Intrinsic feedback regulations in Orai channels

A rapid change in cytosolic calcium levels is one of the main mechanisms to stimulate cellular processes including secretion, contraction and gene activation. In immune cells, Ca2+ entry is mediated by the Ca2+ release activated Ca2+ (CRAC) current, conducted through Orai channels. Upon depletion of endoplasmic reticulum (ER) stores, the luminal Ca2+ sensor Stim1 couples to and activates the three highly Ca2+ selective Orai channels. In this project I aim to elucidate domains of Orai proteins that fine-tune intracellular Ca2+ entry, particularly focusing on Ca2+ feedback regulation. The patch clamp technique together with molecular biology methods will reveal insight how sequence variations among Orai proteins lead to distinct current modulation, including Ca2+ dependent fast inactivation as well as reactivation. Therefore chimera and mutant Orai constructs will be designed. I will further examine a potential cooperative inactivation mechanism of several Orai domains. The results will provide a first picture of the interplay of Orai inactivation domains at the cytosolic entrance of the channels pore. Moreover the interplay of distinct Orai subunits in a heteromeric Orai1/3 channel will be examined for their unique inactivation profile. At a research stay in the laboratory of Prof. M. Trebak, I will characterise native cells for their in-/re- activation profiles of endogenous store-operated Ca2+ currents that involve either Orai1 or Orai3. This project will provide a substantial mechanistic insight into the molecular plasticity of feedback mechanism of homo- and heteromeric Orai channels.

Calcium pores can occlude the blood streamA leading cause of death in western industrialized countries is atherosclerosis, a decrease or occlusion of the blood flow. Within this project, granted by the Austrian science fund (FWF), biophysicists from the University of Linz in collaboration with US scientists discovered that an extensive up-regulation of the Orai protein results in unregulated growth of muscle cells. This disease related cell proliferation is often slow, however can cause heart attack or stroke in the worst case. Inhibition of the Orai protein stops the cell growth. Currently, new drugs against Orai are examined to protect human blood vessels.How is it possible that a protein of a few nanometers affects our health so drastically? Human cells receive and generate a variety of chemical and molecular signals: to proliferate, release substances or migrate into a tissue. One of the main signals is calcium. In many cells a calcium influx is generated by two proteins, STIM1 and Orai that have been examined in my research project. Orai forms a calcium permeable pore, regulated by STIM1.Atomic structure of a calcium trigger:A collaboration of scientist from the university of Toronto and Linz enabled visualizing the interplay of these two proteins with atomic resolution. A combined approach of genetics and measurement of calcium influx revealed how the binding of the two proteins triggers the influx of calcium into cells. Our results are published in the prestigious journal Nature Communications. Orai causes uncontrolled proliferation:Certain rare gene defects modulate the coupling of these two proteins and result in severe diseases of the immune system, muscle or in bleedings. A research stay at the Albany Medical Center allowed me to examine diseases related with the protein Orai. In collaboration with my US colleagues we discovered that Orai proteins are highly present in disease related muscle cells. These cells showed uncontrolled proliferation and can occlude the blood flow. In a mouse model we were already able to successfully control the Orai protein that avoided the restriction of the aorta (published in Circulation Research). In collaboration with the pharmaceutical company GlaxoSmithKline we recently managed to suppress Orai proteins with new blockers.These experiments represent a first step to develop new medications and stop the uncontrolled proliferation of muscle cells. Our results clearly show that Orai is a key protein for the disease related occlusion of the blood flow.