Interactions between the skeletal dihydropyridine receptor {u03B2} subunit, the {u03B1}{u2081}s II-III loop and the ryanodine receptor

Abstract

Excitation-contraction (EC) coupling in skeletal muscle requires a physical coupling between the dihydropyridine receptor (DHPR) Ca2+ channel in the surface membrane and the ryanodine receptor (RyRl) Ca2+ release channel in the sarcoplasmic reticulum (SR) Ca2+ store. However, the exact molecular mechanism of this interaction remains unresolved. Both the {u03B1}{u2081}s and {u03B2}{u2081}a subunits of the DHPR are essential for the skeletal BC coupling process and a central critical region of the {u03B1}{u2081}s II-III loop has been shown to be important for this interaction. The {u03B2} subunit plays an essential role in the targeting of the pore-forming {u03B1}{u2081} subunit to the t-tubular membrane and in the modulation of the DHPR Ca2+ channel. In addition the skeletal isoform, {u03B2}{u2081}a, supports tetrad formation of the DHPRs opposite the ryanodine receptors. {u03B2}{u2081}a has a modular structure consisting of N, C termini and SH3/guanylate kinase (OK) domains separated by a Hook region. A high affinity interaction between the {u03B1}{u2081}s I-II loop and the {u03B2}{u2081}a-OK domain is responsible for the targeting function of the {u03B2} subunit. However the functional significance of the SH3 domain remains unclear. SH3 domains are protein interaction domains that typically bind to proline rich motifs in their interacting partners. The critical region of the {u03B1}{u2081}s II-III loop contains at least two such proline rich motifs. Therefore this study investigated the possibility of an interaction between the {u03B2}{u2081}a-SH3 domain and the {u03B1}{u2081}s II-III loop. The {u03B2}{u2081}a subunit and its SH3 domain bound to the critical region of the {u03B1}{u2081}s II-III loop with an affinity of ~2 {u03BC}M. One of these interactions was narrowed down to the first proline-rich motif of the critical region which encompasses four skeletal specific residues (A739, F741, P742 and D744) that have been previously shown to be important for skeletal type ECcoupling in-vivo. Mutation of these residues to their cardiac counterparts showed residues P742 and D744 to be important for the binding of the {u03B2}{u2081}a-SH3 domain to the critical region of the {u03B1}{u2081}s II-III loop. The C-terminus of the {u03B2}{u2081}a subunit binds to RyRl in vitro and the end 35 residues of the {u03B2}{u2081}a C-terminus is important for skeletal type BC coupling. This study investigated the structure of a peptide corresponding to this region by NMR and identified a nascent helical region extending from residues L<super>493 to G<super>506 . Three hydrophobic residues (L<super>496, L<super>500 and W<super>503 ) within this helical region form a hydrophobic surface which could be a putative binding surface for the skeletal ryanodine receptor. Mutation of these residues to alanines partially disrupts the helical surface and decreases the ability of the mutant peptides to activate the ryanodine receptor. In conclusion this study shows that the SH3 domain of the skeletal {u03B2} subunit is able to bind to the critical region of the {u03B1}{u2081}s II-III loop in-vitro. This study also identifies a quasi-structured helical region in the C terminal tail of the {u03B2}{u2081}a subunit that affects its interaction with the skeletal ryanodine receptor. Based on these findings, a model is proposed where the {u03B2}{u2081}a subunit acts as a conduit in the transformation of the BC coupling signal from the skeletal DHPR to RyRl. Show more