The Orai1 pore entrance is modulated by its third loop

Ca2+ release activated Ca2+ (CRAC) channels represent the primary Ca2+ entry pathway in a vast majority of cell- types. After depletion of Ca2+ stores within the endoplasmic reticulum (ER), robust Ca2+ influx is activated via CRAC channels. The two main components involved are the Ca2+ sensor STIM1, located in the ER membrane, and the plasma-membrane channel Orai1. STIM1 senses the luminal Ca2+ content and after store-depletion it oligomerizes into punctuate clusters close to the plasma-membrane. At these sites it directly interacts with and activates Orai1 channels thereby triggering Ca2+ influx from the extracellular milieu. In this project I aim to elucidate domains within Orai1 that regulate the pore entry. The role of the first and third extracellular loop will be examined by biochemical techniques as well as patch-clamp methods to reveal which residues are essential for gating and permeation of CRAC channels. A potential cooperative interplay of the first and third loop will be investigated with regard to electrostatic interactions between negatively charged aminoacids in the first and positively charged ones in the third loop. The results aim to provide a new model for Orai1 gating at the extracellular entrance of the channels pore. Moreover, the elucidation of residues controlling the permeation at the pore entrance will also explain the altered gating of a recently described Orai1-loop3 mutant that is associated with atopic dermatitis. At a research stay in the lab of Dr. Juan Rosado, I will characterize this phenotype in native cell lines. This project will provide a substantial mechanistic insight into the molecular backgrounds of the regulation of the Orai1 pore entry.

Proteins are bio-molecules that are essential for our bodys immune defense mechanism. This project enabled us to discover a so far unknown function of the immune-protein Orai1.Our immune system needs the element calcium for the generation of antibodies. Calcium can enter immune cells via specific ports within the cells membrane. Inside the cell, calcium fulfils several essential functions. The ports via which calcium can enter are generated by Orai1 proteins, that guide calcium from the extracellular side to the inside of the cell. These Orai1 proteins do not solely act as a port but can also bind calcium - a novel fact that was previously unknown and discovered during this project. We used computer simulations, biophysical and biochemical approaches and together with our partners from the University of Nove Hrady (Czech Republic) and the University of Homburg (Germany) we are proud to show how calcium is bound to the Orai1 protein. This is a vitally important mechanism: If Orai1 lacks the calcium-binding structure, too less calcium is transported into the cell which subsequently leads disrupted immune response.