Activation of transcription factors by carcinogenic Orai mutants

Activation of transcription factors by carcinogenic Orai mutants Rainer Schindl Johannes Kepler University Linz Austria

Ca 2+ through Orai 1 channels activates immune cells Ca 2+

Store-operated Ca 2+ channel 2 m. M Ca 2+ 100 n. M Ca 2+ gene regulation 1 m. M Ca 2+ Vesicle release Proposed in 1986: store-operated channel is dependent on the [Ca 2+] in the endoplasmic reticulum • store depletion triggers channel activation • triggers T-cell activation and mast cell degranulation

Discovery of STIM 1 and Orai proteins Orai 1 Patient with Severe Combined immunodeficiency in Orai 1: caused by a single point mutation in Orai 1 Feske, et al. (2006) Nature Roos, et al. (2005) J Cell Biol Liou et al. (2005) Curr Biol STIM 1

Localisation of STIM 1 and Orai proteins Orai 1 plasma membrane STIM 1 endoplasmic reticulum Lioudyno, et al. (2008) PNAS

STIM 1 endoplasmic reticulum Ca 2+ store Stathopoulos et al. (2008) Cell; Zheng, Stathopulos, Schindl et al. (2011) PNAS

FRET technique detects close distance between proteins Close distance of CFP and YFP results in a energy transfer to YFP Only within 10 nm

STIM 1 couples to ORAI 1 channel upon store-depletion ORAI CFP STIMY Overlay FP FRET CFP STIM 1 YFP PM FRET Muik, Frischauf, Derler et al. (2008) J. Biol. Chem. Endoplamic Reticulum

Time-dependent STIM 1 coupling with ORAI 1 ORAI CFP STIMY Overlay FP FRET Stim 1 to Orai 1 coupling is temporally as well as spatially correlated with an increase in FRET Muik, Frischauf, Derler et al. (2008) J. Biol. Chem.

The Patch Clamp Technique Ca 2+ Orai channel Ca 2+ A tiny glass pipette is fused to a single cell to form a stable Gigaseal (~ GΩ resistance) to record single ion channels 11

Activation of store-operated currents by overexpression of STIM 1 and Orai 1 10 m. M Ca 2+ 10 m. M EGTA -95 m. V -86 m. V Schindl, et al. (2008) PNAS

STIM 1 and ORAI 1 required for channel activity Reconstitution of a store-operated current in a HEK cell ORAI 1 STIM 1 calcium current 10 m. M EGTA STIM 1+ORAI 1 Schindl, Frischauf, Bergsmann et al. (2009) PNAS

Ca 2+ accumulation domain CAR in Orai 1 a b Ca 2+ accumulating region (CAR) D 110 Selectivitiy fiter E 106 Frischauf et al. (2015) Science Signal.

Molecular dynamics simulations predict CAR in Orai 1 Frischauf et al. (2015) Science Signal.

Orai 1 CAR increases Ca 2+ currents at physiological environmental Ca 2+ levels a b c STIM 1 + Orai 1 d STIM 1 + Orai 1 -D 110 A Frischauf et al. (2015) Science Signal.

Thanks to: Linz, Austria: current colleagues: Irene Frischauf, Martin Muik, Marc Fahrner, Isabella Derler, Adela Krizova, Michael Stadlbauer, Carmen Butorac, Victoria Lunz, Christoph Romanin My team: Monika Litviňuková, Romana Schober Toronto, Canada: Peter Stathopulos, Li Zeng, Mitsuiko Ikura Nove Hrady, Czech Republic: Vasilina Zayats, Saurabh K. Pandey, Rüdiger H. Ettrich Homburg, Germany: Ivan Bogeski Supported by: