Introduction The endoplasmic reticulum of most eukaryotic cells contains relatively high levels of Ca 2+ and represent an important source for Ca 2+ release into cytosol in response to stimulation of cells by a variety of agonists (Berridge et al., 2000). There are many components involved in the organisation of intracellular Ca 2+ stores. Ca 2+ pumps of the sarcoplasmic endoplasmic reticulum Ca 2+ ATPases (SERCAs) family accumulate Ca 2+ into the lumen of the endoplasmic reticulum, where specific Ca 2+ - binding proteins (such as calsequestrin and calreticulin) provide an efficient mechanisms of storage, mediated by their high capacity and low affinity for Ca 2+ (Pozzan et al., 1994; Sorrentino and Rizzuto, 2001). Ca 2+ accumulated in the intracellular stores of the endoplasmic reticulum is released into the cytosol through Ca 2+ -release-specific channels in connection with intracellular signalling mechanisms. Two distinct families of intracellular Ca 2+ -release channels, ryanodine receptors (RyRs) and inositol (1,4,5)-trisphosphate receptors [Ins(1,4,5)P 3 Rs] are known (Sorrentino et al., 2000; Patel et al., 1999). In cells, like neurons and muscle cells, where Ca 2+ -mediated signalling is highly developed, these Ca 2+ -release proteins are selectively localised to specific regions of the cells (Franzini-Armstrong and Protasi, 1997; Golovina and Blaustein, 1997; Petersen et al., 2001). The sarcoplasmic reticulum of skeletal muscle cells is a highly organised network of tubules that is well characterised with respect to the redistribution of proteins involved in Ca 2+ homeostasis. In this context, RyRs are selectively localised in the region of the junctional membranes of the terminal cisternae (i.e. the part of the sarcoplasmic reticulum facing the t-tubule). RyRs appear to be part of a large multi-protein complex, where calsequestrin, triadin, junctin are the best- characterised components. By contrast, SERCAs are mainly excluded from junctional membranes and are found mostly in the longitudinal SR (Franzini-Armstrong and Protasi, 1997). Selective redistribution of intracellular Ca 2+ -release channels within the endoplasmic reticulum of neurons has been reported (Berridge, 1998). In pancreatic acinar cells, co-localisation of SERCA pumps and intracellular Ca 2+ -release channels seems to be discontinuous in most of the endoplasmic reticulum, with evidence for clustering in some regions (Petersen et al., 2001). Obviously, the subcellular localisation of intracellular Ca 2+ - release channels may significantly affect their contribution to Ca 2+ signalling (Berridge et al., 1998; Berridge et al., 2000). However, in many other cells, intracellular Ca 2+ -release channels are less evidently restricted in their localisation within the endoplasmic reticulum. We report here on experiments with human HEK 293 cells transfected with either the RyR type 1 (RYR1) or the type 3 (RyR3) isoforms. Since epithelial HEK 293 are not a cell type specialised for RyR-mediated Ca 2+ release, we reasoned that they might represent a good model to study the localisation and the activity of RyR channels in a non-specialised endoplasmic reticulum (i.e. in contrast to the sarcoplasmic reticulum). In 2497 Ryanodine receptors (RyRs) are expressed on the endoplasmic reticulum of many cells, where they form intracellular Ca 2+ -release channels that participate in the generation of intracellular Ca 2+ signals. Here we report studies on the intracellular localisation and functional properties of transfected RyR1 or RyR3 channels in HEK 293 cells. Immunofluorescence studies indicated that both RyR1 and RyR3 did not form clusters but were homogeneously distributed throughout the endoplasmic reticulum. Ca 2+ release experiments showed that transfected RyR1 and RyR3 channels responded to caffeine, although with different sensitivity, generating a global release of Ca 2+ from the entire endoplasmic reticulum. However, video imaging and confocal microscopy analysis revealed that, in RyR3-expressing cells, local spontaneous Ca 2+ release events were observed. No such spontaneous activity was observed in RyR1- expressing cells or in control cells. Interestingly, the spontaneous release events observed in RyR3-expressing cells were restricted to one or two regions of the endoplasmic reticulum, suggesting the formation of a further subcellular organisation of RyR3 in Ca 2+ release units. These results demonstrate that different RyR isoforms can engage in the generation of distinct intracellular Ca 2+ signals in HEK 293 cells. Key words: Ryanodine receptor channels, Calcium signalling, Calcium stores, Endoplasmic sarcoplasmic reticulum, Calcium release Summary RyR1 and RyR3 isoforms provide distinct intracellular Ca 2+ signals in HEK 293 cells Daniela Rossi 1 , Ilenia Simeoni 1 , Marcella Micheli 1 , Martin Bootman 2 , Peter Lipp 2 , Paul D. Allen 3 and Vincenzo Sorrentino 1, * 1 Molecular Medicine Section, Department of Neuroscience, University of Siena, via Aldo Moro 5, 53100 Siena, Italy 2 Laboratory of Molecular Signalling, The Babraham Institute, Babraham, Cambridge CB2 4AT, UK 3 Department of Anesthesia Brigham and Women’s Hospital, Boston, MA 02115, USA *Author for correspondence (e-mail: v.sorrentino@unisi.it) Accepted 18 March 2002 Journal of Cell Science 115, 2497-2504 (2002) © The Company of Biologists Ltd Research Article