Local cytosolic Ca2+ elevations are required for stromal interaction molecule 1 (STIM1) de-oligomerization and termination of store-operated Ca2+ entry

The Ca(2+) depletion of the endoplasmic reticulum (ER) activates the ubiquitous store-operated Ca(2+) entry (SOCE) pathway that sustains long-term Ca(2+) signals critical for cellular functions. ER Ca(2+) depletion initiates the oligomerization of stromal interaction molecules (STIM) that control SOCE activation, but whether ER Ca(2+) refilling controls STIM de-oligomerization and SOCE termination is not known. Here, we correlate the changes in free luminal ER Ca(2+) concentrations ([Ca(2+)](ER)) and in STIM1 oligomerization, using fluorescence resonance energy transfer (FRET) between CFP-STIM1 and YFP-STIM1. We observed that STIM1 de-oligomerized at much lower [Ca(2+)](ER) levels during store refilling than it oligomerized during store depletion. We then refilled ER stores without adding exogenous Ca(2+) using a membrane-permeable Ca(2+) chelator to provide a large reservoir of buffered Ca(2+). This procedure rapidly restored pre-stimulatory [Ca(2+)](ER) levels but did not trigger STIM1 de-oligomerization, the FRET signals remaining elevated as long as the external [Ca(2+)] remained low. STIM1 dissociation evoked by Ca(2+) readmission was prevented by SOC channel inhibition and was associated with cytosolic Ca(2+) elevations restricted to STIM1 puncta, indicating that Ca(2+) acts on a cytosolic target close to STIM1 clusters. These data indicate that the refilling of ER Ca(2+) stores is not sufficient to induce STIM1 de-oligomerization and that localized Ca(2+) elevations in the vicinity of assembled SOCE complexes are required for the termination of SOCE.     

Epstein-Barr virus latent membrane protein 1 increases calcium influx through store-operated channels in B lymphoid cells

Ca(2+) signaling plays an important role in B cell survival and activation and is dependent on Ca(2+) trapped in the endoplasmic reticulum (ER) and on extracellular Ca(2+). Epstein-Barr virus (EBV) can immortalize B cells and contributes to lymphomagenesis. Previously, we showed that the ER Ca(2+) content of Burkitt lymphoma cell lines was increased following infection with immortalization-competent virus expressing the full set of EBV latency genes (B95-8). In contrast, infection with an immortalization-deficient virus (P3HR-1) not expressing LMP-1 is without effect. LMP-1 protein expression was sufficient to increase the ER Ca(2+) content and to increase the cytosolic Ca(2+) concentration ([Ca(2+)](cyt)). In this follow-up study, we showed that the resting [Ca(2+)](cyt) of P3HR-1-infected cells was decreased, implying that EBV not only modified the ER homeostasis but also affected the cytosolic Ca(2+) homeostasis. Furthermore, even if the store-operated calcium entry (SOCE) of these cells was normal, the [Ca(2+)](cyt) increase after thapsigargin + CaCl(2) stimulation was blunted. In contrast, the resting [Ca(2+)](cyt) of B95-8 infected cells was not changed, even if their SOCE was increased significantly. When expressed alone, LMP-1 induced an increase of the SOCE amplitude and the expression of the protein allowing this influx, Orai1, showing the effect of EBV on SOCE of B cells are mediated by LMP-1. However, other hitherto unidentified EBV processes, unmasked in P3HR-1 infected cells, counteract this LMP-1-dependent increase of SOCE amplitude to impair a general and potentially toxic increase of [Ca(2+)](i). Thus, EBV infection modifies the cellular Ca(2+) homeostasis by acting on the ER and plasma membrane transporters.     

Endoplasmic reticulum Ca2+ release modulates endothelial nitric-oxide synthase via extracellular signal-regulated kinase (ERK) 1/2-mediated serine 635 phosphorylation

