Proper restoration of excitation-contraction coupling in the dihydropyridine receptor beta1-null zebrafish relaxed is an exclusive function of the beta1a subunit

Abstract

The paralyzed zebrafish strain relaxed carries a null mutation for the skeletal muscle dihydropyridine receptor (DHPR) beta(1a) subunit. Lack of beta(1a) results in (i) reduced membrane expression of the pore forming DHPR alpha(1S) subunit, (ii) elimination of alpha(1S) charge movement, and (iii) impediment of arrangement of the DHPRs in groups of four (tetrads) opposing the ryanodine receptor (RyR1), a structural prerequisite for skeletal muscle-type excitation-contraction (EC) coupling. In this study we used relaxed larvae and isolated myotubes as expression systems to discriminate specific functions of beta(1a) from rather general functions of beta isoforms. Zebrafish and mammalian beta(1a) subunits quantitatively restored alpha(1S) triad targeting and charge movement as well as intracellular Ca(2+) release, allowed arrangement of DHPRs in tetrads, and most strikingly recovered a fully motile phenotype in relaxed larvae. Interestingly, the cardiac/neuronal beta(2a) as the phylogenetically closest, and the ancestral housefly beta(M) as the most distant isoform to beta(1a) also completely recovered alpha(1S) triad expression and charge movement. However, both revealed drastically impaired intracellular Ca(2+) transients and very limited tetrad formation compared with beta(1a). Consequently, larval motility was either only partially restored (beta(2a)-injected larvae) or not restored at all (beta(M)). Thus, our results indicate that triad expression and facilitation of 1,4-dihydropyridine receptor (DHPR) charge movement are common features of all tested beta subunits, whereas the efficient arrangement of DHPRs in tetrads and thus intact DHPR-RyR1 coupling is only promoted by the beta(1a) isoform. Consequently, we postulate a model that presents beta(1a) as an allosteric modifier of alpha(1S) conformation enabling skeletal muscle-type EC coupling.

FIGURE 1.

Qualitative and quantitative restoration of α_1S triad expression in relaxed myotubes with all tested β subunit isoforms. A, representative images of double immunofluorescence labeling of the DHPR α_1S subunit (anti-α_1S) and GFP-tagged β subunits (anti-GFP). Normal (1st row) and relaxed (2nd row) myotubes mock-transfected with pure GFP revealed diffuse GFP staining throughout the cell (center images). Relaxed myotubes transfected with zf-β_1a, rb-β_1a, β_2a, or β_M showed correct targeting of the β subunits into triadic clusters (center images) and co-localization (merge) with clusters of the endogenous α_1S subunit (left). B, quantification of α_1S triad expression by measuring average fluorescence intensity along a line across a row of α_1S triadic clusters (exemplified 1st row, left; indicated by a red arrowhead) showed that both, skeletal and non-skeletal muscle β subunit isoforms were able to completely restore α_1S triad expression in relaxed myotubes (^**, p < 0.01; ^***, p < 0.001).

FIGURE 2.

Complete restoration of intramembrane α_1S charge movement in relaxed myotubes with all tested β subunit isoforms. A, representative recordings of intramembrane α_1S charge movement at test potentials of -50, 0, and +50 mV from a holding potential of -80 mV of normal myotubes mock-transfected with GFP, untransfected relaxed myotubes, and relaxed myotubes transfected with zf-β_1a, rb-β_1a, β_2a, or β_M. B, voltage dependence of the integrated ON-component of intramembrane α_1S charge movement (Q_on) were comparable for zf-β_1a-, rb-β_1a-transfected relaxed myotubes and normal myotubes mock-transfected with GFP. Maximal charge movement (Q_max) values were somewhat higher (p < 0.05) for zf-β_1a (♦) and rb-β_1a (⋄) compared with normal myotubes (○). No charge movement at any potential could be recorded from untransfected relaxed myotubes (•). C, heterologous β isoforms, β_2a (▴) and β_M (▿) were likewise able to fully recover intramembrane α_1S charge movement comparable with the two homologous β_1a isoforms.

FIGURE 3.

