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 alpha(1S) subunit with the Ca2+ release channel (RyRl) 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 alpha(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 RyRl. 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. (C) 2004 Elsevier Inc. All rights reserved.