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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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Restoration of EC coupling by targeted and nontargeted Ca2+ channel isoforms. Action potential–induced Ca2+ transients were recorded in transfected dysgenic myotubes loaded with the fluorescent Ca2+ indicator Fluo4-AM, using tetanic electrical stimulation (left, 20 Hz, 2 s, bracket) or low frequency stimulation (center, 0.3–0.5 Hz as marked). 0.5 mM Cd2+/0.1 mM La3+ (gray bar) was applied to block the Ca2+ influx during the low-frequency stimulation protocol. Ca2+ release from the SR could be triggered by the application of 6 mM caffeine (gray bar) to the bath after current block. Myotubes transfected with the skeletal GFP-α1S responded to electrical stimulation with Ca2+ transients independently of Ca2+ influx. The cardiac GFP-α1C also reconstituted EC coupling in dysgenic myotubes; however, Ca2+ transients stopped when the Ca2+ influx was blocked. Cardiac-type Ca2+ transients in response to electrical stimulation were rarely observed in dysgenic myotubes transfected with GFP-α1A (see Table ) and about nine times more often with the targeted GFP-α1Aas(1592-clip). Example traces for each construct were recorded from the same myotubes in sequential order.
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Figure 7: Restoration of EC coupling by targeted and nontargeted Ca2+ channel isoforms. Action potential–induced Ca2+ transients were recorded in transfected dysgenic myotubes loaded with the fluorescent Ca2+ indicator Fluo4-AM, using tetanic electrical stimulation (left, 20 Hz, 2 s, bracket) or low frequency stimulation (center, 0.3–0.5 Hz as marked). 0.5 mM Cd2+/0.1 mM La3+ (gray bar) was applied to block the Ca2+ influx during the low-frequency stimulation protocol. Ca2+ release from the SR could be triggered by the application of 6 mM caffeine (gray bar) to the bath after current block. Myotubes transfected with the skeletal GFP-α1S responded to electrical stimulation with Ca2+ transients independently of Ca2+ influx. The cardiac GFP-α1C also reconstituted EC coupling in dysgenic myotubes; however, Ca2+ transients stopped when the Ca2+ influx was blocked. Cardiac-type Ca2+ transients in response to electrical stimulation were rarely observed in dysgenic myotubes transfected with GFP-α1A (see Table ) and about nine times more often with the targeted GFP-α1Aas(1592-clip). Example traces for each construct were recorded from the same myotubes in sequential order.

Mentions: Recordings of cytoplasmic Ca2+ transients in response to electrical field stimulation showed that GFP-α1S regularly restored skeletal muscle EC coupling in dysgenic myotubes (see Fig. 7, and Powell et al. 1996; Flucher et al. 2000). In contrast, restoration of EC coupling by GFP-α1A was only rarely observed (see below). Thus, both the skeletal muscle GFP-α1S isoform and the neuronal GFP-α1A isoform were functionally expressed in dysgenic myotubes, but only GFP-α1S was targeted into the triad junctions and efficiently restored EC coupling. The differential distribution of GFP-α1S and GFP-α1A as well as their functional differences are in general agreement with observations from a previous study comparing the expression of GFP-α1S, GFP-α1A, and a cardiac GFP-α1C construct in primary cultured dysgenic myotubes (Grabner et al. 1998). In that study, GFP-α1A differed from the muscle isoforms in that its distribution patterns were restricted to near the injection site and that only GFP-α1A failed to respond in a contraction assay. Together, these data support our hypothesis that the skeletal muscle α1S contains a signal for its targeting and selective incorporation into triads, but that such a triad targeting signal is missing from the neuronal α1A subunit isoform.


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)

Restoration of EC coupling by targeted and nontargeted Ca2+ channel isoforms. Action potential–induced Ca2+ transients were recorded in transfected dysgenic myotubes loaded with the fluorescent Ca2+ indicator Fluo4-AM, using tetanic electrical stimulation (left, 20 Hz, 2 s, bracket) or low frequency stimulation (center, 0.3–0.5 Hz as marked). 0.5 mM Cd2+/0.1 mM La3+ (gray bar) was applied to block the Ca2+ influx during the low-frequency stimulation protocol. Ca2+ release from the SR could be triggered by the application of 6 mM caffeine (gray bar) to the bath after current block. Myotubes transfected with the skeletal GFP-α1S responded to electrical stimulation with Ca2+ transients independently of Ca2+ influx. The cardiac GFP-α1C also reconstituted EC coupling in dysgenic myotubes; however, Ca2+ transients stopped when the Ca2+ influx was blocked. Cardiac-type Ca2+ transients in response to electrical stimulation were rarely observed in dysgenic myotubes transfected with GFP-α1A (see Table ) and about nine times more often with the targeted GFP-α1Aas(1592-clip). Example traces for each construct were recorded from the same myotubes in sequential order.
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Related In: Results  -  Collection

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Figure 7: Restoration of EC coupling by targeted and nontargeted Ca2+ channel isoforms. Action potential–induced Ca2+ transients were recorded in transfected dysgenic myotubes loaded with the fluorescent Ca2+ indicator Fluo4-AM, using tetanic electrical stimulation (left, 20 Hz, 2 s, bracket) or low frequency stimulation (center, 0.3–0.5 Hz as marked). 0.5 mM Cd2+/0.1 mM La3+ (gray bar) was applied to block the Ca2+ influx during the low-frequency stimulation protocol. Ca2+ release from the SR could be triggered by the application of 6 mM caffeine (gray bar) to the bath after current block. Myotubes transfected with the skeletal GFP-α1S responded to electrical stimulation with Ca2+ transients independently of Ca2+ influx. The cardiac GFP-α1C also reconstituted EC coupling in dysgenic myotubes; however, Ca2+ transients stopped when the Ca2+ influx was blocked. Cardiac-type Ca2+ transients in response to electrical stimulation were rarely observed in dysgenic myotubes transfected with GFP-α1A (see Table ) and about nine times more often with the targeted GFP-α1Aas(1592-clip). Example traces for each construct were recorded from the same myotubes in sequential order.
Mentions: Recordings of cytoplasmic Ca2+ transients in response to electrical field stimulation showed that GFP-α1S regularly restored skeletal muscle EC coupling in dysgenic myotubes (see Fig. 7, and Powell et al. 1996; Flucher et al. 2000). In contrast, restoration of EC coupling by GFP-α1A was only rarely observed (see below). Thus, both the skeletal muscle GFP-α1S isoform and the neuronal GFP-α1A isoform were functionally expressed in dysgenic myotubes, but only GFP-α1S was targeted into the triad junctions and efficiently restored EC coupling. The differential distribution of GFP-α1S and GFP-α1A as well as their functional differences are in general agreement with observations from a previous study comparing the expression of GFP-α1S, GFP-α1A, and a cardiac GFP-α1C construct in primary cultured dysgenic myotubes (Grabner et al. 1998). In that study, GFP-α1A differed from the muscle isoforms in that its distribution patterns were restricted to near the injection site and that only GFP-α1A failed to respond in a contraction assay. Together, these data support our hypothesis that the skeletal muscle α1S contains a signal for its targeting and selective incorporation into triads, but that such a triad targeting signal is missing from the neuronal α1A subunit isoform.

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