RyR3 in situ regulation by Ca2+ and quercetin and the RyR3-mediated Ca2+ release flux in intact Jurkat cells

Ryanodine receptors are generally thought to possess a high-affinity activating cytosolic Ca2+ site and a low-affinity inhibitory cytosolic Ca2+ site. By performing conformation selective measurements in which quercetin was used as a fluorescent marker for RyR3 (ryanodine receptor type 3) in Jurkat cells, we now find that the rectified RyR3 channel in open conformation may be regulated in situ by two cytosolic activating Ca2+ sites, of low and high affinity, respectively, whereas no inhibitory Ca2+ effect could be delineated. In the closed rectified channel, as well as in the open hindered channel, only the high affinity activating Ca2+ site and the inhibitory Ca2+ site were functional, whereas in the closed hindered channel all three regulatory Ca2+ sites appeared to be operational. RyR3 also seems to possess one activating and two inhibitory quercetin sites. Corresponding Hill coefficients and affinities of these regulatory sites were estimated. Quercetin cellular uptake exhibited an initial rapid phase (∼1.04 min), followed by slow accumulation of free quercetin inside the cytosol (∼34.5 min). The RyR3-mediated Ca2+ release current increased from a baseline of 247 to 287 pA in 1 min. after addition of 50 μM quercetin and then declined slowly to a plateau of 265 pA.