Fig 1: Graphical model. Graphical representation of the role of UNC93B1 as a chaperone that facilitates the early steps of STIM1 activation following ER Ca2+ depletion. UNC93B1 binds to STIM1 at rest and promotes the initial conformational step of its activation cascade, namely the zipping of the TM and proximal coiled-coil domains (dark blue bars) that cause STIM1 to adopt an extended conformation with an exposed channel-activating domain (red ovals) that efficiently traps and activates Orai1 channels (green) at the PM (WT, left panel). In the absence of UNC93B1, the maintenance of optimal STIM1–Orai1 coupling is lost (UNC93B1-/-, right panel). ER, endoplasmic reticulum; PM, plasma membrane; STIM1, stromal interaction molecule 1; TM, transmembrane.
Fig 2: Basal Synaptic Transmission, Circuit Activity, and Presynaptic Function Are Not Affected by Orai1 Deletion(A) Representative recordings of spontaneous EPSCs and IPSCs recorded from a CA1 pyramidal neuron. Inward EPSC currents were recorded at a holding potential of −70 mV, and outward IPSC currents were obtained in the same neuron by switching the holding potential to 0 mM mV.(B and C) Amplitude (B) and frequency of EPSCs in WT (Orai1fl/fl) and Orai1 KO (Orai1fl/− Nes-Cre) slices (n = 14 WT, n = 15 Orai1-KO, p = 0.25, p = 0.09).(D and E) Amplitude (D) and frequency (E) of IPSCs in WT and Orai1 KO slices.(F) Ratio of the amplitudes of EPSCs and IPSCs in WT and Orai1 KO neurons (n = 14 WT, n = 15 Orai1-KO, p = 0.65).(G) EPSC/IPSC frequency ratio in the indicated genotype (n = 14 WT, n = 15 Orai1-KO, p = 0.64).(H) Representative traces showing the evoked AMPAR (inward) and NMDAR (outward) currents in a CA1 pyramidal cell.(I) NMDAR/AMPAR current ratios (n = 17 WT, n = 15 Orai1-KO, p = 0.907).(J and K) Paired EPSCs evoked by stimulation at a 50-ms interval. Paired-pulse ratio (PPR) was calculated as the ratio of the peak current of the second EPSC relative to that of the first EPSC (n = 15 WT, n = 15 Orai1-KO, p = 0.706).(L) mEPSCs in WT (Orai1fl/fl) and Orai1 KO (Orai1fl/fl CaMKIIa-Cre) slices in 1 μM TTX.(M–P) Comparison of the mean amplitude (p = 0.558) and frequencies (p = 0.478) of mEPSCs in WT (n = 8) and Orai1 KO mice (n = 6) and representative cumulative distributions of the interevent intervalsand amplitudes. ns, not significant.
Fig 3: Deletion of Orai1 Impairs Synaptically Evoked Ca2+ Signals in Dendritic Spines(A) A section of a secondary apical dendrite of a hippocampal neuron expressing mCherry (red) and jGCaMP7f (yellow). MNI-Glu was uncaged at t = 5 s adjacent to the spine indicated by the arrowhead (4-ms pulse). Scale bar: 2 μm.(B) Dendritic spine jGCaMP7f fluorescence changes in response to Glu uncaging (4-ms pulses indicated by the arrows and vertical dotted lines) in neurons from WT (n = 8 cells, 67 spines) mice. The solid line shows the average of the individual spine Ca2+ responses (shown in lightly shaded lines).(C and D) The NMDAR and AMPAR antagonists, D-APV(100 μM) and NBQX(100 μM), respectively, block Glu-uncaging-evoked spine Ca2+ signals. (D-APV: n = 4 cells, 33 spines; NBQX: n = 5 cells, 42 spines).(E–G) Dendritic spine Ca2+ transients are strongly attenuated in Orai1 KO (Orai1fl/fl Nes-Cre) (n = 7 cells, 68 spines), Orai1fl/fl CaMKIIa-Cre (n = 7 cells, 73 spines), and heterozygous (Orai1fl/+ CaMKIIa-Cre) mice (n = 9 cells, 108 spines).(H) Cumulative distribution of spine Ca2+ responses. Loss of Orai1 increases the fraction of failures (i.e., no Ca2+ response to stimulation).(I–K) In the absence of extracellular Mg2+, dendritic spine Ca2+ signals recover significantly in Orai1 KO (Orai1fl/fl CaMKIIa-Cre) neurons (WT, n = 6 cells, 61 spines; KO n = 9 cells, 98 spines).(L) Expression of WT Orai1 into Orai1 KO (Orai1fl/fl CaMKIIa-Cre) neurons restores dendritic spine Ca2+ signals (n = 5 cells, 43 spines).(M) Expression of the non-conducting E106D Orai1 pore mutant abrogates synaptically evoked spine Ca2+ signals in WT neurons (n = 9 cells, 97 spines).(N) Summary of the peak ΔF/F0 changes (mean ± SEM) in the indicated conditions (**p < 0.01, ***p < 0.001 from WT Orai1).(O) Summary of the peak ΔF/F0 changes (mean ± SEM) in the absence of extracellular Mg2+ (**p = 0.0038).
