The Triad Targeting Signal of the Skeletal Muscle Calcium Channel Is Localized in the Cooh Terminus of the α1S Subunit

Abstract

The specific localization of L-type Ca2+ channels in skeletal muscle triads is critical for their normal function in excitation–contraction (EC) coupling. Reconstitution of dysgenic myotubes with the skeletal muscle Ca2+ channel α1S subunit restores Ca2+ currents, EC coupling, and the normal localization of α1S in the triads. In contrast, expression of the neuronal α1A subunit gives rise to robust Ca2+ currents but not to triad localization. To identify regions in the primary structure of α1S involved in the targeting of the Ca2+ channel into the triads, chimeras of α1S and α1A were constructed, expressed in dysgenic myotubes, and their subcellular distribution was analyzed with double immunofluorescence labeling of the α1S/α1A chimeras and the ryanodine receptor. Whereas chimeras containing the COOH terminus of α1A were not incorporated into triads, chimeras containing the COOH terminus of α1S 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 α1S. Transferring this triad targeting signal to α1A was sufficient for targeting and clustering the neuronal isoform into skeletal muscle triads and caused a marked restoration of Ca2+-dependent EC coupling.

Fluorescence Resonance Energy Transfer (FRET) Indicates That Association with the Type I Ryanodine Receptor (RyR1) Causes Reorientation of Multiple Cytoplasmic Domains of the Dihydropyridine Receptor (DHPR) α1S Subunit

“…A second role that has been described for the C terminus is in targeting. In particular, C-terminal residues 1607-1661 (42) have been reported to be important for the targeting of ␣ 1S to the junctional membrane, and the mutation V1642D in this region was reported to impair targeting (43). However, we found that targeting of ␣ 1S could still occur when a fluorescent protein was attached to the C terminus truncated at 1636.…”

Computer modeling of siRNA knockdown effects indicates an essential role of the Ca 2+ channel α 2 δ-1 subunit in cardiac excitation–contraction coupling

“…Previously we demonstrated that, in the absence of ␣ 1 subunits, ␣ 2 ␦-1 is misstargeted (14,18). Thus, for proper targeting, the ␣ 2 ␦-1 subunit needs to associate with an ␣ 1 subunit, which contains a C-terminal triad-targeting signal (19), but ␣ 2 ␦-1 itself does not contribute to triad targeting of the Ca 2ϩ channel complex.…”

Accessibility of Targeted DHPR Sites to Streptavidin and Functional Effects of Binding on EC Coupling

“…In particular, portions of the C terminus of α 1C (Gao et al, 2000) and α 1S (Proenza et al, 2000) may be important both for traffi cking to the plasma membrane, and for targeting to plasma membrane junctions with the SR (Flucher et al, 2000b;Proenza et al, 2000). Moreover, biochemical studies have suggested that the C-terminal residues 1393-1527 of α 1S bind calmodulin and can bind to RyR1 (Sencer et al, 2001).…”