Ryanodine receptors (RyRs) are Ca2+ release channels, gated by Ca2+ in the cytosol
and the sarcoplasmic reticulum lumen. Their regulation is impaired in certain cardiac
and muscle diseases. Although a lot of data is available on the luminal Ca2+ regulation
of RyR, its interpretation is complicated by the possibility that the divalent ions
used to probe the luminal binding sites may contaminate the cytoplasmic sites by crossing
the channel pore. In this study, we used Eu3+, an impermeable agonist of Ca2+ binding
sites, as a probe to avoid this complication and to gain more specific information
about the function of the luminal Ca2+ sensor. Single-channel currents were measured
from skeletal muscle and cardiac RyRs (RyR1 and RyR2) using the lipid bilayer technique.
We show that RyR2 is activated by the luminal addition of Ca2+, whereas RyR1 is inhibited.
These results were qualitatively reproducible using Eu3+. The luminal regulation of
RyR1 carrying a mutation associated with malignant hyperthermia was not different
from that of the wild-type.
RyR1 inhibition by Eu3+ was extremely voltage dependent, whereas RyR2 activation did
not depend on the membrane potential. These results suggest that the RyR1 inhibition
site is in the membrane’s electric field (channel pore), whereas the RyR2 activation
site is outside. Using in silico analysis and previous results, we predicted putative
Ca2+ binding site sequences. We propose that RyR2 bears an activation site, which
is missing in RyR1, but both isoforms share the same inhibitory Ca2+ binding site
near the channel gate.
