A critical component of the receptor-evoked Ca2+ signal is Ca2+ influx mediated by the TRPC and Orai channels and is regulated by the ER luminal Ca2+ sensor STIM1. Ca2+ influx is essential for all cell functions, yet excess Ca2+ influx is highly toxic and is involved in numerous cellular pathologies. A key question is how STIM1 interacts and open these channels. Most aspects of STIM1 interaction with the TRPC and Orai channels have been studied in model system, while the behavior of the native proteins is not known, in particular in polarized cells. We used model system and gene deletion in mice to study regulation of Orai and TRPC channels by STIM1. We identified several critical domains in STIM1 that are required for regulation of the channels. In particular, the STIM1 SOAR domain mediates interaction of STIM1 with both channel type. The newly discovered CTID domain downstream of SOAR regulates access of the inhibitor SARAF to SOAR. SOAR also mediates interaction of STIM1 with the C terminus of TRPC channels to present the basic lysine-rich domain of STIM1 to the TRPC channels to gate them. The localization of the native Orai1, TRPCs and STIM1 in polarized cells provide further clues as to the regulation and potential role of TRPC channels in cellular pathology. Finally, deletion of TRPC channels in mice markedly affect acinar cells receptor and store mediated Ca2+ influx and protects the cells from Ca2+-mediated toxicity brought about by cell stress.