The beta 1a subunit is essential for the assembly of dihydropyridine-receptor arrays in skeletal muscle.
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 depolarization-induced but not caffeine-induced Ca(2+) transients suggested a defect in the skeletal muscle dihydropyridine receptor (DHPR). Sequencing of DHPR cDNAs indicated that the alpha(1S) subunit is normal, whereas the beta(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 beta(1a) protein in mutant muscle. Thus, the immotile zebrafish relaxed is a beta(1a)-null mutant. Interestingly, immunocytochemistry showed correct triad targeting of the alpha(1S) subunit in the absence of beta(1a). Freeze-fracture analysis of the DHPR clusters in relaxed myotubes revealed an approximately 2-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 beta(1a) subunit does not prevent triad targeting of the DHPR alpha(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 excitation-contraction coupling in beta(1)-null model organisms.
Schredelseker
Johann
J
Department of Medical Genetics, Molecular and Clinical Pharmacology, Innsbruck Medical University, A-6020 Innsbruck, Austria.
Di Biase
Valentina
V
Obermair
Gerald J
GJ
Felder
E Tatiana
ET
Flucher
Bernhard E
BE
Franzini-Armstrong
Clara
C
Grabner
Manfred
M
D000818:Animals; D020746:Calcium Channels, L-Type / Q000172:deficiency / Q000235:genetics / Q000502:physiology*; D005838:Genotype; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000737:chemistry / Q000378:metabolism*; D010641:Phenotype; D011499:Protein Processing, Post-Translational / Q000235:genetics; D020836:Protein Structure, Quaternary; D021122:Protein Subunits / Q000172:deficiency / Q000235:genetics / Q000502:physiology*; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism / Q000502:physiology; D015027:Zebrafish
D020746:Calcium Channels, L-Type; D021122:Protein Subunits; D019837:Ryanodine Receptor Calcium Release Channel