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 (TM 1 ) 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.

Calcium acts as an essential signaling agent in the human body, regulating a variety of vital functions. Calcium acts as important signal to regulate many aspects in a cell live and mediates signals that are essential for the maintenance and proper functioning of the human immune system. In the immune system, calcium plays an important role in allergic reactions, cell growth, activation of immune cells, the killing of infected or cancerous cells and the control of gene expression. The calcium flow into immune cells is regulated by the calcium transport protein Orai1. The current scientific FWF project of PD Dr. med. Rainer Schindl (MedUni Graz) and Prof. Christoph Romanin (JKU Linz) investigated how Orai1 opens a pore to guides calcium into the cell. They were able to engineer mutations in the Orai1 protein, which open the channel permanently and allow calcium to constitutively flow into the cell. This calcium influx could be measured live in single cells. With the help of multi-disciplinary biochemical and molecular biological experiments as well as molecular dynamics simulations, the research team was also able to clarify the gating mechanism of the Orai1 protein. To turn on the calcium influx, the pore widens and charged side chains fold away from the pore center. This calcium influx subsequently turns on molecular gene activation programs. The current research funded by the FWF represents a first, important step in the elucidation of the calcium-dependent switching mechanism of immune cells.