Absence of DHPR tetrad restoration in β₄-expressing relaxed myotubes. (A) Freeze-fracture replicas of peripheral couplings in tail myotomes of 27- to 30-hpf zebrafish. Control myotomes (Top) show arrangement of DHPR particles in tetrads (center indicated by red dots), organized in orthogonal arrays. In β₄-expressing relaxed zebrafish (Bottom) DHPR tetrads show a lack of tetrad formation. (Scale bar, 50 nm.) (B) Quantification of voltage dependence of cytoplasmic Ca²⁺ transients yielded (ΔF/F₀)_max values that are significantly lower (P < 0.001) in β₄ (n = 13)- compared to β_1a (n = 9)-expressing relaxed myotubes. ΔF/F₀ values recorded from untransfected relaxed myotubes were below detection level (n = 10). (C) Similarly, plots of voltage dependence of the integral of the ΔF/F₀ transients in response to 200-ms test depolarizations indicate a highly significant difference (P < 0.001) in the total amount of Ca²⁺ released between relaxed myotubes expressing β_1a (n = 9) or β₄ (n = 12) subunit. (Right) Representative ΔF/F₀ recordings from relaxed myotubes expressing β_1a or β₄. (Scale bars, 50 ms [horizontal], ΔF/F₀ = 1 [vertical].) Error bars indicate SEM. P determined by unpaired Student’s t test.

Loss-of-function β_1a/β₄ chimeras revealed the importance of the β_1a C terminus in skeletal muscle DHPR–RyR1 coupling. (A) Block schemes of domain organization of putative loss-of-function β_1a/β₄ chimeras with systematic exchange of N terminus (N), SH3 domain (SH3), HOOK region (H), GK domain (GK), or C terminus (C) of β_1a (blue) by β₄ sequences (orange). Homologous SH3 and GK domains are represented by hatched boxes. (B, Left) Analyses of voltage dependence of integrated outward gating currents normalized to cell capacitance exhibited maximum charge movement (Q_max) values indistinguishable (P > 0.05) between relaxed myotubes expressing β_1a (n = 16), β₄ (n = 12), β_1a/β₄(N) (n = 21), β_1a/β₄(SH3) (n = 19), β_1a/β₄(H) (n = 24), β_1a/β₄(GK) (n = 15), or β_1a/β₄(C) (n = 13). Q_max values from untransfected relaxed myotubes were slightly above detection level (P < 0.001, n = 11). (Right) Representative Q recordings from relaxed myotubes expressing either β_1a or β₄. (Scale bars, 5 ms [horizontal], 3 pA/pF [vertical].) (C and D) Cytoplasmic Ca²⁺ transient restoration was comparable (P > 0.05) between relaxed myotubes expressing β_1a (n = 9), β_1a/β₄(SH3) (n = 17), β_1a/β₄(GK) (n = 11), β_1a/β₄(N) (n = 14), or β_1a/β₄(H) (n = 16). By contrast, ΔF/F₀ values were significantly lower (P < 0.001) for chimera β_1a/β₄(C) (n = 12) and similar (P > 0.05) to those of β₄ (n = 13). Exemplar Ca²⁺ transient recordings from relaxed myotubes expressing β_1a/β₄(SH3) (C, Right) or β_1a/β₄(C) (D, Right). (Scale bars, 50 ms [horizontal], ΔF/F₀ = 1 [vertical].) Error bars indicate SEM. P determined by unpaired Student’s t test, ***P < 0.001.

Length of the β₄ C terminus is not crucial for skeletal muscle DHPR–RyR1coupling. (A) Amino acid sequence alignment depicting variable lengths of the C termini of β_1a and β₄ subunits (GenBank accession nos.: rabbit β_1a, M25514; rat β₄, L02315). To determine whether the length of the C terminus was functionally critical, the last 51 amino acids from the β₄ C terminus were deleted, yielding mutant β₄(Δ51). (B) Q_max values were indistinguishable (P > 0.05) between relaxed myotubes expressing the deletion mutant β₄(Δ51) (n = 17), β_1a (n = 16), or β₄ (n = 12). (C) Maximal Ca²⁺ transients (ΔF/F₀)_max for β₄(Δ51) expressing relaxed myotubes (n = 10) was similar (P > 0.05) to that of β₄ (n = 13). (D) Similarly, total maximal Ca²⁺ transients (intg.(ΔF/F₀)_max) for β₄(Δ51) expressing relaxed myotubes was statistically indistinguishable (P > 0.05) from that of β₄. Error bars indicate SEM. P determined by unpaired Student’s t test.

