FAILURE TO MAKE NORMAL ALPHA-RYANODINE RECEPTOR IS AN EARLY EVENT ASSOCIATED WITH THE CROOKED NECK DWARF (CN) MUTATION IN CHICKEN

We have investigated the molecular basis of the Crooked Neck Dwarf (cn) mutation in embryonic chickens. Using biochemical and pharmacological techniques we are unable to detect normal alpha ryanodine receptor (RyR) protein in intact cn/cn skeletal muscle. Extremely low levels of alphaRyR immunoreactivity can be observed in mutant muscles, but the distribution of this staining differs from that in normal muscle and colocalizes with the rough endoplasmic reticulum immunoglobulin binding protein, BiP. This suggests the existence of an abnormal alphaRyR protein in mutant muscle. In day E12 cn/cn muscle the levels of RyR mRNA are reduced by approximately 80%, while the levels of other muscle proteins, including the alpha1 subunit of the dihydropyridine receptor, the SR Ca2+-ATPase, calsequestrin, and glyceraldehyde-3-phosphate dehydrogenase, and their associated mRNAs are essentially normal in cn/cn muscle. There is also a failure to express alphaRyR in cn/cn cerebellar Purkinje neurons. Expression of the betaRyR, a second RyR isoform, is not initiated in normal skeletal muscle until day E18. In cn/cn skeletal muscle significant muscle degeneration has occurred by this time and the betaRyR is found at low levels in only a subset of fibers suggesting the reduced levels of this isoform are a secondary consequence of the mutation. The cardiac RyR isoform is found in cn/cn cardiac muscle, which contracts in a vigorous manner. In summary, a failure to make normal alphaRyR receptor appears to be an event closely associated with the cn mutation and one which may he largely responsible for development of the cn/cn phenotype in embryonic skeletal muscle. (C) 1993 Wiley-Liss, Inc.