Endothelial nitric-oxide synthase (eNOS) plays a central role in cardiovascular regulation. eNOS function is critically modulated by Ca(2+) and protein phosphorylation, but the interrelationship between intracellular Ca(2+) mobilization and eNOS phosphorylation is poorly understood. Here we show that endoplasmic reticulum (ER) Ca(2+) release activates eNOS by selectively promoting its Ser-635/633 (bovine/human) phosphorylation. With bovine endothelial cells, thapsigargin-induced ER Ca(2+) release caused a dose-dependent increase in eNOS Ser-635 phosphorylation, leading to elevated NO production. ER Ca(2+) release also promoted eNOS Ser-633 phosphorylation in mouse vessels in vivo. This effect was independent of extracellular Ca(2+) and selective to Ser-635 because the phosphorylation status of other eNOS sites, including Ser-1179 or Thr-497, was unaffected in thapsigargin-treated cells. Blocking ERK1/2 abolished ER Ca(2+) release-induced eNOS Ser-635 phosphorylation, whereas inhibiting protein kinase A or Ca(2+)/calmodulin-dependent protein kinase II had no effect. Protein phosphorylation assay confirmed that ERK1/2 directly phosphorylated the eNOS Ser-635 residue in vitro. Further studies demonstrated that ER Ca(2+) release-induced ERK1/2 activation mediated the enhancing action of purine or bradykinin receptor stimulation on eNOS Ser-635/633 phosphorylation in bovine/human endothelial cells. Mutating the Ser-635 to nonphosphorylatable alanine prevented ATP from activating eNOS in cells. Taken together, these studies reveal that ER Ca(2+) release enhances eNOS Ser-635 phosphorylation and function via ERK1/2 activation. Because ER Ca(2+) is commonly mobilized by agonists or physicochemical stimuli, the identified ER Ca(2+)-ERK1/2-eNOS Ser-635 phosphorylation pathway may have a broad role in the regulation of endothelial function.     

Modulation of Endoplasmic Reticulum Ca2+ Store Filling by Cyclic ADP-ribose Promotes Inositol Trisphosphate (IP3)-evoked Ca2+ Signals

In addition to its well established function in activating Ca²⁺ release from the endoplasmic reticulum (ER) through ryanodine receptors (RyR), the second messenger cyclic ADP-ribose (cADPR) also accelerates the activity of SERCA pumps, which sequester Ca²⁺ into the ER. Here, we demonstrate a potential physiological role for cADPR in modulating cellular Ca²⁺ signals via changes in ER Ca²⁺ store content, by imaging Ca²⁺ liberation through inositol trisphosphate receptors (IP₃R) in Xenopus oocytes, which lack RyR. Oocytes were injected with the non-metabolizable analog 3-deaza-cADPR, and cytosolic [Ca²⁺] was transiently elevated by applying voltage-clamp pulses to induce Ca²⁺ influx through expressed plasmalemmal nicotinic channels. We observed a subsequent potentiation of global Ca²⁺ signals evoked by strong photorelease of IP₃, and increased numbers of local Ca²⁺ puffs evoked by weaker photorelease. These effects were not evident with cADPR alone or following cytosolic Ca²⁺ elevation alone, indicating that they did not arise through direct actions of cADPR or Ca²⁺ on the IP₃R, but likely resulted from enhanced ER store filling. Moreover, the appearance of a new population of puffs with longer latencies, prolonged durations, and attenuated amplitudes suggests that luminal ER Ca²⁺ may modulate IP₃R function, in addition to simply determining the size of the available store and the electrochemical driving force for release.     

Calsequestrin accumulation in rough endoplasmic reticulum promotes perinuclear Ca2+ release

Molecular mechanisms underlying Ca(2+) regulation by perinuclear endoplasmic/sarcoplasmic reticulum (ER/SR) cisternae in cardiomyocytes remain obscure. To investigate the mechanisms of changes in cardiac calsequestrin (CSQ2) trafficking on perinuclear Ca(2+) signaling, we manipulated the subcellular distribution of CSQ2 by overexpression of CSQ2-DsRed, which specifically accumulates in the perinuclear rough ER. Adult ventricular myocytes were infected with adenoviruses expressing CSQ2-DsRed, CSQ2-WT, or empty vector. We found that perinuclear enriched CSQ2-DsRed, but not normally distributed CSQ2-WT, enhanced nuclear Ca(2+) transients more potently than cytosolic Ca(2+) transients. Overexpression of CSQ2-DsRed produced more actively propagating Ca(2+) waves from perinuclear regions than did CSQ2-WT. Activities of the SR/ER Ca(2+)-ATPase and ryanodine receptor type 2, but not inositol 1,4,5-trisphosphate receptor type 2, were required for the generation of these perinuclear initiated Ca(2+) waves. In addition, CSQ2-DsRed was more potent than CSQ2-WT in inducing cellular hypertrophy in cultured neonatal cardiomyocytes. Our data demonstrate for the first time that CSQ2 retention in the rough ER/perinuclear region promotes perinuclear Ca(2+) signaling and predisposes to ryanodine receptor type 2-mediated Ca(2+) waves from CSQ2-enriched perinuclear compartments and myocyte hypotrophy. These findings provide new insights into the mechanism of CSQ2 in Ca(2+) homeostasis, suggesting that rough ER-localized Ca(2+) stores can operate independently in raising levels of cytosolic/nucleoplasmic Ca(2+) as a source of Ca(2+) for Ca(2+)-dependent signaling in health and disease.     

