Gating mechanism of cancer related Orai channel mutants

Ca2+ is an important second messenger that regulates a plethora of cellular functions. It maintains cell homeostasis and provides long-term Ca2+ signals. These signals regulate diverse signaling cascades indispensable for correct function of our immune system like mast cell degranulation, cytokine secretion, T- and B-cell differentiation, lysis of infected or cancerous target cells, gene transcription or cell proliferation. Ca2+ enters the cell via so called store-operated Ca2+ release activated Ca2+ (CRAC) channels that activate upon Ca2+ depletion from internal cell stores. The molecular components of the CRAC channel have just recently been defined. The ER located Ca2+- sensor, STIM1 and the Ca2+-selective ion pore, Orai1 in the membrane are sufficient to fully reconstitute CRAC currents. The identification of STIM1 and Orai1 and recently the structural resolution of both proteins by X-ray crystallography substantiated many findings from structure- function studies which has substantially improved the understanding of CRAC channel activation. Altered Ca2+ influx via CRAC channels has furthermore been implicated in many pathophysiological processes including atherosclerosis and cancer progression. Nowadays many studies emphasize a pivotal role of Orai proteins in tumorigenesis like in prostate cancer, glioblastoma, cervical cancer and in the progression and metastasis. CRAC channels therefore play an essential role in a variety of diseases and have important clinical applications. Within the current project entitled Gating mechanism of cancer related Orai channel mutants, I aim to systematically evaluate reported Orai single point mutations derived from currently 89 studies from cancer patients. What influence do these mutations have on Ca2+ homeostasis and downstream signaling? I aim to characterize these cancer related Orai mutants in terms of altered localization within the cell, interaction with STIM1, Ca2+ gating and permeation. In addition, down-stream implications of these mutants will be investigated for their physiological effect on gene transcription in immune and cancer cells. This project will give fundamental insight into the unique Ca2+ permeation mechanism through cancer related Orai mutants. The physiological and pathophysiological functions of STIM/Orai channels in cancer cells and the medical treatment of cancer patients will highly benefit from the mechanism of channel activation/permeation and down-stream Ca2+ signaling.

The Ca2+ ion maintains cell homeostasis and provides long-term Ca2+ signals. These signals regulate diverse signalling cascades indispensable for correct function of the human immune system. Ca2+ enters the cell via so called store-operated Ca2+ release activated Ca2+ (CRAC) channels that activate upon Ca2+ depletion from internal cell stores. The molecular key players of the CRAC channel are the ER - located Ca2+-sensor, STIM1 and the Ca2+-selective ion pore, Orai1 in the membrane. Besides its role in mast cell degranulation, immune-cell differentiation and gene transcription, an alteration of Ca2+ influx via CRAC channels has also been implicated in many pathophysiological processes including atherosclerosis and cancer progression. Nowadays many studies emphasize a pivotal role of Orai proteins in tumorigenesis like in prostate cancer, glioblastoma, cervical cancer and in its progression and metastasis. CRAC channels therefore play an essential role in a variety of diseases and have important clinical applications. Within the project entitled Gating mechanism of cancer related Orai channel mutants, I aimed at systematically evaluating reported Orai single point mutations derived from currently 89 studies from cancer patients. My research question was: What influence do these mutations have on Ca2+ homeostasis and downstream signalling? I characterized these cancer related Orai mutants in terms of altered localization within the cell, interaction with STIM1, Ca2+ gating and permeation. In addition, down-stream implications of these mutants was investigated for their physiological effect on gene transcription in immune and cancer cells. This project gave fundamental insight into the unique Ca2+ permeation mechanism through cancer related Orai mutants. In detail, we could show that cancer data derived Orai1 mutants can lead to enhanced Ca2+ influx into cells. This Ca2+ overload leads to the activation of several transcription factors which, among other things, can activate genes responsible for cell proliferation. Uncontrolled cell proliferation is a hallmark of cancer. In cooperation with scientists from the Czech Republic we carried out molecular dynamics simulations and found that within open Orai mutants, the ion conducting pore undergoes two major rearrangements: side-chains of one specific amino acid are displaced and a chain of water molecules is formed; both steps are necessary for releasing barriers within the pore allowing for Ca2+ influx. Furthermore, this project enabled us to show that the transmembrane 2 region of the Orai1 protein is a really important site for channel gating, as cancer-derived mutations cluster within this region. Therefore, we were the first to show a new gating model for the Orai1 channel based on bioinformatical and functional screens of mutations occurring in human cancer tissues. The physiological and pathophysiological functions of STIM/Orai channels in cancer cells and the medical treatment of cancer patients will highly benefit from the mechanism of channel activation/permeation and down-stream Ca2+-signalling.