The extended transmembrane Orai1 N-terminal (ETON) region combines binding interface and gate for Orai1 activation by STIM1.
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 the N terminus and the C terminus of Orai. 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. To identify "hot spot" residues in the ETON binding interface for STIM1 interaction, 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 STIM1-dependent Orai1 function. Loss of Orai1 function resulted from neither an impairment of plasma membrane targeting nor pore damage, but from a disruption of STIM1 interaction. In a complementary approach, we monitored STIM1-Orai interaction via Orai1 V102A by determining restored Ca(2+) selectivity as a consequence of STIM1 coupling. 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 Orai1 activation. 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. We suggest that several hot spot residues in the ETON region contribute in aggregate to the binding of STIM1, which in turn is coupled to a conformational reorientation of the gate.
From the Institute of Biophysics, Johannes Kepler University of Linz, Gruberstrasse 40, 4020 Linz and.
Isabella
I
Derler
Peter
P
Plenk
Marc
M
Fahrner
Martin
M
Muik
Isaac
I
Jardin
Rainer
R
Schindl
Hermann J
HJ
Gruber
Klaus
K
Groschner
Christoph
C
Romanin
D000200:Action Potentials; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D001665:Binding Sites; D015220:Calcium Channels / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D015640:Ion Channel Gating; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000378:metabolism; D009363:Neoplasm Proteins / Q000378:metabolism*; D000071740:ORAI1 Protein; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017384:Sequence Deletion / Q000235:genetics; D000071737:Stromal Interaction Molecule 1; D013329:Structure-Activity Relationship
D000596:Amino Acids; D015220:Calcium Channels; D008565:Membrane Proteins; D050505:Mutant Proteins; D009363:Neoplasm Proteins; D000071740:ORAI1 Protein; C510746:ORAI1 protein, human; C103530:STIM1 protein, human; D000071737:Stromal Interaction Molecule 1
Calcium Channels; Calcium Intracellular Release; Calcium Signaling; Electrophysiology; Ion Channels