Skeletal muscle excitation-contraction coupling is independent of a conserved heptad repeat motif in the C-terminus of the DHPRbeta(1a) subunit

Abstract

In skeletal muscle excitation-contraction (EC) coupling the sarcolemmal L-type Ca(2+) channel or 1,4-dihydropyridine receptor (DHPR) transduces the membrane depolarization signal to the sarcoplasmic Ca(2+) release channel RyR1 via protein-protein interaction. While it is evident that the pore-forming and voltage-sensing DHPRalpha(1S) subunit is essential for this process, the intracellular DHPRbeta(1a) subunit was also shown to be indispensable. We previously found that the beta(1a) 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 (Lcdots, three dots, centeredVcdots, three dots, centeredV) in the C-terminus of beta(1a) 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 Ca(2+) release. Therefore, we wanted to address the question if the proposed beta(1a) 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 beta(1)-null zebrafish relaxed larvae and isolated myotubes upon expression of a beta(1a)-specific heptad repeat mutant (LVV to AAA) and thus contradict the earlier results.

Fig. 1

A conserved leucine-valine heptad repeat motif in the DHPRβ_1a C-terminus. (A) Cartoon of the domain organization of the β_1a subunit based on crystal structure models [28–30]. (B) Sequence alignment of β_1a C-termini from different vertebrate classes, from fish to human, showed the conservation of the β_1a-specific leucine-valine heptad repeat motif (boxed). To test for the contribution of the heptad repeat motif in skeletal muscle EC coupling, the LVV residues (boxed) were substituted by AAA (β_1aAAA) in zebrafish and rabbit β_1a subunits to be expressed in the zebrafish β₁-null mutant relaxed system. Sequence for human (Homo sapiens) β_1a, anole lizard (Anolis carolinensis) β_1a, and Xenopus (Xenopus tropicalis) β_1a were extracted from genomic assemblies at http://www.ensembl.org using a BLAST search with zf-β_1a or rb-β_1a cDNA sequences. GeneBank accession numbers for all other sequences used are: mouse (Mus musculus) β_1a, NM_031173; rabbit (Oryctolagus cuniculus) β_1a, NM_001082279 and zebrafish (Danio rerio) β_1a, AY952462.

Fig. 2

Intact DHPR tetrad formation with β_1aAAA mutant expressed in relaxed myotubes. Freeze-fracture replicas of tail muscle tissue of normal zebrafish larvae (left panel) revealed the arrangement of DHPR particles in tetrads (indicated by red dots) organized in orthogonal arrays. In contrast, the β₁-null mutant relaxed (middle panel) lacks DHPR tetrad formation. Comparable to normal larvae, relaxed larvae zygote-injected with in vitro synthesized rb-β_1aAAA RNA, displayed correct assembly of DHPR particles in tetradic arrays (right panel). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

Fig. 3

Full restoration of motility in relaxed larvae zygote-injected with β_1aAAA mutant RNA. (A) Representative plots of total dynamic pixels per frame of 2-min video recordings of spontaneous larval movements. Relaxed larvae zygote-injected with either rb-β_1a (left) or rb-β_1aAAA RNA (right) revealed a similar movement profile. (B) To quantify larval movement extent, mean values of cumulative dynamic pixels per movement for each experimental group were calculated and standardized to those of normal larvae. Relaxed larvae zygote-injected with either zf-β_1aAAA or rb-β_1aAAA mutant RNA showed full recovery of larval movement extent, indistinguishable (p > 0.05) from normal larvae or from relaxed larvae injected with either zf-β_1a or rb-β_1a. One-way ANOVA revealed overall non-significance (p = 0.62, F_(4,274) = 0.66). Uninjected relaxed larvae never showed any motility (nd, not detectable).

Fig. 4

The β_1aAAA mutation has only a minor effect on intracellular Ca²⁺ transients. (A) Representative intracellular Fluo-4 Ca²⁺ recordings from relaxed myotubes expressing zebrafish and rabbit WT β_1a (upper panel) or β_1aAAA mutant subunits (lower panel). Pronounced intracellular Ca²⁺ transients in response to 200-ms test pulses with similar kinetics were recorded with all constructs. Differences in transient size with β_1aAAA can be explained by slight differences in expression levels as indicated by differences in intramembrane charge movement recordings (insets). (B) Voltage dependence curves of ΔF/F₀ were corrected for differences in expression levels by normalizing ΔF/F₀ to Q_max. Transients were slightly shifted towards more positive potentials for the β_1aAAA constructs.