Hexameric CRAC channels (red) bind STIM1 dimers (blue) upon entering ER-PM junctions, but show little activity until five sites become occupied. The quintuplybound channels have altered properties, including reduced Po, Ca2+ blocking affinity, and CDI, and elevated unitary conductance and PCs/PNa compared to the fully bound channel or native CRAC channels. Binding to all six subunits is needed to produce the effective activation and permeation characteristics of native CRAC channels.