Microsoft Word - ms_O1_NT_revised_submitted_text_all_Figs

STIM1 and Orai1 represent the two molecular key components of the Ca 2+ release-activated Ca 2+ -channels. Their activation involves STIM1 C-terminus coupling to both Orai-Nand -C-terminus. Here we focused on the extended transmembrane Orai1 N-terminal (ETON, aa73-90) region, conserved among the Orai family forming an elongated helix of TM1 as recently shown by X-ray crystallography. In order to identify “hot spot”-residues in the ETON binding interface for STIM1interaction, numerous Orai1-constructs with N-terminal truncations or point mutations within the ETON region were generated. N-terminal truncations of the first four residues of the ETON region or beyond completely abolished STIM1dependent Orai1-function. Loss of Orai1function resulted from neither an impairment of plasma membrane targeting nor pore damage, but from a disruption of STIM1-interaction. Complementary we monitored STIM1-Orai-interaction via Orai1-V102A by determining restored Ca 2+ selectivity as a consequence of STIM1coupling. Orai1 N-terminal truncations that led to a loss of function consistently failed to restore Ca 2+ -selectivity of Orai1-V102A in the presence of STIM1 demonstrating impairment of STIM1-binding. Hence, the major portion of the ETON region (aa76-90) is essential for STIM1-binding and Orai1activation. Mutagenesis within the ETON region revealed several hydrophobic and basic “hot spot”-residues that appear to control STIM1-coupling to Orai1 in a concerted manner. Moreover, we identified two basic residues, which protrude into the elongated pore to redound to Orai1-gating. In aggregate, we suggest that several “hot spot”-residues in the ETON region contribute to the binding of STIM1, which in turn is coupled to a conformational reorientation of the gate. Introduction The CRAC signaling machinery consists of two key components, STIM1 (1,2) and Orai (35). STIM1 acts as an ER-located Ca sensor and mediates CRAC channel activation (1,2,6). After ER-Ca-store-depletion, STIM1 multimerizes and redistributes into punctae close to the plasma membrane (7,8). It http://www.jbc.org/cgi/doi/10.1074/jbc.M113.501510 The latest version is at JBC Papers in Press. Published on August 13, 2013 as Manuscript M113.501510 Copyright 2013 by The American Society for Biochemistry and Molecular Biology, Inc. by gest on M ay 2, 2016 hp://w w w .jb.org/ D ow nladed from by gest on M ay 2, 2016 hp://w w w .jb.org/ D ow nladed from by gest on M ay 2, 2016 hp://w w w .jb.org/ D ow nladed from by gest on M ay 2, 2016 hp://w w w .jb.org/ D ow nladed from by gest on M ay 2, 2016 hp://w w w .jb.org/ D ow nladed from by gest on M ay 2, 2016 hp://w w w .jb.org/ D ow nladed from by gest on M ay 2, 2016 hp://w w w .jb.org/ D ow nladed from by gest on M ay 2, 2016 hp://w w w .jb.org/ D ow nladed from by gest on M ay 2, 2016 hp://w w w .jb.org/ D ow nladed from by gest on M ay 2, 2016 hp://w w w .jb.org/ D ow nladed from by gest on M ay 2, 2016 hp://w w w .jb.org/ D ow nladed from by gest on M ay 2, 2016 hp://w w w .jb.org/ D ow nladed from by gest on M ay 2, 2016 hp://w w w .jb.org/ D ow nladed from by gest on M ay 2, 2016 hp://w w w .jb.org/ D ow nladed from