The β _1a subunit is essential for the assembly of dihydropyridine-receptor arrays in skeletal muscle

Abstract

Homozygous zebrafish of the mutant relaxed (red ts25 ) are paralyzed and die within days after hatching. A significant reduction of intramembrane charge movements and the lack of depolarizationinduced but not caffeine-induced Ca 2؉ transients suggested a defect in the skeletal muscle dihydropyridine receptor (DHPR). Sequencing of DHPR cDNAs indicated that the ␣1S subunit is normal, whereas the ␤1a subunit harbors a single point mutation resulting in a premature stop. Quantitative RT-PCR revealed that the mutated gene is transcribed, but Western blot analysis and immunocytochemistry demonstrated the complete loss of the ␤1a protein in mutant muscle. Thus, the immotile zebrafish relaxed is a ␤1a-null mutant. Interestingly, immunocytochemistry showed correct triad targeting of the ␣1S subunit in the absence of ␤1a. Freeze-fracture analysis of the DHPR clusters in relaxed myotubes revealed an Ϸ2-fold reduction in cluster size with a normal density of DHPR particles within the clusters. Most importantly, DHPR particles in the junctional membranes of the immotile zebrafish mutant relaxed entirely lacked the normal arrangement in arrays of tetrads. Thus, our data indicate that the lack of the ␤1a subunit does not prevent triad targeting of the DHPR ␣1S subunit but precludes the skeletal muscle-specific arrangement of DHPR particles opposite the ryanodine receptor (RyR1). This defect properly explains the complete deficiency of skeletal muscle excitationcontraction coupling in ␤1-null model organisms.calcium channels ͉ excitation-contraction coupling ͉ tetrads ͉ zebrafish E xcitation-contraction (EC) coupling is understood as the signal transduction process connecting membrane depolarization to the contraction of muscle cells. This process is initiated by the concerted action of two Ca 2ϩ channels, the plasmalemmal voltage-gated dihydropyridine receptor (DHPR) and the sarcoplasmic reticulum (SR) ryanodine receptor (RyR). In junctions of the SR with the plasma membrane (peripheral couplings) or with the transverse tubules (triads), membrane depolarization is sensed by the DHPR, which then triggers RyR opening and Ca 2ϩ release from the SR. In skeletal muscle cells, this signaltransduction is independent of Ca 2ϩ influx through the DHPR (1) but depends on protein-protein interaction between the DHPR and the RyR1 (2, 3). This physical coupling requires the coordinated arrangement of DHPRs and RyR1s in the junctions. In skeletal muscle triads and peripheral couplings, groups of four DHPRs (tetrads) are arranged in orthogonal arrays matching the opposing RyR1 arrays (4). Formation of DHPR tetrads requires the presence of RyR1.The skeletal muscle DHPR complex is composed of the voltage-sensing and pore-forming ␣ 1S subunit and the auxiliary subunits ␤ 1a , ␣ 2 ␦-1, and ␥ (5). Targeted deletions of the ␣ 2 ␦-1 and ␥ subunits do not critically interfere with EC coupling function (6, 7). In contrast, ␣ 1S and ␤ 1a subunit null-mutant mice display a lack of EC coupling and, thus, lethal muscle paralysis (8, 9). Although failure o...

Fluorescence Resonance Energy Transfer (FRET) Indicates That Association with the Type I Ryanodine Receptor (RyR1) Causes Reorientation of Multiple Cytoplasmic Domains of the Dihydropyridine Receptor (DHPR) α1S Subunit

“…As for other instances of in vitro binding of DHPR domains to RyR1, however, it is uncertain that this binding occurs in vivo. In particular, even though some ␣ 1S is pres- ent in the membrane of ␤ 1 null zebrafish muscle cells, it is not arrayed as tetrads (19), indicating that ␣ 1S does not bind to RyR1 in the absence of the ␤ subunit. Conversely, in ␣ 1S null myotubes, fluorescently tagged ␤ 1a does not bind to RyR1 (18) and is freely diffusible (39).…”

“…The ␤ 1a subunit is a soluble protein which binds to ␣ 1S and promotes its membrane trafficking (18). Moreover, ␤ 1a is coequal in importance with the ␣ 1S II-III loop for tetrad formation (19) and skeletal EC coupling (20,21). Among the possible explanations for these results is that ␤ 1a and/or the II-III loop bind to RyR1, perhaps in close proximity to one another.…”

Reciprocal Interactions Regulate Targeting of Calcium Channel β Subunits and Membrane Expression of α1 Subunits in Cultured Hippocampal Neurons

“…In skeletal muscle of the immotile zebra fish mutant relaxed, which lacks the ␤ subunit, this is not the case. In the absence of the ␤ subunit Ca V 1.1, channels were not only incorporated into the membrane but even correctly targeted to the triadic junctions (34). Evidently, the requirement of ␤-AID interactions for Ca V membrane targeting differs between nerve and muscle cells.…”

“…In skeletal muscle of a ␤-null zebrafish mutant, for example, this is not the case. There the Ca V s are inserted in the membrane and normally target into the triads in the absence of a ␤ subunit (34). Due to the expression of multiple channel isoforms in pre-and postsynaptic compartments, subcellular targeting of Ca V s in neurons is highly complex.…”

Identification and expression of voltage‐gated calcium channel β subunits in Zebrafish

“…In excitable cells, such as muscles, neurons and endocrine cells, voltage-gated calcium channels play important roles in a variety of processes, including excitation-contraction coupling, synaptic transmission, and hormone secretion (Sheng et al, 1994;Rettig et al, 1997;Schredelseker et al, 2005). In addition, Ca 2ϩ entering through voltagegated calcium channels can serve as a second messenger in signaling pathways to regulate gene expression in developmental processes, including mesoderm patterning, neural induction, process outgrowth, neuronal migration, and cardiac cell differentiation (Komuro and Rakic, 1992;Moorman and Hume, 1993;Moreau et al, 1994;Leclerc et al, 1995Leclerc et al, , 1997Leclerc et al, , 2000Brosenitsch et al, 1998;Haase et al, 2000;Palma et al, 2001;Rottbauer et al, 2001).…”