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

Abstract

Interactions of the II–III loop of the voltage-gated Ca 2+ channel α 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 α 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 α 1S, α 1C, α 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 α 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.