Dysferlin’s C2A domain supports normal Ca²⁺ signaling and membrane repair in dysferlin-null myofibers

Dysferlin is required for the health of skeletal muscle, where it mediates at least two seemingly distinct processes, repair of the sarcolemmal membrane and stabilization of Ca²⁺ release after injury. Dysferlin is a ∼230-kDa protein comprised of multiple C2 domains, Fer and Dysf domains, and a transmembrane domain that anchors it to the transverse tubule membrane at the triad junction (TJ). Here we show that replacing C2A with other C2 domains yields fusion proteins that are less active in supporting Ca²⁺ signaling than WT dysferlin. We also show that the most N-terminal of dysferlin’s C2 domains, C2A (DysfC2A), is sufficient to support normal Ca²⁺ signaling and sarcolemmal repair in dysferlin-null muscle, though it accumulates at TJs inefficiently. The C2A domains of myoferlin and PKCα, though homologous, are less active. The C2 domain of PKCα (PKCαC2) targets the TJ more efficiently than DysfC2A, however. Fusion proteins containing one or two PKCαC2 domains accumulate at the TJ and are active in sarcolemmal membrane repair, but they do not support normal Ca²⁺ signaling unless linked to DysfC2A Control of Ca²⁺ signaling is fully restored when the PKCαC2 domain, alone or in pairs, is linked to DysfC2A. This suggests that the DysfC2A is sufficient to support two major functions of dysferlin in skeletal muscle, and that its effect on Ca²⁺ signaling is specific. We propose that the PKCαC2–PKCαC2-DysfC2A fusion protein is a promising candidate for future viral gene therapy treatment for dysferlinopathy.