The monoclonal antibody mAB 1A binds to the excitation--contraction coupling domain in the II-III loop of the skeletal muscle calcium channel alpha(1S) subunit.
Interactions of the II-III loop of the voltage-gated Ca(2+) channel alpha(1S) subunit with the Ca(2+) release channel (RyR1) are essential for skeletal-type excitation-contraction (EC) coupling. Here, we characterized the binding site of the monoclonal alpha(1S) antibody mAB 1A and used it to probe the structure of the II-III loop in chimeras with different EC coupling properties. Phage-display epitope mapping of mAB 1A revealed a minimal consensus binding sequence X-P-X-X-D-X-P. Immunofluorescence labeling of (1S), alpha(1C), alpha(1D), and of II-III loop chimeras expressed in dysgenic myotubes established that mAB 1A reacted specifically with amino acids 737-744 in the II-III loop of alpha(1S), which is within the domain (D734-L764) critical for bidirectional coupling with RyR1. Comparing mAB 1A immunoreactivity with known structural and functional properties of II-III loop chimeras in which the non-conserved skeletal residues were systematically mutated to their cardiac counterparts indicated a correlation of mAB 1A immunoreactivity and skeletal-type EC coupling.
Kugler
Gerlinde
G
Department of Biochemical Pharmacology, Innsbruck Medical University, A-6020 Innsbruck, Austria.
Grabner
Manfred
M
Platzer
Josef
J
Striessnig
Jörg
J
Flucher
Bernhard E
BE
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D017104:Bacteriophage M13 / Q000235:genetics; D001666:Binding Sites, Antibody; D015220:Calcium Channels / Q000737:chemistry / Q000378:metabolism*; D002460:Cell Line; D000939:Epitopes / Q000235:genetics / Q000276:immunology; D018482:Muscle, Skeletal / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D000911:Antibodies, Monoclonal; D015220:Calcium Channels; D000939:Epitopes