Visualising the local single-channel underpinnings of calcium sparks in excitable cells

The elementary release of calcium (Ca2+) from a cell’s internal store is known as a Ca2+ spark. The spatiotemporal summation of a cell’s Ca2+ signalling underpins its function. The junctional membrane complex (JMC) is a structural unit which contains clusters of the ryanodine receptor (RyR) or inositol triphosphate receptor (IP3R) responsible for Ca2+ spark generation. The impact of a cell’s Ca2+ signalling due to single protein orientation has only been studied experimentally in isolation. This thesis outlines the development of a correlative imaging protocol that has allowed Ca2+ signalling within primary cells to be visualised in relation to the local nanoscale structure of the JMC. This was achieved by combining total internal reflection fluorescence imaging of Ca2+ sparks, with protein distribution using DNA-PAINT and dSTORM super-resolution microscopies. Examining Ca2+ sparks and RyR arrays in ventricular myocytes taken from the monocrotaline-induced rat model of right ventricular heart failure (RV HF) revealed a relationship between RyR orientation and spark mass. Remodelling of the RyR cluster pattern was observed within RV HF. Specifically, a fragmentation of the RyR cluster pattern, represented by an increased distance between puncta, was accompanied by a decreased spark mass. Therefore, at a local level the structural remodelling of the JMC has an impact upon functional Ca2+ signalling within the failing heart. Versatility of the correlative imaging protocol was evidenced by its application to the dorsal root ganglion sensory neurones to evaluate the role of IP3R and RyR in Ca2+ spark generation. Application of the protocol revealed that both IP3R1 and RyR3 were present in regions where Ca2+ sparks occur. However, the IP3R1 was considered to have a more fragmented pattern compared to RyR3 in regions where Ca2+ sparks were generated. Overall, the project highlights the importance of studying local Ca2+ signalling in relation to a cell’s ultrastructure.