Xiangyu Cai

STIM1 dimers undergo unimolecular coupling to activate Orai1 channels

The endoplasmic reticulum (ER) Ca2+ sensor, STIM1, becomes activated when ER-stored Ca2+ is depleted and translocates into ER–plasma membrane junctions where it tethers and activates Orai1 Ca2+ entry channels. The dimeric STIM1 protein contains a small STIM-Orai-activating region (SOAR)—the minimal sequence sufficient to activate Orai1 channels. Since SOAR itself is a dimer, we constructed SOAR concatemer–dimers and introduced mutations at F394, which is critical for Orai1 coupling and activation. The F394H mutation in both SOAR monomers completely blocks dimer function, but F394H introduced in only one of the dimeric SOAR monomers has no effect on Orai1 binding or activation. This reveals an unexpected unimolecular coupling between STIM1 and Orai1 and argues against recent evidence suggesting dimeric interaction between STIM1 and two adjacent Orai1 channel subunits. The model predicts that STIM1 dimers may be involved in crosslinking between Orai1 channels with implications for the kinetics and localization of Orai1 channel opening.

The Orai1 Store-operated Calcium Channel Functions as a Hexamer

Orai channels mediate store-operated Ca2+ signals crucial in regulating transcription in many cell types, and implicated in numerous immunological and inflammatory disorders. Despite their central importance, controversy surrounds the basic subunit structure of Orai channels, with several biochemical and biophysical studies suggesting a tetrameric structure yet crystallographic evidence indicating a hexamer. We systematically investigated the subunit configuration of the functional Orai1 channel, generating a series of tdTomato-tagged concatenated Orai1 channel constructs (dimers to hexamers) expressed in CRISPR-derived ORAI1 knock-out HEK cells, stably expressing STIM1-YFP. Surface biotinylation demonstrated that the full-length concatemers were surface membrane-expressed. Unexpectedly, Orai1 dimers, trimers, tetramers, pentamers, and hexamers all mediated similar and substantial store-operated Ca2+ entry. Moreover, each Orai1 concatemer mediated Ca2+ currents with inward rectification and reversal potentials almost identical to those observed with expressed Orai1 monomer. In Orai1 tetramers, subunit-specific replacement with Orai1 E106A “pore-inactive” subunits revealed that functional channels utilize only the N-terminal dimer from the tetramer. In contrast, Orai1 E106A replacement in Orai1 hexamers established that all the subunits can contribute to channel formation, indicating a hexameric channel configuration. The critical Ca2+ selectivity filter-forming Glu-106 residue may mediate Orai1 channel assembly around a central Ca2+ ion within the pore. Thus, multiple E106A substitutions in the Orai1 hexamer may promote an alternative “trimer-of-dimers” channel configuration in which the C-terminal E106A subunits are excluded from the hexameric core. Our results argue strongly against a tetrameric configuration for Orai1 channels and indicate that the Orai1 channel functions as a hexamer.

Protein Kinase D3 Is a Pivotal Activator of Pathological Cardiac Hypertrophy by Selectively Increasing the Expression of Hypertrophic Transcription Factors

Background: Post-translational modulation of preexisting translational factors is a well-established cardiac hypertrophic mechanism. Results: The abundance of NFATc4, Nkx2.5 and GATA4 is up-regulated by newly-expressed protein kinase D3 (PKD3) to induce pathological cardiac hypertrophy. Conclusion: PKD3 is a pivotal mediator of cardiac hypertrophic signaling cascade. Significance: PKD3 is a potential new drug target for pathological cardiac hypertrophy. Fetal cardiac gene reactivation is a hallmark of pathological cardiac hypertrophy (PCH) driven by cardiac transcription factors (TFs) such as nuclear factor of activated T-cells (NFATs). Nuclear import of dephosphorylated NFATs catalyzed by calcineurin (CaN) is a well-established hypertrophic mechanism. Here we report that NFATc4 expression is also up-regulated by newly expressed protein kinase D3 (PKD3) to induce PCH. In both in vitro and in vivo cardiac hypertrophic models, the normally undetectable PKD3 was profoundly up-regulated by isoproterenol followed by overt expression of cardiac TFs including NFATc4, NK family of transcription factor 2.5 (Nkx2.5), GATA4 and myocyte enhancer factor 2 (MEF2). Using gene silencing approaches, we demonstrate PKD3 is required for increasing the expression of NFATc4, Nkx2.5, and GATA4 while PKD1 is required for the increase in MEF2D expression. Upstream induction of PKD3 is driven by nuclear entry of CaN-activated NFATc1 and c3 but not c4. Therefore, PKD3 is a pivotal mediator of the CaN-NFATc1/c3-PKD3-NFATc4 hypertrophic signaling cascade and a potential new drug target for the PCH.

