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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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Targeting properties and current properties of wild-type α1 subunit isoforms and COOH-terminal chimeras. (a) Isoform sequence composition of COOH termini in the studied chimeras, with sequences of α1S in gray and α1A in black. Bar graph indicates the percentages of transfected myotubes showing triad targeting in immunofluorescence analysis. Alignment of the 55 α1S amino acids containing the targeting signal with the corresponding sequences of α1C and α1A. (b) Representative current traces recorded from dysgenic myotubes transfected with wild-type GFP-α1S (in 10 mM Ca2+), with GFP-α1A or with the targeted chimera GFP-α1As (both in 3 mM Ca2+). Currents from GFP-α1Sa (in 10 mM Ca2+) were too small for systematic analysis; see frequency distribution of current densities. (c) Representative current trace of the targeted chimera GFP-α1Aas(1592-clip) and comparison of peak current densities recorded from GFP-α1A, GFP-α1Aas(1592-clip), and GFP-α1Aas(1524-1591). Substituting COOH-terminal α1A sequence with the skeletal sequence 1592–1661 results in a twofold increase of current density compared with GFP-α1A, whereas substituting for skeletal sequence 1524–1591 reduces the current densities to near the detection level and could be analyzed only after increasing the Ca2+ concentration to 10 mM (n = 7–17).
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Figure 6: Targeting properties and current properties of wild-type α1 subunit isoforms and COOH-terminal chimeras. (a) Isoform sequence composition of COOH termini in the studied chimeras, with sequences of α1S in gray and α1A in black. Bar graph indicates the percentages of transfected myotubes showing triad targeting in immunofluorescence analysis. Alignment of the 55 α1S amino acids containing the targeting signal with the corresponding sequences of α1C and α1A. (b) Representative current traces recorded from dysgenic myotubes transfected with wild-type GFP-α1S (in 10 mM Ca2+), with GFP-α1A or with the targeted chimera GFP-α1As (both in 3 mM Ca2+). Currents from GFP-α1Sa (in 10 mM Ca2+) were too small for systematic analysis; see frequency distribution of current densities. (c) Representative current trace of the targeted chimera GFP-α1Aas(1592-clip) and comparison of peak current densities recorded from GFP-α1A, GFP-α1Aas(1592-clip), and GFP-α1Aas(1524-1591). Substituting COOH-terminal α1A sequence with the skeletal sequence 1592–1661 results in a twofold increase of current density compared with GFP-α1A, whereas substituting for skeletal sequence 1524–1591 reduces the current densities to near the detection level and could be analyzed only after increasing the Ca2+ concentration to 10 mM (n = 7–17).

Mentions: To exclude the possibility that the absence of GFP-α1A resulted from improper folding or lack of plasma membrane incorporation of the GFP-α1A construct rather than lack of a triad targeting signal, we performed patch-clamp recordings of myotubes expressing this construct. Even though a plasma membrane stain was not detected with immunocytochemistry in GFP-α1A–transfected myotubes, the whole-cell recordings showed large Ca2+ currents with the macroscopic properties of class-A Ca2+ channels expressed in heterologous mammalian expression systems (example shown in Fig. 6, below) (Adams et al. 1994). Thus, GFP-α1A expressed in dysgenic myotubes formed functional channels in the cell membrane. But instead of becoming locally concentrated in the triads, GFP-α1A was distributed diffusely in the plasma membrane at densities below detectability with immunocytochemistry.


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)

Targeting properties and current properties of wild-type α1 subunit isoforms and COOH-terminal chimeras. (a) Isoform sequence composition of COOH termini in the studied chimeras, with sequences of α1S in gray and α1A in black. Bar graph indicates the percentages of transfected myotubes showing triad targeting in immunofluorescence analysis. Alignment of the 55 α1S amino acids containing the targeting signal with the corresponding sequences of α1C and α1A. (b) Representative current traces recorded from dysgenic myotubes transfected with wild-type GFP-α1S (in 10 mM Ca2+), with GFP-α1A or with the targeted chimera GFP-α1As (both in 3 mM Ca2+). Currents from GFP-α1Sa (in 10 mM Ca2+) were too small for systematic analysis; see frequency distribution of current densities. (c) Representative current trace of the targeted chimera GFP-α1Aas(1592-clip) and comparison of peak current densities recorded from GFP-α1A, GFP-α1Aas(1592-clip), and GFP-α1Aas(1524-1591). Substituting COOH-terminal α1A sequence with the skeletal sequence 1592–1661 results in a twofold increase of current density compared with GFP-α1A, whereas substituting for skeletal sequence 1524–1591 reduces the current densities to near the detection level and could be analyzed only after increasing the Ca2+ concentration to 10 mM (n = 7–17).
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Related In: Results  -  Collection

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Figure 6: Targeting properties and current properties of wild-type α1 subunit isoforms and COOH-terminal chimeras. (a) Isoform sequence composition of COOH termini in the studied chimeras, with sequences of α1S in gray and α1A in black. Bar graph indicates the percentages of transfected myotubes showing triad targeting in immunofluorescence analysis. Alignment of the 55 α1S amino acids containing the targeting signal with the corresponding sequences of α1C and α1A. (b) Representative current traces recorded from dysgenic myotubes transfected with wild-type GFP-α1S (in 10 mM Ca2+), with GFP-α1A or with the targeted chimera GFP-α1As (both in 3 mM Ca2+). Currents from GFP-α1Sa (in 10 mM Ca2+) were too small for systematic analysis; see frequency distribution of current densities. (c) Representative current trace of the targeted chimera GFP-α1Aas(1592-clip) and comparison of peak current densities recorded from GFP-α1A, GFP-α1Aas(1592-clip), and GFP-α1Aas(1524-1591). Substituting COOH-terminal α1A sequence with the skeletal sequence 1592–1661 results in a twofold increase of current density compared with GFP-α1A, whereas substituting for skeletal sequence 1524–1591 reduces the current densities to near the detection level and could be analyzed only after increasing the Ca2+ concentration to 10 mM (n = 7–17).
Mentions: To exclude the possibility that the absence of GFP-α1A resulted from improper folding or lack of plasma membrane incorporation of the GFP-α1A construct rather than lack of a triad targeting signal, we performed patch-clamp recordings of myotubes expressing this construct. Even though a plasma membrane stain was not detected with immunocytochemistry in GFP-α1A–transfected myotubes, the whole-cell recordings showed large Ca2+ currents with the macroscopic properties of class-A Ca2+ channels expressed in heterologous mammalian expression systems (example shown in Fig. 6, below) (Adams et al. 1994). Thus, GFP-α1A expressed in dysgenic myotubes formed functional channels in the cell membrane. But instead of becoming locally concentrated in the triads, GFP-α1A was distributed diffusely in the plasma membrane at densities below detectability with immunocytochemistry.

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