The monoclonal antibody mAB 1A binds to the excitation–contraction coupling domain in the II–III loop of the skeletal muscle calcium channel α1S subunit

Interactions of the II–III loop of the voltage-gated Ca2+ channel α1S subunit with the Ca2+ release channel (RyR1) are essential for skeletal-type excitation-contraction (EC) coupling. Here, we characterized the binding site of the monoclonal α1S 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 α1S, α1C, α1D, 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 α1S, 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.