The distal C terminus of β_1a is crucial for skeletal muscle EC coupling. (A) Block scheme of domain organization of gain-of-function chimera β₄/β_1a(C), where the the C terminus of β₄ (orange) was replaced by a corresponding β_1a sequence (blue). (B) Q_max values in relaxed myotubes expressing either chimera β₄/β_1a(C) (n = 18) or β_1a (n = 16) were comparable (P > 0.05). (C) Quantification of voltage dependence of cytoplasmic Ca²⁺ transients yielded significantly higher (P < 0.001) (ΔF/F₀)_max values for chimera β₄/β_1a(C) (n = 12) compared to β₄ (n = 13) but indistinguishable (P > 0.05) from that of β_1a (n = 9) expressing relaxed myotubes. (Right) Exemplar cytoplasmic Ca²⁺ transient recordings from relaxed myotubes expressing chimera β₄/β_1a(C). (Scale bars, 50 ms [horizontal], ΔF/F₀ = 1 [vertical].) (D) Amino acid sequence alignment of C termini of β_1a and β₄ depicting the homologous proximal C terminus (green box) and heterologous distal C terminus (blue box). Red bracket indicates the highly homologous sequence in the distal C terminus of β_1a revealed from sequence alignments of β_1a from several vertebrate species (fish to mammals) (SI Appendix, Fig. S2B). (E) Block scheme of domain organization of chimeras β₄/β_1a(prox.C) and β₄/β_1a(dist.C), where the proximal and distal C terminus of β₄ (orange) were exchanged by corresponding β_1a sequences (blue). (F) Q_max values were indistinguishable (P > 0.05) between relaxed myotubes expressing chimera β₄/β_1a(prox.C) (n = 11), β₄/β_1a(dist.C) (n = 19), or β_1a (n = 16). (G) Quantification of voltage dependence of cytoplasmic Ca²⁺ transients yielded (ΔF/F₀)_max values that were significantly lower (P < 0.001) for chimera β₄/β_1a(prox.C) (n = 15)- compared to β_1a (n = 9)-expressing relaxed myotubes. However, relaxed myotubes expressing chimera β₄/β_1a(dist.C) (n = 14) exhibited pronounced Ca²⁺ transients, equivalent (P > 0.05) to β_1a transfected myotubes (n = 13). (Right) Exemplar Ca²⁺ transient recordings from relaxed myotubes expressing chimera β₄/β_1a(dist.C) or β₄/β_1a(prox.C). (Scale bars, 50 ms [horizontal], ΔF/F₀ = 1 [vertical].) (H) Quantification of spontaneous or touch-evoked coiling of 27- to 30-hpf relaxed zebrafish injected with β_1a (n = 35), β₄ (n = 202), β₄/β_1a(C) (n = 79), and β₄/β_1a(dist.C) (n = 58) mRNA. Degree of motility was indistinguishable (P > 0.05) between relaxed zebrafish expressing β₄/β_1a(C) or β_1a. Relaxed zebrafish expressing β₄/β_1a(dist.C) displayed robust spontaneous coiling only slightly lower (P = 0.02) than β_1a. Conversely, β₄-injected relaxed zebrafish showed either no (n = 151) or very weak (n = 51) coiling following tactile stimulation and thus, highly significantly lower motility compared to (P < 0.001) β_1a-expressing relaxed zebrafish. Uninjected relaxed zebrafish displayed neither spontaneous nor tactile-induced motility (P < 0.001, n = 28). Error bars indicate SEM. P determined by unpaired Student’s t test, *P < 0.05; ***P < 0.001.

