Skeletal muscle excitation–contraction coupling is independent of a conserved heptad repeat motif in the C-terminus of the DHPRβ1a subunit

Abstract

In skeletal muscle excitation–contraction (EC) coupling the sarcolemmal L-type Ca2+ channel or 1,4-dihydropyridine receptor (DHPR) transduces the membrane depolarization signal to the sarcoplasmic Ca2+ release channel RyR1 via protein–protein interaction. While it is evident that the pore-forming and voltage-sensing DHPRα1S subunit is essential for this process, the intracellular DHPRβ1a subunit was also shown to be indispensable. We previously found that the β1a subunit is essential to target the DHPR into groups of four (tetrads) opposite the RyR1 homotetramers, a prerequisite for skeletal muscle EC coupling. Earlier, a unique hydrophobic heptad repeat motif (L⋯V⋯V) in the C-terminus of β1a was postulated by others to be essential for skeletal muscle EC coupling, as substitution of these residues with alanines resulted in 80% reduction of RyR1 Ca2+ release. Therefore, we wanted to address the question if the proposed β1a heptad repeat motif could be an active element of the DHPR–RyR1 signal transduction mechanism or already contributes at the ultrastructural level i.e. DHPR tetrad arrangement. Surprisingly, our experiments revealed full tetrad formation and an almost complete restoration of EC coupling in β1-null zebrafish relaxed larvae and isolated myotubes upon expression of a β1a-specific heptad repeat mutant (LVV to AAA) and thus contradict the earlier results.

An α‐helical C‐terminal tail segment of the skeletal L‐type Ca 2+ channel β 1a subunit activates ryanodine receptor type 1 via a hydrophobic surface

“…The heptad repeat has been shown to be important for skeletal type EC coupling in mouse myotubes, where alanine substitution of the 3 hydrophobic heptad repeat residues abolished voltage‐dependent Ca 2+ release (8). However, the heptad repeat does not alter voltage‐dependent Ca 2+ release in a zebrafish model (9). Although the different results could be explained by species differences, the question of the role of the heptad repeat in skeletal EC coupling and interactions between the β 1a C‐terminal tail and RyR1 remains.…”

“…Mutation of the hydrophobic heptad repeat residues (L478A/V485A/V492A) reduces depolarization‐induced Ca 2+ release in mouse myotubes, implicating these residues in EC coupling (8). However, mutation of the hydrophobic heptad repeat residues in the β 1a ‐null zebrafish model does not depress EC coupling (9). Thus, the role of the heptad repeat remains unclear and is also addressed in this study.…”

CaV1.1: The atypical prototypical voltage-gated Ca2+ channel

“…Within this stretch of residues, a hydrophobic heptad motif unique to β 1a was identified as being an important element for EC coupling and triple mutation of three of these residues (L478, V485 and V492) to alanine impaired the ability of rabbit β 1a to support EC coupling in mouse β 1 null myotubes [80]. However, the same mutations were inconsequential to EC coupling and tetrad formation when expressed in zebrafish relaxed (β 1 null) myotubes [81] and β 1a -based peptides with these substitutions enhanced RyR1 P o to the same extent as peptides with wild-type sequence [43]. Though the requirement for the presence of the β 1a carboxyl-terminus for the EC coupling is widely accepted, these diametrically opposed results obtained in divergent (mammalian vs. osteoicthyes and lipid bilayers) systems have given rise to uncertainty of the role of the hydrophobic heptad repeat in coupling with RyR1.…”

Domain cooperativity in the β1asubunit is essential for dihydropyridine receptor voltage sensing in skeletal muscle

“…In vitro transcription and zygote injection of RNA were carried out as described previously (19,28). Tails of 27-30 hpf injected homozygous relaxed zebrafish were fractured and replicas analyzed in an electron microscope as illustrated earlier (28).…”