Fig 4: siRNA-mediated inhibition of Orai1 and SARAF attenuates HUVEC tube formation, proliferation, and migration. (A) Fluorescence images (×4 objective; scale bar = 500 μm) and (B) summary data (% of meshes number normalized to scramble) obtained from HUVECs embedded on Matrigel and stained with Calcein-AM of control (white) and transfected with scramble (pink), siRNA Orai1 (green), and siRNA SARAF (orange) (n = 5 to 6). (C) Merged representative images (×20 objective; scale bar = 100 μm) of HUVECs stained with Ki67+ (red) and DAPI (blue) in control and in cells transfected with scramble, or siRNA Orai1 and SARAF (n = 5). (D) Bar graph shows the percentage of Ki67+ control (white bar) and transfected HUVECs with siOrai1 and siSARAF, normalized to scramble. (E) Phase-contrast imaging (×10 objective; scale bar = 200 μm) of the HUVEC wound healing assay modified with ImageJ. Images were taken at 0, 12, and 24 h after the scratch from control HUVECs and form cells transfected with scramble siRNA, and siRNA against Orai1 and SARAF. (F) Graph shows summary data of the evolution of the wound area in experiments as in (E) (n = 4). Values are presented as the means ± S.E.M. Significance is indicated by (*) and (**) for p < 0.05 and p < 0.01, respectively.
Fig 5: Selective Deletion of Orai1 in Excitatory Neurons Also Impairs Learning and Memory(A) Summary of the spontaneous alternations in the Y-maze for male and female WT (Orai1fl/fl) and Orai1 KO (Orai1fl/fl CaMKIIa-Cre) mice. Male mice: n = 10 WT; n = 8 Orai1 KO. p = 0.018. Female mice: n = 14 WT; n = 9 Orai1 KO. p = 0.00589. Dashed line denotes chance (50% alternation).(B) Summary of the freezing behaviors of WT (Orai1fl/fl) and Orai1 KO (Orai1fl/fl CaMKIIa-Cre) mice in the fear-conditioning protocol. Fear conditioning was quantified using Freezeframe software. Male mice: n = 8 WT; n = 5 Orai1 KO. p values are as follows: context2, p = 0.946; tone, p = 0.0028; trace, p = 0.0044; context1, p = 0.00331. Female mice: n = 6 WT; n = 5 Orai1 KO. p values are as follows: context2, p = 0.795; tone, p = 0.0081; trace, p = 0.0288; context1, p = 0.00647.(C) Y-maze in Gad2-Cre Orai1 KOs. Summary of the alternations in WT (Orai1fl/fl) and inhibitory neuron-specific Orai1 KOs (Orai1fl/fl Gad2-Cre) mice. Male mice: n = 10 WT; n = 6 Orai1 KO. p = 0.854. Female mice: n = 14 WT; n = 7 Orai1 KO, p = 0.816.(D) Summary of the freezing behavior in the fear-conditioning protocol in WT (Orai1fl/fl) and inhibitory neuron-specific (Orai1fl/fl Gad2-Cre) mice. Male mice: n = 8WT; n = 6 Orai1 KO. p values are as follows: context2, p = 0.551; tone, p = 0.1905; trace, p = 0.446; context1, p = 0.316. Female mice: n = 6WT; n = 6 Orai1 KO. p values are as follows: context2, p = 0.335; tone, p = 0.0775; trace, p = 0.2661; context1, p = 0.931.
Supplier Page from Abcam for Anti-Orai1 antibody [266.1]