Differential rescue of voltage-dependent intracellular Ca²⁺ release in relaxed myotubes with skeletal muscle and non-skeletal muscle β subunit isoforms. A, representative recordings of intracellular Ca²⁺ release in response to 200-ms depolarizing step pulses to -50, 0, and +60 mV. Intracellular Ca²⁺ transients recorded from relaxed myotubes transfected with the skeletal muscle isoforms zf-β_1a or rb-β_1a were identical to those recorded from normal myotubes mock-transfected with GFP and during the 200 ms of depolarization displayed a rapid upstroke that was followed by a constant plateau of intracellular Ca²⁺ release that finally declined due to Ca²⁺ re-uptake into the sarcoplasmic reticulum. In contrast, Ca²⁺ transients recorded from relaxed myotubes transfected with the heterologous β_2a or β_M subunits were not able to sustain a plateau but showed a decline in intracellular Ca²⁺ immediately after initiation of the pulse. B, size and voltage-dependence of intracellular Ca²⁺ transients were indistinguishable (p > 0.05) between normal myotubes mock-transfected with GFP (○) and relaxed myotubes transfected with zf-β_1a (♦, note, both graphs are superimposed) or rb-β_1a (⋄). Dashed lines indicate half-maximal activation potentials of all three groups. C, voltage dependence of intracellular Ca²⁺ transients obtained from myotubes transfected with β_2a (▴) or β_M (▿) were significantly (p < 0.001) shifted toward more positive potentials compared with GFP-transfected normal myotubes (dashed lines, half-maximal activation). Furthermore, β_2a or β_M were unable to restore maximum ΔF/F₀ values like normal myotubes.

FIGURE 4.

Impaired tetrad formation with non-skeletal muscleβ isoforms in relaxed myotubes. Freeze-fracture electron microscopy on tail muscle tissue of 30-32 hpf relaxed larvae, zygote-injected with in vitro synthesized RNA coding for different β isoforms, revealed assembly of DHPRs in triadic clusters, indicated by yellow ellipses. In control experiments on normal larvae (upper row, left) DHPR particles were predominantly found in tetrad-like groups of 3 or 4 (indicated by red dots), indistinguishable (p > 0.05) from relaxed larvae injected with zf-β_1a (upper row, right). No particles could be found between the tetrads. In contrast, arrangement of DHPRs in β_2a-(center row) or β_M-(bottom row) injected larvae was less organized. Arrangement of DHPR particles in tetrads was lacking in many of the DHPR clusters (β_2a and β_M, left images) or was very limited (right images).

FIGURE 5.

Full restoration of larval motility in relaxed larvae injected with β_1a RNA but only very weak or no motility with β_2a and β_M, respectively. To analyze larval motility, 2-min video sequences of normal larvae and relaxed larvae zygote-injected with β subunit RNAs were recorded and converted into sequences of differential images. A, representative sequences of differential images (only every 3rd frame is shown) of a single spontaneous larval movement, and B, the resulting plot of the total number of dynamic pixels per frame as described under “Experimental Procedures.” The first peak represents muscle contraction, the trough is tension maintenance, and the second peak is relaxation. C, representative recordings of relaxed larvae zygote-injected with zf-β_1a (left) or β_2a (right). Larvae injected with β_2a displayed fewer and weaker movements, than larvae injected with zf-β_1a. D, to quantify larval movement extent, the mean value of cumulative dynamic pixels per movement for each experimental group was calculated and standardized to normal larvae. Full recovery of larval movement extent was obtained in relaxed larvae when zygote-injected with either zf-β_1a or rb-β_1a. However, movement extent of β_2a-injected relaxed larvae was rescued to only 26 ± 2% of normal larvae, whereas β_M was unable to recover any motility in relaxed larvae (^***, p < 0.001).

FIGURE 6.

Model of β-induced DHPR α_1S-RyR1 interactions, incorporating results of the present and previous studies. A, hypothetical situation of the lack of any DHPR α_1S-RyR1 interaction due to the lack of the β_1a subunit in the relaxed zebrafish triad. Without β the conformation of the α_1S subunit is severely distorted, which hampers α_1S charge movement (Q) and also inactivates presumptive α_1S-RyR1 interaction sites (indicated by arrows). Bold arrow represents the primary interaction site in the α_1S II-III loop (53, 57), and small arrows represent an unspecific number of additional α_1S-specific interaction sites as previously postulated (53). The deficiency of direct α_1S-RyR1 interaction correlates with the lack of tetrad formation (19) (Tetrads). Consequently, by the lack of charge movement and tetrad formation skeletal-type EC coupling (sk-ECC) is completely hampered. In B, the situation in normal muscle or muscle from the mutant relaxed reconstituted with β_1a is depicted. The β_1a subunit leads to full and correct restoration of α_1S conformation, allowing charge movement and appropriate targeting of the α_1S into tetrads opposite the RyR1. If the β subunit adds interaction sites with RyR1 (red arrow) (62), or not, is irrelevant for our model according to which β_1a acts as an allosteric modifier of α_1S conformation and thus function. C, if heterologousβ subunits like the cardiac/neuronalβ_2a or the ancestral, neuronal Musca β_M are expressed in relaxed muscle, a partial restoration of α_1S conformation takes place. Charge movement (Q) is now fully possible, but tetrad formation and thus proper α_1S-RyR1 protein-protein interaction is still impaired. This “fuzzy targeting” of α_1S opposite the RyR1 leads to unstable Ca²⁺ release and thus to very weak (in the case of β_2a) or even no (for β_M) muscle motility.