STIM1 couples to ORAI1 via an intramolecular transition into an extended conformationUpon depletion of ER calcium stores, STIM1 and ORAI1 associate and induce calcium release-activated calcium (CRAC) currents. This study reveals that STIM1 undergoes an intramolecular transition into an extended conformation that is involved in ORAI1 binding and activation.

Background: STIM1 and Orai1, reconstituting a main cellular Ca2+ entry pathway, interact via their cytosolic strands.Results: The extended transmembrane Orai1 N-terminal (ETON) region combines binding interface and gate for Orai1 activation by STIM1.Conclusion: Several “hot spot” residues in the ETON region mediate STIM1 interaction, enabling conformational reorientation of the gate.Significance: Identification of critical residues for protein-protein interaction are fundamental to therapeutic drug development.

Ca2+ release-activated Ca2+ (CRAC) channels constitute the major Ca2+ entry pathway into the cell. They are fully reconstituted via intermembrane coupling of the Ca2+-selective Orai channel and the Ca2+-sensing protein STIM1. In addition to the Orai C terminus, the main coupling site for STIM1, the Orai N terminus is indispensable for Orai channel gating. Although the extended transmembrane Orai N-terminal region (Orai1 amino acids 73–91; Orai3 amino acids 48–65) is fully conserved in the Orai1 and Orai3 isoforms, Orai3 tolerates larger N-terminal truncations than Orai1 in retaining store-operated activation. In an attempt to uncover the reason for these isoform-specific structural requirements, we analyzed a series of Orai mutants and chimeras. We discovered that it was not the N termini, but the loop2 regions connecting TM2 and TM3 of Orai1 and Orai3 that featured distinct properties, which explained the different, isoform-specific behavior of Orai N-truncation mutants. Atomic force microscopy studies and MD simulations suggested that the remaining N-terminal portion in the non-functional Orai1 N-truncation mutants formed new, inhibitory interactions with the Orai1-loop2 regions, but not with Orai3-loop2. Such a loop2 swap restored activation of the N-truncation Orai1 mutants. To mimic interactions between the N terminus and loop2 in full-length Orai1 channels, we induced close proximity of the N terminus and loop2 via cysteine cross-linking, which actually caused significant inhibition of STIM1-mediated Orai currents. In aggregate, maintenance of Orai activation required not only the conserved N-terminal region but also permissive communication of the Orai N terminus and loop2 in an isoform-specific manner.

Orai1 occurs via a physical interaction with the Ca2þ sensor protein STIM1 when ER Ca2þ stores are depleted. We have recently shown (Muik et al., 2011) that this coupling process correlates with a refolding of STIM1 into an extended conformation by utilizing a STIM1-derived FRET sensor. Furthermore, such extended conformations have been engineered by single point mutations within the cytosolic coiled -coil domains of STIM1. Hence intramolecular interaction domains are likely involved in keeping STIM1 in a resting, tight state. For further investigations, a new system called ''FRETbased Interactions in Restricted Environments (FIRE)'' was engineered for mapping such putative domains. This new developed system revealed not only a coiled-coil related shielding effect in the tight conformation of STIM1, but also a change in the accessibility of certain domains within STIM1 upon intramolecular transitions into an extended conformation. Hence, this new FIRE represents a promising tool for dissecting STIM1 into interaction domains responsible for STIM1 conformational states. (Supported by Austrian Science Fund (FWF): P22565) 1597-Pos Board B367 Gated Regulation of CRAC Channel Ion Selectivity by STIM1 Two defining functional features of ion channels are ion selectivity and channel gating. Ion selectivity is generally considered an immutable property of the open channel structure, whereas gating involves transitions between open and closed channel states typically without changes in ion selectivity.In store-operated Ca2þ release-activated Ca2þ (CRAC) channels, the molecular mechanism of channel gating by the CRAC channel activator, STIM1 (stromal interaction molecule 1) remains unknown. CRAC channels are distinguished by an extraordinarily high Ca2þ selectivity and are instrumental in generating sustained [Ca2þ]i elevations necessary for gene expression and effector function in many eukaryotic cells. Here, we probed the central features of the STIM1 gating mechanism in the CRAC channel protein, Orai1, and identified V102, a residue located in the extracellular region of the pore, as a candidate for the channel gate. Mutations at V102 produced constitutively active CRAC channels that were open even in the absence of STIM1. Although STIM1free V102 mutant channels were not Ca2þ-selective, their Ca2þ selectivity was dose-dependently increased by interactions with STIM1. Similar enhancement of Ca2þ selectivity also occurred in WT Orai1 channels by increasing the number of STIM1 activation domains directly tethered to Orai1 channels. Thus, exquisite Ca2þ selectivity is not an intrinsic property of CRAC channels, but rather a tunable feature bestowed on otherwise non-selective Orai1 channels by STIM1. Our results demonstrate that STIM1-mediated gating of CRAC channels occurs through an unusual mechanism wherein permeation and gating are closely coupled.

potassium (BK Ca ) channels and inducing membrane hyperpolarization and vasorelaxation. In Fluo-4-AM-loaded mesenteric myocytes, application of the Epac-specific cAMP analogue 8-pCPT-2'-O-Me-cAMP-AM (10mM) increased spark frequency from 0.045 5 0.008 sparks/s/mm under basal conditions to 0.103 5 0.022 sparks/s/mm (p<0.05). Importantly this increase also occurred in the presence of myristoylated PKI amide (14-22), a potent and selective inhibitor of PKA. Application of 8-pCPT-2'-O-Me-cAMP-AM (5mM) reversibly increased both the frequency (0.94 5 0.25 to 2.30 5 0.72 s À1 ) and amplitude (23.9 5 3.3 to 35.8 5 7.7 pA) of spontaneous transient outward currents (STOCs) recorded in isolated mesenteric myocytes (n=7; p<0.05). These currents were sensitive to the selective BK Ca channel blocker, iberiotoxin (100nM), and to ryanodine (30mM). In addition, current clamp recordings of isolated myocytes showed a 7.43 5 0.96 mV (n=4) hyperpolarization in response to exposure to 8-pCPT-2'-O-Me-cAMP-AM (5mM). Our data suggest a novel cAMP-dependent mechanism in mesenteric smooth muscle cells whereby activation of Epac facilitates localized Ca 2þ release which activates surface BK Ca channels to modulate membrane potential and vascular tone.

446-Pos Board B246The Relationship Between Ip 3 Level and the Frequency of Ca 2þ Oscillations Toru Matsu-ura, Sachiko Ishida, Takayiki Michikawa, Katsuhiko Mikoshiba. Many cellular stimuli induce oscillations of cytosolic calcium concentration ([Ca 2þ ]). Ca 2þ signals are generated by a cascade involving agonist binding to G protein-coupled receptor (GPCR), generation of inositol 1,4,5-trisphosphate (IP 3 ), release of Ca 2þ from endoplasmic reticulum through IP 3 receptor (IP 3 R). In the previous study, we have developed Förster resonance energy transfer based IP 3 sensors and monitored the IP 3 dynamics during Ca 2þ oscillations. In stimulated HeLa cells, IP 3 started to increase at a relatively constant rate before the pacemaker Ca 2þ rise, and IP 3 gradually accumulated in the cytosol with a little fluctuations during cytosolic Ca 2þ oscillations. In this study, we compared the relationship between IP 3 concentration ([IP 3 ]) and the frequency of Ca 2þ oscillations, and found they are well correlated but the relationships are different among the types of stimulated GPCRs. This result shows the frequency of Ca 2þ oscillations are not only determined by [IP 3 ]. To explain the result,