The Triad Targeting Signal of the Skeletal Muscle Calcium Channel Is Localized in the Cooh Terminus of the α_1S Subunit

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 α_1S subunit restores Ca²⁺ currents, EC coupling, and the normal localization of α_1S in the triads. In contrast, expression of the neuronal α_1A subunit gives rise to robust Ca²⁺ currents but not to triad localization. To identify regions in the primary structure of α_1S involved in the targeting of the Ca²⁺ channel into the triads, chimeras of α_1S and α_1A were constructed, expressed in dysgenic myotubes, and their subcellular distribution was analyzed with double immunofluorescence labeling of the α_1S/α_1A chimeras and the ryanodine receptor. Whereas chimeras containing the COOH terminus of α_1A were not incorporated into triads, chimeras containing the COOH terminus of α_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 α_1S. Transferring this triad targeting signal to α_1A was sufficient for targeting and clustering the neuronal isoform into skeletal muscle triads and caused a marked restoration of Ca²⁺-dependent EC coupling.