Abstract
The skeletal muscle dihydropyridine receptor (DHPR) β1a subunit is indispensable for full trafficking of DHPRs into triadic junctions (i.e., the close apposition of transverse tubules and sarcoplasmic reticulum [SR]), facilitation of DHPRα1S voltage sensing, and arrangement of DHPRs into tetrads as a consequence of their interaction with ryanodine receptor (RyR1) homotetramers. These three features are obligatory for skeletal muscle excitation–contraction (EC) coupling. Previously, we showed that all four vertebrate β isoforms (β1–β4) facilitate α1S triad targeting and, except for β3, fully enable DHPRα1S voltage sensing [Dayal et al., Proc. Natl. Acad. Sci. U.S.A. 110, 7488–7493 (2013)]. Consequently, β3 failed to restore EC coupling despite the fact that both β3 and β1a restore tetrads. Thus, all β-subunits are able to restore triad targeting, but only β1a restores both tetrads and proper DHPR–RyR1 coupling [Dayal et al., Proc. Natl. Acad. Sci. U.S.A. 110, 7488–7493 (2013)]. To investigate the molecular region(s) of β1a responsible for the tetradic arrangement of DHPRs and thus DHPR–RyR1 coupling, we expressed loss- and gain-of-function chimeras between β1a and β4, with systematically swapped domains in zebrafish strain relaxed (β1-null) for patch clamp, cytoplasmic Ca2+ transients, motility, and freeze-fracture electron microscopy. β1a/β4 chimeras with either N terminus, SH3, HOOK, or GK domain derived from β4 showed complete restoration of SR Ca2+ release. However, chimera β1a/β4(C) with β4 C terminus produced significantly reduced cytoplasmic Ca2+ transients. Conversely, gain-of-function chimera β4/β1a(C) with β1a C terminus completely restored cytoplasmic Ca2+ transients, DHPR tetrads, and motility. Furthermore, we found that the nonconserved, distal C terminus of β1a plays a pivotal role in reconstitution of DHPR tetrads and thus allosteric DHPR–RyR1 interaction, essential for skeletal muscle EC coupling.