Plasma Membrane Ca2+-ATPase Overexpression Depletes Both Mitochondrial and Endoplasmic Reticulum Ca2+ Stores and Triggers Apoptosis in Insulin-secreting BRIN-BD11 Cells

Ca²⁺ may trigger apoptosis in β-cells. Hence, the control of intracellular Ca²⁺ may represent a potential approach to prevent β-cell apoptosis in diabetes. Our objective was to investigate the effect and mechanism of action of plasma membrane Ca²⁺-ATPase (PMCA) overexpression on Ca²⁺-regulated apoptosis in clonal β-cells. Clonal β-cells (BRIN-BD11) were examined for the effect of PMCA overexpression on cytosolic and mitochondrial [Ca²⁺] using a combination of aequorins with different Ca²⁺ affinities and on the ER and mitochondrial pathways of apoptosis. β-cell stimulation generated microdomains of high [Ca²⁺] in the cytosol and subcellular heterogeneities in [Ca²⁺] among mitochondria. Overexpression of PMCA decreased [Ca²⁺] in the cytosol, the ER, and the mitochondria and activated the IRE1α-XBP1s but inhibited the PRKR-like ER kinase-eIF2α and the ATF6-BiP pathways of the ER-unfolded protein response. Increased Bax/Bcl-2 expression ratio was observed in PMCA overexpressing β-cells. This was followed by Bax translocation to the mitochondria with subsequent cytochrome c release, opening of the permeability transition pore, and apoptosis. In conclusion, clonal β-cell stimulation generates microdomains of high [Ca²⁺] in the cytosol and subcellular heterogeneities in [Ca²⁺] among mitochondria. PMCA overexpression depletes intracellular [Ca²⁺] stores and, despite a decrease in mitochondrial [Ca²⁺], induces apoptosis through the mitochondrial pathway. These data open the way to new strategies to control cellular Ca²⁺ homeostasis that could decrease β-cell apoptosis in diabetes.     

Ablation of skeletal muscle triadin impairs FKBP12/RyR1 channel interactions essential for maintaining resting cytoplasmic Ca2+

Previously, we have shown that lack of expression of triadins in skeletal muscle cells results in significant increase of myoplasmic resting free Ca(2+) ([Ca(2+)](rest)), suggesting a role for triadins in modulating global intracellular Ca(2+) homeostasis. To understand this mechanism, we study here how triadin alters [Ca(2+)](rest), Ca(2+) release, and Ca(2+) entry pathways using a combination of Ca(2+) microelectrodes, channels reconstituted in bilayer lipid membranes (BLM), Ca(2+), and Mn(2+) imaging analyses of myotubes and RyR1 channels obtained from triadin-null mice. Unlike WT cells, triadin-null myotubes had chronically elevated [Ca(2+)](rest) that was sensitive to inhibition with ryanodine, suggesting that triadin-null cells have increased basal RyR1 activity. Consistently, BLM studies indicate that, unlike WT-RyR1, triadin-null channels more frequently display atypical gating behavior with multiple and stable subconductance states. Accordingly, pulldown analysis and fluorescent FKBP12 binding studies in triadin-null muscles revealed a significant impairment of the FKBP12/RyR1 interaction. Mn(2+) quench rates under resting conditions indicate that triadin-null cells also have higher Ca(2+) entry rates and lower sarcoplasmic reticulum Ca(2+) load than WT cells. Overexpression of FKBP12.6 reverted the null phenotype, reducing resting Ca(2+) entry, recovering sarcoplasmic reticulum Ca(2+) content levels, and restoring near normal [Ca(2+)](rest). Exogenous FKBP12.6 also reduced the RyR1 channel P(o) but did not rescue subconductance behavior. In contrast, FKBP12 neither reduced P(o) nor recovered multiple subconductance gating. These data suggest that elevated [Ca(2+)](rest) in triadin-null myotubes is primarily driven by dysregulated RyR1 channel activity that results in part from impaired FKBP12/RyR1 functional interactions and a secondary increased Ca(2+) entry at rest.     

