The paralyzed zebrafish strain relaxed carries a null mutation for the skeletal muscle dihydropyridine receptor (DHPR) ␤ 1a subunit. Lack of ␤ 1a results in (i) reduced membrane expression of the pore forming DHPR ␣ 1S subunit, (ii) elimination of ␣ 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 muscletype excitation-contraction (EC) coupling. In this study we used relaxed larvae and isolated myotubes as expression systems to discriminate specific functions of ␤ 1a from rather general functions of ␤ isoforms. Zebrafish and mammalian ␤ 1a subunits quantitatively restored ␣ 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 ␤ 2a as the phylogenetically closest, and the ancestral housefly ␤ M as the most distant isoform to ␤ 1a also completely recovered ␣ 1S triad expression and charge movement. However, both revealed drastically impaired intracellular Ca 2؉ transients and very limited tetrad formation compared with ␤ 1a . Consequently, larval motility was either only partially restored (␤ 2a -injected larvae) or not restored at all (␤ M ). Thus, our results indicate that triad expression and facilitation of 1,4-dihydropyridine receptor (DHPR) charge movement are common features of all tested ␤ subunits, whereas the efficient arrangement of DHPRs in tetrads and thus intact DHPR-RyR1 coupling is only promoted by the ␤ 1a isoform. Consequently, we postulate a model that presents ␤ 1a as an allosteric modifier of ␣ 1S conformation enabling skeletal muscle-type EC coupling. Excitation-contraction (EC)3 coupling in skeletal muscle is critically dependent on the close interaction of two distinct Ca 2ϩ channels. Membrane depolarizations of the myotube are sensed by the voltage-dependent 1,4-dihydropyridine receptor (DHPR) in the sarcolemma, leading to a rearrangement of charged amino acids (charge movement) in the transmembrane segments S4 of the pore-forming DHPR ␣ 1S subunit (1, 2). This conformational change induces via protein-protein interaction (3, 4) the opening of the sarcoplasmic type-1 ryanodine receptor (RyR1) without need of Ca 2ϩ influx through the DHPR (5). The release of Ca 2ϩ from the sarcoplasmic reticulum via RyR1 consequently induces muscle contraction. The protein-protein interaction mechanism between DHPR and RyR1 requires correct ultrastructural targeting of both channels. In Ca 2ϩ release units (triads and peripheral couplings) of the skeletal muscle, groups of four DHPRs (tetrads) are coupled to every other RyR1 and hence are geometrically arranged following the RyR-specific orthogonal arrays (6).The skeletal muscle DHPR is a heteromultimeric protein complex, composed of the voltage-sensing and pore-forming ␣ 1S subunit and auxiliary subunits ␤ 1a , ␣ 2 ␦-1, and ␥ 1 (7). While gene knock-out of t...

During skeletal muscle excitation-contraction (EC) coupling, membrane depolarizations activate the sarcolemmal voltage-gated L-type Ca 2+ channel (Ca V 1.1). Ca V 1.1 in turn triggers opening of the sarcoplasmic Ca 2+ release channel (RyR1) via interchannel protein-protein interaction to release Ca 2+ for myofibril contraction. Simultaneously to this EC coupling process, a small and slowly activating Ca 2+ inward current through Ca V 1.1 is found in mammalian skeletal myotubes. The role of this Ca 2+ influx, which is not immediately required for EC coupling, is still enigmatic. Interestingly, whole-cell patch clamp experiments on freshly dissociated skeletal muscle myotubes from zebrafish larvae revealed the lack of such Ca 2+ currents. We identified two distinct isoforms of the pore-forming Ca V 1.1α 1S subunit in zebrafish that are differentially expressed in superficial slow and deep fast musculature. Both do not conduct Ca 2+ but merely act as voltage sensors to trigger opening of two likewise tissue-specific isoforms of RyR1. We further show that non-Ca 2+ conductivity of both Ca V 1.1α 1S isoforms is a common trait of all higher teleosts. This non-Ca 2+ conductivity of Ca V 1.1 positions teleosts at the most-derived position of an evolutionary trajectory. Though EC coupling in early chordate muscles is activated by the influx of extracellular Ca 2+ , it evolved toward Ca V 1.1-RyR1 protein-protein interaction with a relatively small and slow influx of external Ca 2+ in tetrapods. Finally, the Ca V 1.1 Ca 2+ influx was completely eliminated in higher teleost fishes.calcium conductivity | evolution | ion channels | slow and fast muscle | zebrafish

Skeletal muscle excitation–contraction (EC) coupling is initiated by sarcolemmal depolarization, which is translated into a conformational change of the dihydropyridine receptor (DHPR), which in turn activates sarcoplasmic reticulum (SR) Ca2+ release to trigger muscle contraction. During EC coupling, the mammalian DHPR embraces functional duality, as voltage sensor and l-type Ca2+ channel. Although its unique role as voltage sensor for conformational EC coupling is firmly established, the conventional function as Ca2+ channel is still enigmatic. Here we show that Ca2+ influx via DHPR is not necessary for muscle performance by generating a knock-in mouse where DHPR-mediated Ca2+ influx is eliminated. Homozygous knock-in mice display SR Ca2+ release, locomotor activity, motor coordination, muscle strength and susceptibility to fatigue comparable to wild-type controls, without any compensatory regulation of multiple key proteins of the EC coupling machinery and Ca2+ homeostasis. These findings support the hypothesis that the DHPR-mediated Ca2+ influx in mammalian skeletal muscle is an evolutionary remnant.

The dihydropyridine receptor (DHPR) β 1a subunit is crucial for enhancement of DHPR triad expression, assembly of DHPRs in tetrads, and elicitation of DHPRα 1S charge movement-the three prerequisites of skeletal muscle excitation-contraction coupling. Despite the ability to fully target α 1S into triadic junctions and tetradic arrays, the neuronal isoform β 3 was unable to restore considerable charge movement (measure of α 1S voltage sensing) upon expression in β 1 -null zebrafish relaxed myotubes, unlike the other three vertebrate β-isoforms (β 1a , β 2a , and β 4 ). Thus, we used β 3 for chimerization with β 1a to investigate whether any of the five distinct molecular regions of β 1a is dominantly involved in inducing the voltage-sensing function of α 1S . Surprisingly, systematic domain swapping between β 1a and β 3 revealed a pivotal role of the src homology 3 (SH3) domain and C terminus of β 1a in charge movement restoration. More interestingly, β 1a SH3 domain and C terminus, when simultaneously engineered into β 3 sequence background, were able to fully restore charge movement together with proper intracellular Ca 2+ release, suggesting cooperativity of these two domains in induction of the α 1S voltage-sensing function in skeletal muscle excitation-contraction coupling. Furthermore, substitution of a proline by alanine in the putative SH3-binding polyproline motif in the proximal C terminus of β 1a (also of β 2a and β 4 ) fully obstructed α 1S charge movement. Consequently, we postulate a model according to which β subunits, probably via the SH3-C-terminal polyproline interaction, adapt a discrete conformation required to modify the α 1S conformation apt for voltage sensing in skeletal muscle.