Analysis of Ca v1.2 and Ryanodine Receptor Clusters in Rat Ventricular Myocytes

Abstract

We analyzed the distribution of ryanodine receptor (RyR) and Ca v1.2 clusters in adult rat ventricular myocytes using three-dimensional object-based colocalization metrics. We found that ∼75% of the Ca v1.2 clusters and 65% of the RyR clusters were within couplons, and both were roughly two and a half times larger than their extradyadic counterparts. Within a couplon, Ca v1.2 was concentrated near the center of the underlying RyR cluster and accounted for ∼67% of its size. These data, together with previous findings from binding studies, enable us to estimate that a couplon contains 74 RyR tetramers and 10 copies of the α-subunit of Ca v1.2. Extradyadic clusters of RyR contained ∼30 tetramers, whereas the extradyadic Ca v1.2 clusters contained, on average, only four channels. Between 80% and 85% of both RyR and Ca v1.2 molecules are within couplons. RyR clusters were in the closest proximity, with a median nearest-neighbor distance of 552 nm; comparable values for Ca v1.2 clusters and couplons were 619 nm and 735 nm, respectively. Extradyadic RyR clusters were significantly closer together (624 nm) and closer to the couplons (674 nm) than the couplons were to each other. In contrast, the extradyadic clusters of Ca v1.2 showed no preferential localization and were broadly distributed. These results provide a wealth of morphometric data that are essential for understanding intracellular Ca 2+ regulation and modeling Ca 2+ dynamics.