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 |
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Authors: | Kugler Gerlinde Grabner Manfred Platzer Josef Striessnig Jörg Flucher Bernhard E |
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Institution: | Department of Biochemical Pharmacology, Innsbruck Medical University, A-6020 Innsbruck, Austria. |
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Abstract: | 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. |
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Keywords: | Ca2+ channel Skeletal muscle EC coupling Dihydropyridine receptor Ryanodine receptor Monoclonal antibody Phage-display epitope mapping Immunofluorescence microscopy |
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