The C2 domains of dysferlin: roles in membrane localization, Ca²⁺ signalling and sarcolemmal repair

Abstract: Dysferlin is an integral membrane protein of the transverse tubules of skeletal muscle that is mutated or absent in limb girdle muscular dystrophy 2B and Miyoshi myopathy. Here we examine the role of dysferlin's seven C2 domains, C2A through C2G, in membrane repair and Ca²⁺ release, as well as in targeting dysferlin to the transverse tubules of skeletal muscle. We report that deletion of either domain C2A or C2B inhibits membrane repair completely, whereas deletion of C2C, C2D, C2E, C2F or C2G causes partial loss of membrane repair that is exacerbated in the absence of extracellular Ca²⁺. Deletion of C2C, C2D, C2E, C2F or C2G also causes significant changes in Ca²⁺ release, measured as the amplitude of the Ca²⁺ transient before or after hypo-osmotic shock and the appearance of Ca²⁺ waves. Most deletants accumulate in endoplasmic reticulum. Only the C2A domain can be deleted without affecting dysferlin trafficking to transverse tubules, but Dysf-ΔC2A fails to support normal Ca²⁺ signalling after hypo-osmotic shock. Our data suggest that (i) every C2 domain contributes to repair; (ii) all C2 domains except C2B regulate Ca²⁺ signalling; (iii) transverse tubule localization is insufficient for normal Ca²⁺ signalling; and (iv) Ca²⁺ dependence of repair is mediated by C2C through C2G. Thus, dysferlin's C2 domains have distinct functions in Ca²⁺ signalling and sarcolemmal membrane repair and may play distinct roles in skeletal muscle. Key points: Dysferlin, a transmembrane protein containing seven C2 domains, C2A through C2G, concentrates in transverse tubules of skeletal muscle, where it stabilizes voltage-induced Ca²⁺ transients and participates in sarcolemmal membrane repair. Each of dysferlin's C2 domains except C2B regulate Ca²⁺ signalling. Localization of dysferlin variants to the transverse tubules is not sufficient to support normal Ca²⁺ signalling or membrane repair. Each of dysferlin's C2 domains contributes to sarcolemmal membrane repair. The Ca²⁺ dependence of membrane repair is mediated by C2C through C2G. Dysferlin's C2 domains therefore have distinct functions in Ca²⁺ signalling and sarcolemmal membrane repair.