Preferential targeting of a signal recognition particle-dependent precursor to the Ssh1p translocon in yeast

Protein translocation across the endoplasmic reticulum membrane occurs via a "translocon" channel formed by the Sec61p complex. In yeast, two channels exist: the canonical Sec61p channel and a homolog called Ssh1p. Here, we used trapped translocation intermediates to demonstrate that a specific signal recognition particle-dependent substrate, Sec71p, is targeted exclusively to Ssh1p. Strikingly, we found that, in the absence of Ssh1p, precursor could be successfully redirected to canonical Sec61p, demonstrating that the normal targeting reaction must involve preferential sorting to Ssh1p. Our data therefore demonstrate that Ssh1p is the primary translocon for Sec71p and reveal a novel sorting mechanism at the level of the endoplasmic reticulum membrane enabling precursors to be directed to distinct translocons. Interestingly, the Ssh1p-dependent translocation of Sec71p was found to be dependent upon Sec63p, demonstrating a previously unappreciated functional interaction between Sec63p and the Ssh1p translocon.     

Deregulation of microRNAs by HIV-1 Vpr protein leads to the development of neurocognitive disorders

Studies have shown that HIV-infected patients develop neurocognitive disorders characterized by neuronal dysfunction. The lack of productive infection of neurons by HIV suggests that viral and cellular proteins, with neurotoxic activities, released from HIV-1-infected target cells can cause this neuronal deregulation. The viral protein R (Vpr), a protein encoded by HIV-1, has been shown to alter the expression of various important cytokines and inflammatory proteins in infected and uninfected cells; however the mechanisms involved remain unclear. Using a human neuronal cell line, we found that Vpr can be taken up by neurons causing: (i) deregulation of calcium homeostasis, (ii) endoplasmic reticulum-calcium release, (iii) activation of the oxidative stress pathway, (iv) mitochondrial dysfunction and v- synaptic retraction. In search for the cellular factors involved, we performed microRNAs and gene array assays using human neurons (primary cultures or cell line, SH-SY5Y) that we treated with recombinant Vpr proteins. Interestingly, Vpr deregulates the levels of several microRNAs (e.g. miR-34a) and their target genes (e.g. CREB), which could lead to neuronal dysfunctions. Therefore, we conclude that Vpr plays a major role in neuronal dysfunction through deregulating microRNAs and their target genes, a phenomenon that could lead to the development of neurocognitive disorders.     

Endoplasmic Reticulum (ER) Localization Is Critical for DsbA-L Protein to Suppress ER Stress and Adiponectin Down-regulation in Adipocytes

Adiponectin is an adipokine with insulin-sensitizing and anti-inflammatory functions. We previously reported that adiponectin multimerization and stability are promoted by the disulfide bond A oxidoreductase-like protein (DsbA-L) in cells and in vivo. However, the precise mechanism by which DsbA-L regulates adiponectin biosynthesis remains elusive. Here we show that DsbA-L is co-localized with the endoplasmic reticulum (ER) marker protein disulfide isomerase and the mitochondrial marker MitoTracker. In addition, DsbA-L interacts with the ER chaperone protein Ero1-Lα in 3T3-L1 adipocytes. In silico analysis and truncation mapping studies revealed that DsbA-L contains an ER targeting signal at its N terminus. Deletion of the first 6 residues at the N terminus greatly impaired DsbA-L localization in the ER. Overexpression of the wild type but not the ER localization-defective mutant of DsbA-L protects against thapsigargin-induced ER stress and adiponectin down-regulation in 3T3-L1 adipocytes. In addition, overexpression of the wild type but not the ER localization-defective mutant of DsbA-L promotes adiponectin multimerization. Together, our results reveal that DsbA-L is localized in both the mitochondria and the ER in adipocytes and that its ER localization plays a critical role in suppressing ER stress and promoting adiponectin biosynthesis and secretion.     

Beta-subunits promote the expression of Ca(V)2.2 channels by reducing their proteasomal degradation

The β-subunits of voltage-gated calcium channels regulate their functional expression and properties. Two mechanisms have been proposed for this, an effect on gating and an enhancement of expression. With respect to the effect on expression, β-subunits have been suggested to enhance trafficking by masking an unidentified endoplasmic reticulum (ER) retention signal. Here we have investigated whether, and how, β-subunits affect the level of Ca(V)2.2 channels within somata and neurites of cultured sympathetic neurons. We have used YFP-Ca(V)2.2 containing a mutation (W391A), that prevents binding of β-subunits to its I-II linker and found that expression of this channel was much reduced compared with WT CFP-Ca(V)2.2 when both were expressed in the same neuron. This effect was particularly evident in neurites and growth cones. The difference between the levels of YFP-Ca(V)2.2(W391A) and CFP-Ca(V)2.2(WT) was lost in the absence of co-expressed β-subunits. Furthermore, the relative reduction of expression of Ca(V)2.2(W391A) compared with the WT channel was reversed by exposure to two proteasome inhibitors, MG132 and lactacystin, particularly in the somata. In further experiments in tsA-201 cells, we found that proteasome inhibition did not augment the cell surface Ca(V)2.2(W391A) level but resulted in the observation of increased ubiquitination, particularly of mutant channels. In contrast, we found no evidence for selective retention of Ca(V)2.2(W391A) in the ER, in either the soma or growth cones. In conclusion, there is a marked effect of β-subunits on Ca(V)2.2 expression, particularly in neurites, but our results point to protection from proteasomal degradation rather than masking of an ER retention signal.     

