In the thirteenth year of support from the Jain Foundation, we focused on refining our understanding of the role of the C2A domain of dysferlin in regulating Ca2+ signaling in myofibers before and after a mild injury. Our previous results indicated that the C2A domain has a unique role in sustaining Ca2+ signaling after mild hypoosmotic shock. To assess its specificity, we introduced inactivating point mutations as well as polymorphisms, substituted other, homologous C2 domains for C2A, and examined the alternatively spliced variant, C2Av1. The variant and polymorphic mutations sustained normal activity. Other constructs failed to do so. We found, however, that the C2 domain of PKCα targets the triad junction regions, where dysferlin normally concentrates. We therefore created constructs of C2A and C2PKCα and showed that these are more active at lower levels of expression than any other constructs we have studied. Additional evidence suggested that C2PKCα-C2A was likely to function because it concentrates at triad junctions, where it can bind Ca2+ effectively.
In an extension of our ongoing characterization of the C2 domains of dysferlin, we also completed our studies of point mutations that are thought to inactivate the protein’s role in vivo. Our results indicate that nearly all of them also inactivate dysferlin’s role in regulating Ca2+ signaling. This supports our earlier conclusion that all the C2 domains of dysferlin, with the possible exception of C2B, are required for normal Ca2+ homeostasis.