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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 α ryanodine receptor (RyR) protein in intact cn/cn skeletal muscle. Extremely low levels of αRyR 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 αRyR protein in mutant muscle. In day E12 cn/cn muscle the levels of RyR mRNA are reduced by ∼80%, while the levels of other muscle proteins, including the α1 subunit of the dihydropyridine receptor, the SRCa2+‐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 αRyR in cn/cn cerebellar Purkinje neurons. Expression of the βRyR, 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 βRyR 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 αRyR receptor appears to be an event closely associated with the cn mutation and one which may be largely responsible for development of the cn/cn phenotype in embryonic skeletal muscle. © 1993 Wiley‐Liss, Inc.

1. We identified and studied the function of ryanodine receptors in neurons isolated from dorsal root ganglia (DRG) of 10-day-old chick embryos. 2. A monoclonal antibody (mAb 34C) that recognizes all known ryanodine receptor isoforms in skeletal and cardiac muscle and CNS identified ryanodine receptor-like immunoreactivity in cultured DRG neurons. 3. Using the permeabilized patch technique to record membrane currents, we found that calcium currents were followed by a current with characteristics of a Ca(2+)-activated Cl- current (ICl(Ca)) in approximately two-thirds of the neurons. In these cells, acute application of 10 mM caffeine activated a similar ICl(Ca) and this effect was inhibited by 10 microM ryanodine. The activation of ICl(Ca) by caffeine was not dependent on extracellular Ca2+. These data suggest that caffeine raises intracellular free Ca2+ (Cai2+) by activating the release of Ca2+ from an intracellular store and that this Ca2+ activates the membrane conductance responsible for ICl(Ca). 4. The magnitude of ICl(Ca) activated by depolarization was not affected by ryanodine, implying that the Ca2+ that activates ICl(Ca) in this protocol is supplied by the Ca2+ current without amplification by a ryanodine-sensitive mechanism such as Ca(2+)-induced Ca2+ release. 5. We also used indo-1 to measure Cai2+ in DRG neurons. Ten millimolar caffeine caused a transient increase in Cai2+ that was inhibited by 10 microM ryanodine. 6. The ability of caffeine to elevate Cai2+ and activate ICl(Ca) was reduced at higher temperatures, suggesting increased Ca2+ sequestration. 7. These data demonstrate the existence of an intracellular store of Ca2+ that can be mobilized by a caffeine- and ryanodine-sensitive mechanism. The release of Ca2+ from this store can elevate Cai2+ and modulate membrane conductances.

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