Super-resolution analysis of the origins of the elementary events of ER calcium release in dorsal root ganglion neurons

Coordinated events of calcium (Ca2+) released from the endoplasmic reticulum (ER) are key second messengers in excitable cells. In pain-sensing dorsal root ganglion (DRG) neurons, these events can be observed as Ca2+ sparks, produced by a combination of ryanodine receptors (RyR) and inositol 1,4,5- triphosphate receptors (IP3R1). These microscopic signals offer the neuronal cells with a possible means of modulating the sub-plasmalemmal Ca2+ handling, initiating vesicular exocytosis. With super-resolution dSTORM and expansion microscopies, we visualised the nanoscale distributions of both RyR and IP3R1 that featured loosely organised clusters in the sub-plasmalemmal regions of cultured, rat DRG somata. We adapted a novel correlative microscopy protocol to examine the nanoscale patterns of RyR and IP3R1 in the locality of each Ca2+ spark. We found that most subplasmalemmal sparks correlated with relatively small groups of RyR whilst larger sparks were often associated with larger groups of IP3R1. These data also showed spontaneous Ca2+ sparks in < 30% of the sub-plasmalemmal cell area but consisted of both these channel species at a 3.8-5 times higher density than in non-active regions of the cell. Taken together, these observations reveal distinct patterns and length-scales of RyR and IP3R1 co-clustering at contact sites between the ER and the surface plasmalemma that encode the positions and the quantity of Ca2+ released at each Ca2+ spark.