Abstract
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.
Highlights
STIM1 and Orai1, reconstituting a main cellular Ca2ϩ entry pathway, interact via their cytosolic strands
We suggest that several hot spot residues in the extended transmembrane Orai1 N-terminal (ETON) region contribute in aggregate to the binding of STIM1, which in turn is coupled to a conformational reorientation of the gate
For a thorough evaluation of the impact of this ETON region on STIM1 binding and Orai1 activation, we initially generated several Orai1 mutants with increasing N-terminal deletions in an attempt to uncover the minimal stretch of the ETON region that retains store-operated activation
Summary
STIM1 and Orai, reconstituting a main cellular Ca2ϩ entry pathway, interact via their cytosolic strands. Gating Determinants within the N Terminus of Orai glycine hinge (Gly-98) [18], which may enable flexion of the upstream pore-lining region to reduce the impedance of Ca2ϩ flow after passing the selectivity filter [16] This part of the cytosolic N-terminal strand upstream of the first transmembrane helix (TM1) forms a helical [19], extended transmembrane Orai N-terminal (ETON) region that comprises the N-terminal residues aa, which are fully conserved among the three human homologues of Orai proteins and protrudes about 20 Å into the cytosol [16]. The arginine Arg-91 inhibits store-operated current activation upon its mutation to a hydrophobic residue [20, 21] This barrier of the three positively charged residues must be released to let Ca2ϩ pass into the cell, which may be accomplished by an interaction of STIM1 with the conserved ETON regions forming the elongated pore [16]. It contains electrostatic gating elements that fine-tune and shape the elongated pore for controlled STIM1-dependent Ca2ϩ entry
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