UMB Digital Archive
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The UMB Digital Archive is a service of the Health Sciences and Human Services Library (HSHSL) that collects, preserves, and distributes the academic works of the University of Maryland, Baltimore. It is a place that digitally captures the historical record of the campus.
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Item Item Founders Week 2024(2024)Item Dysferlin’s C2a Domain, Ca2+ Binding and Pkcα Suppress Ryr1-mediated Ca2+ Leak Into the Triad Junction(2025-02-15)Dysferlin is an integral membrane protein of the triad junction (TJ). It absence in skeletal muscle leads to Limb Girdle Muscular Dystrophy R2, an autosomal recessive disease. Our studies of dysferlin-null mouse myofibers in vitro show the following. (i) Mild injury by hypoosmotic shock decreases the amplitude of the voltage-induced Ca2+ transient and generates Ca2+ waves via Ca2+-induced Ca2+ release (CICR). (ii) These effects are suppressed by drugs that stabilize the DHPR-RyR1 couplon or inhibit RyR1 Ca2+ leak. (iii) They are also suppressed by low concentrations (10nM) of BAPTA-AM in the bathing medium; (iv) BAPTAAM also increases the voltage-induced Ca2+ transient amplitude in dysferlin-null fibers, bringing them to control levels. (v) Dysferlin’s C2A domain binds Ca2+. A chimera in which C2A is replaced with GCaMP6fu, which binds Ca2+ rapidly, targets TJs normally and suppresses abnormal Ca2+ signaling. (vi) Expression of dysferlin’s C2A domain via transfection also rescues Ca2+ signaling. These results suggest that dysferlin -- specifically its C2A domain -- stabilizes DHPR -RyR1 coupling to suppress Ca2+ leak and CICR, which is likely pathogenic. We also found a role for PKCα. (vii) PKCα concentrates at TJs. (viii) Dysferlin and PKCα co-immunoprecipitate. (ix) Targeting dysferlin’s C2A domain to TJs as a chimera with the C2 domain of PKCα increases its ability to rescue Ca2+ signaling. (x) Activation of PKCα with PMA suppresses abnormal Ca2+ signaling in injured dysferlin-null fibers. (xi) Inhibition of PKCα with Gö6976 induces abnormal Ca2+ signaling, including Ca2+ waves, in injured wild type fibers. These results suggest that dysferlin stabilizes DHPR-RyR1 coupling and suppresses Ca2+ leak by facilitating targeted phosphorylation by PKCα, but that activation of PKCα can compensate for dysferlin’s absence to shut down RyR1-mediated leak.Item Item