Association of calcium channel α(1S) and β(1a) subunits is required for the targeting of β(1a) but not of α(1S) into skeletal muscle triads

The skeletal muscle L-type Ca(2+) channel is a complex of five subunits that is specifically localized in the triad. Its primary function is the rapid activation of Ca(2+) release from cytoplasmic stores in a process called excitation-contraction coupling. To study the role of α(1S)–β(1a) interactions in the incorporation of the functional channel complex into the triad, α(1S) and β(1a) [or a β(1a)-green fluorescent protein (GFP) fusion protein] were expressed alone and in combination in myotubes of the dysgenic cell line GLT. βGFP expressed in dysgenic myotubes that lack the skeletal muscle α(1S) subunit was diffusely distributed in the cytoplasm. On coexpression with the α(1S) subunit βGFP distribution became clustered and colocalized with α(1S) immunofluorescence. Based on the colocalization of βGFP and α(1S) with the ryanodine receptor the clusters were identified as T-tubule/sarcoplasmic reticulum junctions. Expression of α(1S) with and without β(1a) restored Ca(2+) currents and depolarization-induced Ca(2+) release. The translocation of βGFP from the cytoplasm into the junctions failed when βGFP was coexpressed with α(1S) mutants in which the β interaction domain had been altered (α(1S)-Y366S) or deleted (α(1S)-Δ351–380). Although α(1S)-Y366S did not associate with βGFP it was incorporated into the junctions, and it restored Ca(2+) currents and depolarization-induced Ca(2+) release. Thus, β(1a) requires the association with the β interaction domain in the I–II cytoplasmic loop of α(1S) for its own incorporation into triad junctions, but stable α(1S)–β(1a) association is not necessary for the targeting of α(1S) into the triads or for its normal function in Ca(2+) conductance and excitation-contraction coupling.