encoding the skeletal muscle sarcoplasmic reticulum calcium release channel (ryanodine receptor, RyR1), which is localized on human chromosome 19 (RYR11). Central core disease is a relatively mild, slowly progressive autosomal dominant myopathy, characterized histologically by the presence of centrally located cores running the length of the muscle fibres. The subclinical myopathy malignant hyperthermia is a potentially fatal pharmacogenetic disorder occurring in predisposed individuals when they are exposed to volatile anaesthetics and depolarising muscle relaxants such as suxamethonium. The study of the functional properties of ryanodine receptor channels carrying mutations linked to neuromuscular disorders is important not only from a diagnostic point of view, but also to understand the basic pathophysiological mechanism leading to these different diseases. To date there are no effective therapies for the treatment of muscle weakness in central core disease and multi-minicore disease patients, while for malignant hyperthermia presymptomatic diagnosis is fundamental. The overall objective of this project is to further our understanding of the molecular pathophysiology of neuromuscular disorders linked to mutations in the skeletal muscle ryanodine receptor gene. We think that the decrease of sarco(endo)plasmic reticulum Ca2+ load via leaky RyR1 channels may also result from alterations of Ca2+ influx, via excitation-coupled Ca2+ entry (ECCE) and/or SOCE. These events may account for the phenotype of CCD patients, which includes muscle weakness and abnormal secretion of inflammatory cytokines from muscle cells and from cells of the immune system. In this project we will set out to test the validity of our hypothesis by investigating the extent of Ca2+ influx in skeletal muscle cells and immune cells (B cells and/or dendritic cells) from normal and RYR1 mutation bearing individuals. The aims of this project are: 1) directly assess Ca2+ influx in cells expressing naturally occurring RYR1 mutations, from CCD and MH patients, and compare it with that from cells obtained from control individuals; 2) evaluate the downstream effects of RYR1 mutations, on calcium handling by the sarcoplasmic reticulum. Calcium influx in myotubes expressing naturally occurring RYR1 mutations from CCD and MH patients will be monitored by exploiting the state of the art technology, i.e. TIRF (Total Internal Reflection Fluorescence) microscopy. TIRF microscopy is the best optical technology available to monitor membrane-associated events at very high resolution. It offers several advantages over conventional line scanning confocal microscopy, including the possibility of acquiring data from an ultrathin optical section such as [Ca2+]i signals occurring on, or very close to, the plasma membrane (TIRF microscopes offer a spatial resolution of 100nm which is approx. 5 times that of a confocal microscope), with a high signal-to-noise ratio.