The distal C terminus of β_1a is crucial for DHPR tetrad formation. (A and B) Representative freeze-fracture replicas from tail muscle tissue of 27- to 30-hpf relaxed zebrafish expressing β₄/β_1a(C) (A) or β₄/β_1a(dist.C) (B) reveal accurate arrangement of DHPR particles in tetrads. The red dots (Bottom) indicate the centers of three- or four-particle tetrads and additional particles that are in the expected position for an orthogonal array. (Scale bar, 50 nm.) (C) Numbers of tetrads (three or four particles) determined by two independent investigators from 95 anonymized freeze-fracture images acquired from zebrafish tails, either normal controls (normal), uninjected (relaxed), or injected with β₄, β₄/β_1a(C), or β₄/β_1a(dist.C) mRNA. Each bar represents mean of the counts normalized to normal zebrafish (where the mean of the two investigators’ counts was defined as 100%) and the two arrows (red and green) depict the counts of the two individual investigators (SI Appendix, Table S2). (D) Counts of DHPR particles per junction from zebrafish tails, either uninjected (relaxed), injected with β₄, β₄/β_1a(C), or β₄/β_1a(dist.C) mRNA, or normal controls (normal). Error bars indicate SEM. P determined by unpaired Student’s t test, *P < 0.05; **P < 0.01.

Hydrophobic residues (L₄₉₆L₅₀₀W₅₀₃) in the β_1a distal C terminus are not important for skeletal muscle EC coupling. (A) Amino acid sequence of rabbit β_1a C terminus depicting the position of the three hydrophobic residues LLW (red box with yellow filling), which were exchanged with alanines (AAA). (B) Relaxed myotubes expressing triple mutant β_1a(LLW-AAA) (n = 16) displayed Q_max values similar (P > 0.05) to β_1a (n = 16). (Right) Exemplar charge movement recording from relaxed myotubes expressing β_1a(LLW-AAA). (Scale bars, 5 ms [horizontal], 3 pA/pF [vertical].) (C) Plots of voltage dependence of maximal Ca²⁺ transients were indistinguishable (P > 0.05) between β_1a(LLW-AAA) (n = 13) and β_1a (n = 9)-expressing relaxed myotubes. (Right) Exemplar Ca²⁺ transient recordings from relaxed myotubes expressing mutant β_1a(LLW-AAA). (Scale bars, 50 ms [horizontal], ΔF/F₀ = 1 [vertical].) Error bars indicate SEM. P determined by unpaired Student’s t test.

Model of conformational modification of α_1S by the β_1a distal C terminus—prerequisite for proper skeletal muscle EC coupling. (A) In zebrafish mutant relaxed due to the absence of the DHPRβ_1a subunit, the α_1S subunit is in a distorted conformation. This causes impediment of charge movement (Q) and of arrangement of DHPR into tetrads (tetrads) that accounts for the lack of skeletal muscle EC coupling (ECC). The distorted conformation of the membrane spanning hydrophobic core regions of the four homologous α_1S repeats (I–IV) is depicted by rectangular boxes. The primary and unspecified numbers of secondary α_1S-specific RyR1 interaction sites (32) are indicated with bold and normal black arrows, respectively. (B) β₄ is unable to reinstate full EC coupling [(+)/−] due to impaired DHPR tetrad formation. According to our model, β₄ (symbolized in orange) induces proper conformation of the hydrophobic α_1S core regions (depicted with cylinders) required for charge movement function, but is unable to reconstitute accurate conformation of the intracellular α_1S loops facilitating RyR1 anchoring (tetrad formation). Improper DHPR–RyR1 interaction (tilted arrows) leads to weak EC coupling and impaired tetrad formation. (C) Likewise, chimera β₄/β_1a(prox.C) in which the proximal C terminus of β₄ is swapped with corresponding β_1a sequence (blue), was unable to reinstate intact tetrad formation and thus full ECC. Yellow dots on the proximal C terminus of the β-subunit depict the intramolecular SH3–PXXP interaction sites critical for charge movement function (16). (D) However, the distal C terminus of β_1a (blue) enables proper conformation of the intracellular α_1S loops crucial for RyR1 anchoring (tetrad formation). Consequently, EC coupling is highly restored upon expression of chimera β₄/β_1a(dist.C). The direct DHPR–RyR1 interaction depicted in the model is still obscure. However, it is irrelevant for our conclusions whether the two channels interact directly or via an intermediate protein.