Project Summary The overall objective of this renewal proposal is to better understand how Orai1 channels function in T cells at the molecular, subcellular, and cellular levels. Orai proteins are a novel class of Ca2+ channels in the plasma membrane (PM). They are activated by STIM proteins in the ER, with which they colocalize in puncta at ER-PM junctions following depletion of Ca2+ in the ER lumen. Despite the importance of Orai1 as a potential therapeutic target in T cells, crucial questions remain at single channel, puncta and cellular levels. In large part this is because single-channel recording with the patch-clamp technique cannot be applied to Orai1 or other Orai channels, which have exceptionally low conductance, and because local signalling takes place in STIM1:Orai1 puncta. To circumvent these limitations, our proposal is predicated on our recent development of novel Ca2+- indicator fusion proteins; an Orai1-GCaMP6f channel-indicator that reports single-channel signals, and a ratiometric, red/green cytosolic Ca2+ indicator. Implemented in transgenic mice, these probes are revealing new and unanticipated local Ca2+ signalling events and the first chance to monitor endogenous Orai1 activity in native T cells. In conjunction with T cell-specific Orai1 knockout mice, we use these tools to investigate: 1) how Orai1 is activated at the single-molecule level; 2) how Orai1 channel activity varies in puncta and in native T cells; and 3) how local Ca2+ signals modulate T cell motility during immune surveillance in the lymph node.