Isoform-dependent formation of heteromeric Ca2+ release channels (ryanodine receptors)

Three ryanodine receptor (RyR) isoforms, RyR1, RyR2, and RyR3, are expressed in mammalian tissues. It is unclear whether RyR isoforms are capable of forming heteromeric channels. To investigate their ability to form heteromeric channels, we co-expressed different RyR isoforms in HEK293 cells and examined their interactions biochemically and functionally. Immunoprecipitation studies revealed that RyR2 is able to interact physically with RyR3 and RyR1 in HEK293 cells and that RyR1 does not interact with RyR3. Co-expression of a ryanodine binding deficient mutant of RyR2, RyR2 (14827T), with RyR3 (wt) restored [H-3]ryanodine binding to the mutant. Interactions between RyR isoforms were further assessed by complementation analysis using mutants RyR2 (14827T), RyR2 (E3987A), RyR3 (14732T), RyR3 (E3885A), and RyR1 (E4032A), all of which are deficient in caffeine response. Caffeine-induced Ca2+ release was restored in HEK293 cells co-transfected with mutants RyR2 (14827T) and RyR3 (E3885A), RyR2 (E3987A) and RyR3 (14732T), or RyR2 (14827T) and RyR1 (E4032A), but not with RyR1 (E4032A) and RyR3 (14732T), indicating that mutants of RyR2 and RyR,3, or RyR2 and RyR1, but not RyR1 and RyR3, are able to complement each other. Co-expression of RyR3 (wt) and a pore mutant of RyR2, RyR2 (G4824A), produced regulatable single channels with intermediate unitary conductances. These observations demonstrate that RyR2 is capable of forming functional heteromeric channels with RyR,3 and RyR1, whereas RyR1 is incapable of forming heteromeric channels with RyR3.