The syntrophins are a family of structurally related proteins that contain multiple protein interaction motifs. Syntrophins associate directly with dystrophin, the product of the Duchenne muscular dystrophy locus, and its homologues. We have generated α-syntrophin null mice by targeted gene disruption to test the function of this association. The α-Syn−/− mice show no evidence of myopathy, despite reduced levels of α-dystrobrevin–2. Neuronal nitric oxide synthase, a component of the dystrophin protein complex, is absent from the sarcolemma of the α-Syn−/− mice, even where other syntrophin isoforms are present. α-Syn−/− neuromuscular junctions have undetectable levels of postsynaptic utrophin and reduced levels of acetylcholine receptor and acetylcholinesterase. The mutant junctions have shallow nerve gutters, abnormal distributions of acetylcholine receptors, and postjunctional folds that are generally less organized and have fewer openings to the synaptic cleft than controls. Thus, α-syntrophin has an important role in synapse formation and in the organization of utrophin, acetylcholine receptor, and acetylcholinesterase at the neuromuscular synapse.

α-Dystrobrevin is both a dystrophin homologue and a component of the dystrophin protein complex. Alternative splicing yields five forms, of which two predominate in skeletal muscle: full-length α-dystrobrevin-1 (84 kD), and COOH-terminal truncated α-dystrobrevin-2 (65 kD). Using isoform-specific antibodies, we find that α-dystrobrevin-2 is localized on the sarcolemma and at the neuromuscular synapse, where, like dystrophin, it is most concentrated in the depths of the postjunctional folds. α-Dystrobrevin-2 preferentially copurifies with dystrophin from muscle extracts. In contrast, α-dystrobrevin-1 is more highly restricted to the synapse, like the dystrophin homologue utrophin, and preferentially copurifies with utrophin. In yeast two-hybrid experiments and coimmunoprecipitation of in vitro–translated proteins, α-dystrobrevin-2 binds dystrophin, whereas α-dystrobrevin-1 binds both dystrophin and utrophin. α-Dystrobrevin-2 was lost from the nonsynaptic sarcolemma of dystrophin-deficient mdx mice, but was retained on the perisynaptic sarcolemma even in mice lacking both utrophin and dystrophin. In contrast, α-dystrobrevin-1 remained synaptically localized in mdx and utrophin-negative muscle, but was absent in double mutants. Thus, the distinct distributions of α-dystrobrevin-1 and -2 can be partly explained by specific associations with utrophin and dystrophin, but other factors are also involved. These results show that alternative splicing confers distinct properties of association on the α-dystrobrevins.

Abstract. Two high-affinity mAbs were prepared against Torpedo dystrophin, an electric organ protein that is closely similar to human dystrophin, the gene product of the Duchenne muscular dystrophy locus. The antibodies were used to localize dystrophin relative to acetylcholine receptors (AChR) in electric organ and in skeletal muscle, and to show identity between

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The ryanodine receptor (RyR)-Ca2+ release channels of frog skeletal muscle have been purified as 30S protein complexes comprised of two high molecular weight polypeptides. The upper and lower bands of the frog doublet comigrated on sodium dodecyl sulfate polyacylamide gels with the mammalian skeletal and cardiac RyR polypeptides, respectively. Immunoblot analysis showed that a polyclonal antiserum to the rat skeletal RyR preferentially cross-reacted with the upper band, whereas monoclonal antibodies to the canine cardiac RyR preferentially cross-reacted with the lower band of the frog receptor doublet. Immunoprecipitation studies indicated the presence of two homooligomer 30S RyR complexes comprised of either the lower or upper polypeptide band of the frog doublet, and immunocytochemical staining revealed their colocalization in frog gastrocnemius muscle. After planar lipid bilayer reconstitution of the 30S frog RyR, single-channel currents were observed that exhibited a Na+ and Ca2+ conductance and pharmacological characteristics similar to those of the mammalian skeletal and cardiac Ca2+ release channels. These results suggest that amphibian skeletal muscle expresses two distinct RyR isoforms that share epitopes in common with the mammalian skeletal or cardiac RyR.

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