Stoichiometry of CRAC channel assembly and gating [electronic resource]

Ca2+ release-activated Ca2+ (CRAC) channels are a ubiquitous pathway for Ca2+ entry in mammalian cells, and generate the sustained calcium signals required for T lymphocyte activation. They are assembled from two proteins: STIM1, an ER Ca2+ sensor, and Orai1, which forms the channel pore at the plasma membrane. CRAC channels are opened by binding of STIM1 to the C-terminus of Orai1 at closely apposed ER-PM junctions. While the basic steps in the CRAC activation process have been delineated, there are several fundamental questions that remain controversial or unaddressed. These include the subunit stoichiometry of the CRAC channel, the binding mode of STIM1 to Orai1, and an understanding of how STIM1 binding determines CRAC channel biophysical properties such as open probability and unitary calcium flux. Multiple early studies converged on a tetrameric stoichiometry for Orai1 channels, but this was later contradicted by a hexameric Drosophila Orai crystal structure. To test whether CRAC channels function as hexamers of Orai1 subunits, we generated concatenated Orai1 hexamers (6xOrai1) to encode a complete channel and enable the precise positioning of mutations. The inhibitory effects of L273D mutation indicated that all subunits of 6xOrai1 participated equally in channel assembly, verifying that 6xOrai1 channels function as hexamers. Importantly, hexameric Orai1 channels yielded currents with properties that were equivalent to those of native CRAC channels, including Ca2+-dependent inactivation and pore properties such as ion selectivity, affinity for Ca2+ block and unitary conductance. From these results we conclude that native CRAC channels function as hexamers of Orai1 subunits. FRET measurements between STIM1-YFP and CFP-Orai1-Orai1 tandem dimers were used to study STIM1 binding to the Orai1 C-terminus. While channels made from Orai1-L273D homodimers did not detectably bind STIM1, the L273D C-terminus enhanced STIM1 binding when located adjacent to a wildtype (WT) C-terminus. These results suggest that STIM1 interacts with pairs of Orai1 C-termini. To determine the dependence of CRAC channel activation on STIM1 binding we studied 6xOrai1 channels bearing single binding site mutations. Weakening binding to a single subunit (1xL273D) depressed the current to 35% of WT levels. Surprisingly, noise analysis indicated that the L273D mutation increased the unitary current 3-fold without affecting surface expression, indicating that the average open probability of 1xL273D channels is only ~11% of WT. In addition, the L273D mutation reduced the Ca2+ block affinity by 2-fold. These results show that all six binding sites must be occupied by STIM1 to effectively open the channel as well as to produce characteristic CRAC channel pore properties. Surprisingly, noise analysis also showed that within brief measurement intervals, the 1xL273D open probability was similar to WT while the number of active channels was decreased ~10-fold. These observations are consistent with channels transiting between long-lived periods of silence and bursts of high activity. Thus, STIM1 binding may control CRAC channel activity by regulating the amount of time spent in the high-activity state. In summary, we present the first functional evidence that active CRAC channels are hexamers of Orai1, and that STIM1 interacts with adjacent Orai1 C-termini in the activation process. Our data also indicate that STIM1 is an essential component of the CRAC channel, where maximal STIM1 occupancy is required for significant channel activation and conferring characteristic channel properties such as ion selectivity, Ca2+ affinity and unitary conductance.

Advisor: Lewis Richard (Richard Sheridan)
Butte M. (Manish)
Feng Liang
Goodman Miriam Beth advisor. Thesis advisor