RyR1/RyR3 Chimeras Reveal that Multiple Domains of RyR1 Are Involved in Skeletal-Type E-C Coupling

摘要

Skeletal-type E-C coupling is thought to require a direct interaction between RyR1 and the α1S-DHPR. Most available evidence suggests that the cytoplasmic II–III loop of the dihydropyridine receptor (DHPR) is the primary source of the orthograde signal. However, identification of the region(s) of RyR1 involved in bidirectional signaling with the α1S-DHPR remains elusive. To identify these regions we have designed a series of chimeric RyR cDNAs in which different segments of RyR1 were inserted into the corresponding region of RyR3 and expressed in dyspedic 1B5 myotubes. RyR3 provides a preferable background than RyR2 for defining domains essential for E-C coupling because it possesses less sequence homology to RyR1 than the RyR2 backbone used in previous studies. Our data show that two regions of RyR1 (chimera Ch-10 aa 1681–2641 and Ch-9 aa 2642–3770), were independently able to restore skeletal-type E-C coupling to RyR3. These two regions were further mapped and the critical RyR1 residues were 1924–2446 (Ch-21) and 2644–3223 (Ch-19). These results both support and refine the previous hypothesis that multiple domains of RyR1 combine to functionally interact with the DHPR during E-C coupling.