Transmembrane helix coupling regulates Orai channel gating

Orai channel gating and Ca2+ permeation is an essential mechanistic step for the immune response. A mutation that hinders Orai1 channel gating causes a form of severe combined immune deficiency. Employing a combined approach of patch-clamp, molecular biology, biochemical techniques and structure guided mutagenesis, I aim evaluating the role of trans-membrane helical interactions in the regulation of Orai1 channel gating. In preliminary experiments we discovered a novel key mutation in the second transmembrane (TM1) helix of Orai1, H134A that results in a Ca2+ selective, STIM1 independent, constitutively active current. Our predictions based on a recent crystal structure of Drosophila melanogaster Orai suggest that this histidine 134 connects the TM2 with both TM1 and TM3 via hydrogen bonds, forcing Orai1 channel in a closed conformation. To test this hypothesis, we will use structure-guided mutagenesis to manipulate these hydrogen bonds. Furthermore, employing cysteine mutagenesis, the predicted hydrogen bonds will be replaced by disulfide bonds to control the reversibility of these interactions. Indeed, this will allow us to either induce or break disulfide bonds to switch between store-operated and constitutive active Orai1 currents. In addition, we will take advantage of the constitutively active Orai1-H134A channel, to visualize reorientation of the gate located within the cytosolic region of TM1 helices. Cysteine scanning within the TM1 helix will determine the degree of dimerization necessary for mapping the relative distances in an opened and closed Orai1 channel conformation. In a more physiological context, we will evaluate TM1 helix reorientation by directly linking a STIM1 fragment to Orai1. In summary these experiments aim to reveal how STIM1 binding might uncouple TM helices resulting in a reorientation of TM1 that leads to an opened channel conformation.