The δA isoform of calmodulin kinase II mediates pathological cardiac hypertrophy by interfering with the HDAC4-MEF2 signaling pathway

Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is a new promising target for prevention and treatment of cardiac hypertrophy and heart failure. There are three δ isoforms of CaMKII in the heart and previous studies focused primarily on δB and δC types. Here we report the δA isoform of CaMKII is also critically involved in cardiac hypertrophy. We found that δA was significantly upregulated in pathological cardiac hypertrophy in both neonatal and adult models. Upregulation of δA was accompanied by cell enlargement, sarcomere reorganization and reactivation of various hypertrophic cardiac genes including atrial natriuretic factor (ANF) and β-myocin heavy chain (β-MHC). Studies further indicated the pathological changes were largely blunted by silencing the δA gene and an underlying mechanism indicated selective interference with the HDAC4-MEF2 signaling pathway. These results provide new evidence for selective interfering cardiac hypertrophy and heart failure when CaMKII is considered as a therapeutic target.

CpG-ODN Attenuates Pathological Cardiac Hypertrophy and Heart Failure by Activation of PI3Kα-Akt Signaling

Phosphoinositide-3-kinase α (PI3Kα) represents a potential novel drug target for pathological cardiac hypertrophy (PCH) and heart failure. Oligodeoxynucleotides containing CpG motifs (CpG-ODN) are classic agonists of Toll-like receptor 9 (TLR9), which typically activates PI3K-Akt signaling in immune cells; however, the role of the nucleotide TLR9 agonists in cardiac myocytes is largely unknown. Here we report that CpG-ODN C274 could both attenuate PCH and improve cardiac dysfunction by activating PI3Kα-Akt signaling cascade. In vitro studies indicated that C274 could blunt reactivation of fetal cardiac genes and cell enlargement induced by a hypertrophic agent, isoproterenol. The anti-hypertrophic effect of C274 was suppressed by a pan-PI3K inhibitor, LY294002, or a small interfering RNA targeting PI3Kα. In vivo studies demonstrated that PCH, as marked by increased heart weight (HW) and cardiac ANF mRNA, was normalized by pre-administration with C274. In addition, Doppler echocardiography detected cardiac ventricular dilation, and contractile dysfunction in isoproterenol-treated animals, consistent with massive replacement fibrosis, reflecting cardiac cell death. As expected, pre-treatment of mice with C274 could prevent cardiac dysfunction associated with diminished cardiac cell death and fibrosis. In conclusion, CpG-ODNs are novel cardioprotective agents possessing antihypertrophic and anti-cell death activity afforded by engagement of the PI3Kα-Akt signaling. CpG-ODNs may have clinical use curbing the progression of PCH and preventing heart failure.

Cross-linking of Orai1 channels by STIM proteins

Significance The work presents a unique understanding of the organization and function of two ubiquitously expressed proteins, central in generating calcium signals in all cell types. These are the intracellular calcium sensing “STIM” proteins, and the highly selective cell surface “Orai” calcium channels. We reveal that STIM proteins can cross-link Orai channels, resulting in a reorganized microenvironment within the membrane junctions in which they function, with important consequences in the generation of oscillatory calcium signals. Interestingly, we show a variant STIM protein widely expressed in cells functions to prevent the STIM–Orai cross-linking and clustering of channels. This provides important modulation of calcium signal generation and can serve to protect cells from overstimulation of the calcium signaling machinery. The transmembrane docking of endoplasmic reticulum (ER) Ca2+-sensing STIM proteins with plasma membrane (PM) Orai Ca2+ channels is a critical but poorly understood step in Ca2+ signal generation. STIM1 protein dimers unfold to expose a discrete STIM–Orai activating region (SOAR1) that tethers and activates Orai1 channels within discrete ER–PM junctions. We reveal that each monomer within the SOAR dimer interacts independently with single Orai1 subunits to mediate cross-linking between Orai1 channels. Superresolution imaging and mobility measured by fluorescence recovery after photobleaching reveal that SOAR dimer cross-linking leads to substantial Orai1 channel clustering, resulting in increased efficacy and cooperativity of Orai1 channel function. A concatenated SOAR1 heterodimer containing one monomer point mutated at its critical Orai1 binding residue (F394H), although fully activating Orai channels, is completely defective in cross-linking Orai1 channels. Importantly, the naturally occurring STIM2 variant, STIM2.1, has an eight-amino acid insert in its SOAR unit that renders it functionally identical to the F394H mutant in SOAR1. Contrary to earlier predictions, the SOAR1–SOAR2.1 heterodimer fully activates Orai1 channels but prevents cross-linking and clustering of channels. Interestingly, combined expression of full-length STIM1 with STIM2.1 in a 5:1 ratio causes suppression of sustained agonist-induced Ca2+ oscillations and protects cells from Ca2+ overload, resulting from high agonist-induced Ca2+ release. Thus, STIM2.1 exerts a powerful regulatory effect on signal generation likely through preventing Orai1 channel cross-linking. Overall, STIM-mediated cross-linking of Orai1 channels is a hitherto unrecognized functional paradigm that likely provides an organizational microenvironment within ER–PM junctions with important functional impact on Ca2+ signal generation.