Optical Analysis of Ryanodine Receptor Behavior In Situ: The Role of FKBP12.6 in Cardiomyocytes

Abstract

Since the finding of Ca2+ sparks (and its equivalents), optical recording has become the most important tool to study intracellular Ca2+-permeable channels in situ, which are inaccessible otherwise by electrophysiological means. Due to the mixture of in-focus and out-of-focus events, spontaneous sparks do not quantitatively reflect the ryanodine receptor (RyR) Ca2+ release flux (iRyR). To quantify the iRyR, we activated and recorded in-focus Ca2+ sparks under the loose-seal patch-clamp condition, and calibrate iRyR with Ca2+ sparklets from a single L-type Ca2+ channel (LCC). Using this analytical tool, we studied the role of FKBP12.6 in regulating RyR gating behavior in intact cardiomyocytes, which has been highly controversial over the last decade. We found that, in wild-type mouse ventricular myocytes, iRyR exhibited a distribution with periodic quantal peaks, with each quantum of 1.05 pA representing the iRyR of a single RyR. By contrast, in heart cells from FKBP12.6 knockout (FKO) mice, the quantal property of iRyR was eliminated, indicating that synchronized activation of multiple (2∼4) RyRs basically depends on the existence of FKBP12.6. Notably, FKO cells displayed fractional iRyR of 0.3–0.8 pA. This finding provided direct evidence for the partial opening of a single RyR in situ in the absence of FKBP12.6. The suppression of fractional iRyR events in wild-type cells demonstrated that FKBP12.6 played a key role in coordinating the allosterism among RyR subunits. Taken together, our study demonstrated for the first time that FKBP12.6 mediates both inter- and intra-molecular coordination of RyR gating in intact heart cells.