FIGURE 5.

In concatenated Orai1 tetramers, a single WT intracellular loop restores fast Ca²⁺-dependent inactivation. a, schematic showing concatenated Orai1 tetramers with zero (4 MutA), one, two, three, or four WT inactivation particles. MutA monomers are depicted in red. b, measurement of SOCE in Orai1-null MEFs expressing concatenated tetramers with different stoichiometry of WT and MutA subunits. Store-operated Ca²⁺ entry was measured in Orai1-null MEFs stably expressing either empty vector or concatenated Orai1 tetramers comprising zero, one, two, or three WT inactivation particles. The bar graph (right) shows averages of peak [Ca²⁺]_i from at least three independent experiments. Each trace represents an average from 30–50 GFP-positive cells. c, measurement of CRAC currents from HEK293 cells expressing STIM1 and various tetramers of Orai1 (molar ratio of 1:1). Whole-cell currents were recorded with pipette solution containing 12 mm EGTA and an external solution containing 110 mm Ca²⁺. The traces show currents obtained with WT tetramer (blue), MutA tetramer (red), and 3MutA1WT tetramer (cyan). The individual traces for WT and 3MutA1WT tetramers are fit by two exponentials (black lines). d, Ca²⁺-dependent fast inactivation in concatenated Orai1 tetramers. The extent of inactivation (1 − I_ss/I_p) is plotted as a function of voltage for tetramers comprising four WT subunits (blue), one WT and three MutA subunits (cyan), or four MutA subunits (red). e, steady state currents from concatenated Orai1 tetramers. Data show the average (and S.E.) steady state currents at −160 mV from HEK293 cells expressing tetramers of Orai1 with either four, zero, or one WT monomer (same as in b) in extracellular solution containing 110 mm CaCl₂. Each bar represents an average of 4–6 cells.