Association of calcium channel alpha1S and beta1a subunits is required for the targeting of beta1a but not of alpha1S into skeletal muscle triads.
The skeletal muscle L-type Ca2+ channel is a complex of five subunits that is specifically localized in the triad. Its primary function is the rapid activation of Ca2+ release from cytoplasmic stores in a process called excitation-contraction coupling. To study the role of alpha1S-beta1a interactions in the incorporation of the functional channel complex into the triad, alpha1S and beta1a [or a beta1a-green fluorescent protein (GFP) fusion protein] were expressed alone and in combination in myotubes of the dysgenic cell line GLT. betaGFP expressed in dysgenic myotubes that lack the skeletal muscle alpha1S subunit was diffusely distributed in the cytoplasm. On coexpression with the alpha1S subunit betaGFP distribution became clustered and colocalized with alpha1S immunofluorescence. Based on the colocalization of betaGFP and alpha1S with the ryanodine receptor the clusters were identified as T-tubule/sarcoplasmic reticulum junctions. Expression of alpha1S with and without beta1a restored Ca2+ currents and depolarization-induced Ca2+ release. The translocation of betaGFP from the cytoplasm into the junctions failed when betaGFP was coexpressed with alpha1S mutants in which the beta interaction domain had been altered (alpha1S-Y366S) or deleted (alpha1S-Delta351-380). Although alpha1S-Y366S did not associate with betaGFP it was incorporated into the junctions, and it restored Ca2+ currents and depolarization-induced Ca2+ release. Thus, beta1a requires the association with the beta interaction domain in the I-II cytoplasmic loop of alpha1S for its own incorporation into triad junctions, but stable alpha1S-beta1a association is not necessary for the targeting of alpha1S into the triads or for its normal function in Ca2+ conductance and excitation-contraction coupling.
Neuhuber
B
B
Department of Biochemical Pharmacology, University of Innsbruck, A-6020 Innsbruck, Austria.
Gerster
U
U
Döring
F
F
Glossmann
H
H
Tanabe
T
T
Flucher
B E
BE
D000818:Animals; D002118:Calcium / Q000502:physiology*; D015220:Calcium Channels / Q000737:chemistry*; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D004594:Electrophysiology; D019084:Fluorescent Antibody Technique, Indirect; D046911:Macromolecular Substances; D051379:Mice; D009119:Muscle Contraction; D018482:Muscle, Skeletal / Q000737:chemistry / Q000648:ultrastructure*; D009223:Myotonic Dystrophy; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D014162:Transfection
D015220:Calcium Channels; D046911:Macromolecular Substances; D002118:Calcium