The role of STIM and Orai proteins in the Ca²⁺ release-activated Ca²⁺ channel.

Abstract

Store operated Ca²⁺ channels on the plasma membrane are activated by depletion of intracellular Ca²⁺ stores such as the endoplasmic reticulum. The archetypal example of these channels is the Ca²⁺ release-activated Ca²⁺ (CRAC) channel. CRAC channels are expressed in most mammalian cell types, and are required for T cell activation during immune responses, maintenance of muscle tone, and regulation of cell division. The molecular components of the CRAC channel are: STIM1, located on the endoplasmic reticulum membrane, which acts as a luminal Ca²⁺ sensor and store depletion signal; and Orai proteins on the plasma membrane, tetramers of which form the Ca²⁺ permeable pore of the channel. The first study in this thesis investigated the hypothesis that the relative expression levels of STIM1 and Orai1 determine the biophysical properties of CRAC channels. Changing the transfection ratio of STIM1 to Orai1 containing plasmids resulted in a corresponding change in the relative amount of each protein expressed. Concomitant with this result was a change in a range of channel properties. Most notably, fast Ca²⁺ dependent inactivation (FCDI) of the current was strongest when STIM1 was expressed in relative excess to Orai1. The results of this study demonstrated that STIM1 is a determinant of the CRAC current properties and that it is likely that a variable number of STIM1 peptides can bind to each Orai1 tetramer. Having defined a new role for STIM1 in the kinetics of CRAC current, mutations of the Orai1 predicted pore reported to affect selectivity and/or gating were investigated to determine whether they may also be affected by the relative expression of STIM1. The results of the second study of this thesis showed that V102I and E190Q Orai1 were both dependent on the relative expression of STIM1, similarly to WT Orai1. A third mutant, E106D Orai1 produced currents with very strong, accelerated FCDI, independently of the relative expression level of STIM1. In addition, this mutant displayed altered pH dependence. It was concluded that the selectivity centre of Orai1 is also crucial for channel gating and pH dependence. While the properties and physiological roles of Orai1 have been well described, less is known about its homologues. In order address this, the third study characterised the biophysical properties of channels formed with Orai3. While Orai3 channels were activated by store depletion via STIM1, FCDI was not dependent on the relative expression of STIM1. Surprisingly, an Orai3 current was able to be activated independently of store depletion and STIM1 by external application of the compound 2-APB. Unlike store operated current, the 2-APB activated current was non-selective and displayed no Ca²⁺ dependent kinetics or pH dependence. The results of this thesis have helped to elucidate the molecular basis underlying many CRAC current properties. Most notably, a now widely accepted understanding was developed that in addition to its role as an intracellular Ca²⁺ sensor, STIM1 is a regulator of CRAC current properties. These findings have the potential to be applied in understanding a broad range of Ca²⁺ signaling dependent cellular processes and disease states.