Structure-function link in the DHPR ß1a for muscle motility

The ß1a subunit of the heteromultimeric skeletal muscle dihydropyridine receptor (DHPR) complex is essential for enhancement of DHPR triad expression, elicitation of DHPRa1S charge movement, and assembly of DHPRs in tetrads. These three features are prerequisite for the tight protein-protein interaction between the sarcolemmal DHPR and the sarcoplasmic ryanodine receptor (RyR1) which consequently enables skeletal-muscle type excitation-contraction (EC) coupling. Akin to DHPRa1S-null (dysgenic) and RyR1-null (dyspedic) mice, DHPRß1-null mice and zebrafish (strain relaxed) show a lethal phenotype due to complete absence of skeletal muscle EC coupling. Previously we showed, in reconstitution studies in relaxed myotubes, that all vertebrate -isoforms () and even the ancestral M from housefly are able to fully restore DHPR triad expression. Similarly, DHPRa1S charge movement (Q) restoration is rather a promiscuous function of all subunits, except 3. As recently shown, systematic swapping of molecular domains between ß1a and ß3 revealed a pivotal role of the ß1a SH3 domain and C- terminus in Q restoration. Our results also indicated that this domain-domain interaction is dependent on a SH3-binding polyproline (PXXP) motif in the proximal ß1a C-terminus. Nevertheless, we are just at the beginning of understanding the importance of the distinct molecular domains of the ß1a subunit in skeletal muscle EC coupling. Hence, in this proposed study we aim to thoroughly characterize the third crucial pillar of the skeletal-muscle EC coupling cascade, i.e., identification of ß1a-domains responsible for correct DHPR tetrad formation and thus proper DHPR-RyR1 interaction---basis for induction of SR Ca2+ release and finally of muscle contractility/motility. For this in-depth structure-function analyses, loss--and gain- of-function chimeras with systematically swapped ß1a-ß4 (and/or ßM) domains will be expressed in relaxed myotubes and subjected to rigorous analyses for tetrad formation, accompanied by SR Ca2+-release recordings and sophisticated, advanced motility extent measurements. Selected ß1a-ß3 chimeras, which were so far investigated only for Q restoration, will also be a part of this study. Furthermore, we plan to extend our study to amino acid level to identify putative crucial interaction motives. While the bulk of results from biophysical studies from our and other labs supports models where ß1a facilitates EC coupling via allosteric interactions with the DHPR1S subunit, results from peptide binding studies brought up models postulating ß1a to bind and thus interact directly with RyR1 in promoting EC coupling. Consequently, our proposed study also includes attempts to test one of the apparently most interesting 1a- RyR1 binding hypotheses in a broad physiological experimental context. With our approach to connect in- vivo and in-vitro-results we expect to provide deeper insights into ß1a-1S sub-structures and interactions involved in the mechanism of DHPR tetrad formation. Consequently, this should lead to a comprehensive functional model of this third pillar of the skeletal muscle EC coupling mechanism.