dysgenic myotubes. (a and b) Double immunofluorescence labeling of α1S subunits (a) and RyRs (b) shows that GFP-α1S is colocalized with RyRs in clusters (examples indicated by arrows), presumably representing triad junctions and peripheral couplings. A nontransfected myotube in the same field (#) shows RyR clusters but no α1S labeling. (c and d) Double staining of GFP-α1S with antibodies against α1S and against GFP shows that the fusion protein can be localized using either one of the antibodies. (e and f) As with anti–α1S (a), clusters labeled with anti–GFP (e) are colocalized with RyR clusters (examples indicated by arrows). In poorly differentiated myotubes that lack RyR clusters (*), GFP-α1S accumulates in a reticular membrane system with densities in the perinuclear region, presumably the ER/SR network. N, nuclei. Bar, 10 μm.
Mentions: The triad localization of GFP-α1S can be detected in double immunofluorescence labeling experiments with antibodies against the α1S subunit and against the RyR (Fig. 1, a and b). Immunolabeling results in a punctate distribution pattern of anti–α1S that is colocalized with similar clusters of anti–RyR. This clustered distribution pattern is characteristic of triad proteins in developing myotubes, and the coexistence of the t-tubule protein α1S with the SR protein, the RyR, is indicative of their localization in junctions between the two membrane systems (Flucher et al. 1994). Myotubes not expressing GFP-α1S form RyR clusters (Fig. 1 b), which have previously been shown to correspond to t tubule/SR junctions by electron microscopy (Powell et al. 1996).

Bottom Line: In contrast, expression of the neuronal alpha(1A) subunit gives rise to robust Ca(2+) currents but not to triad localization.Mapping of the COOH terminus revealed a triad-targeting signal contained in the 55 amino-acid sequence (1607-1661) proximal to the putative clipping site of alpha(1S).Transferring this triad targeting signal to alpha(1A) was sufficient for targeting and clustering the neuronal isoform into skeletal muscle triads and caused a marked restoration of Ca(2+)-dependent EC coupling.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemical Pharmacology, University of Innsbruck, A-6020 Innsbruck, Austria. bernhard.e.flucher@uibk.ac.at

ABSTRACT
The specific localization of L-type Ca(2+) channels in skeletal muscle triads is critical for their normal function in excitation-contraction (EC) coupling. Reconstitution of dysgenic myotubes with the skeletal muscle Ca(2+) channel alpha(1S) subunit restores Ca(2+) currents, EC coupling, and the normal localization of alpha(1S) in the triads. In contrast, expression of the neuronal alpha(1A) subunit gives rise to robust Ca(2+) currents but not to triad localization. To identify regions in the primary structure of alpha(1S) involved in the targeting of the Ca(2+) channel into the triads, chimeras of alpha(1S) and alpha(1A) were constructed, expressed in dysgenic myotubes, and their subcellular distribution was analyzed with double immunofluorescence labeling of the alpha(1S)/alpha(1A) chimeras and the ryanodine receptor. Whereas chimeras containing the COOH terminus of alpha(1A) were not incorporated into triads, chimeras containing the COOH terminus of alpha(1S) were correctly targeted. Mapping of the COOH terminus revealed a triad-targeting signal contained in the 55 amino-acid sequence (1607-1661) proximal to the putative clipping site of alpha(1S). Transferring this triad targeting signal to alpha(1A) was sufficient for targeting and clustering the neuronal isoform into skeletal muscle triads and caused a marked restoration of Ca(2+)-dependent EC coupling.

Show MeSH
Related in: MedlinePlus