TMTC1 and TMTC2 Are Novel Endoplasmic Reticulum Tetratricopeptide Repeat-containing Adapter Proteins Involved in Calcium Homeostasis

The endoplasmic reticulum (ER) is organized in part by adapter proteins that nucleate the formation of large protein complexes. Tetratricopeptide repeats (TPR) are well studied protein structural motifs that support intermolecular protein-protein interactions. TMTC1 and TMTC2 were identified by an in silico search as TPR-containing proteins possessing N-terminal ER targeting signal sequences and multiple hydrophobic segments, suggestive of polytopic membrane proteins that are targeted to the secretory pathway. A variety of cell biological and biochemical assays was employed to demonstrate that TMTC1 and TMTC2 are both ER resident integral membrane proteins with multiple clusters of TPR domains oriented within the ER lumen. Proteomic analysis followed by co-immunoprecipitation verification found that both proteins associated with the ER calcium uptake pump SERCA2B, and TMTC2 also bound to the carbohydrate-binding chaperone calnexin. Live cell calcium measurements revealed that overexpression of either TMTC1 or TMTC2 caused a reduction of calcium released from the ER following stimulation, whereas the knockdown of TMTC1 or TMTC2 increased the stimulated calcium released. Together, these results implicate TMTC1 and TMTC2 as ER proteins involved in ER calcium homeostasis.     

Structural and Functional Profiling of the Lateral Gate of the Sec61 Translocon

The evolutionarily conserved Sec61 translocon mediates the translocation and membrane insertion of proteins. For the integration of proteins into the membrane, the Sec61 translocon opens laterally to the lipid bilayer. Previous studies suggest that the lateral opening of the channel is mediated by the helices TM2b and TM7 of a pore-forming subunit of the Sec61 translocon. To map key residues in TM2b and TM7 in yeast Sec61 that modulate lateral gating activity, we performed alanine scanning and in vivo site-directed photocross-linking experiments. Alanine scanning identified two groups of critical residues in the lateral gate, one group that leads to defects in the translocation and membrane insertion of proteins and the other group that causes faster translocation and facilitates membrane insertion. Photocross-linking data show that the former group of residues is located at the interface of the lateral gate. Furthermore, different degrees of defects for the membrane insertion of single- and double-spanning membrane proteins were observed depending on whether the mutations were located in TM2b or TM7. These results demonstrate subtle differences in the molecular mechanism of the signal sequence binding/opening of the lateral gate and membrane insertion of a succeeding transmembrane segment in a polytopic membrane protein.     

Association of the Yeast RNA-binding Protein She2p with the Tubular Endoplasmic Reticulum Depends on Membrane Curvature

Localization of mRNAs contributes to the generation and maintenance of cellular asymmetry in a wide range of organisms. In Saccharomyces cerevisiae, the so-called locasome complex with its core components Myo4p, She2p, and She3p localizes more than 30 mRNAs to the yeast bud tip. A significant fraction of these mRNAs encodes membrane or secreted proteins. Their localization requires, besides the locasome, a functional segregation apparatus of the cortical endoplasmic reticulum (ER), including the machinery that is involved in the movement of ER tubules into the bud. Colocalization of RNA-containing particles with these tubules suggests a coordinated transport of localized mRNAs and the cortical ER to the bud. Association of localized mRNAs to the ER requires the presence of the locasome component She2p. Here we report that She2p is not only an RNA-binding protein but can specifically bind to ER-derived membranes in a membrane curvature-dependent manner in vitro. Although it does not contain any known curvature recognizing motifs, the protein shows a binding preference for liposomes with a diameter resembling that of yeast ER tubules. In addition, membrane binding depends on tetramerization of She2p. In an in vivo membrane-tethering assay, She2p can target a viral peptide GFP fusion protein to the cortical ER, indicating that a fraction of She2p associates with the ER in vivo. Combining RNA- and membrane-binding features makes She2p an ideal coordinator of ER tubule and mRNA cotransport.     

Phosphoregulation of the Ceramide Transport Protein CERT at Serine 315 in the Interaction with VAMP-associated Protein (VAP) for Inter-organelle Trafficking of Ceramide in Mammalian Cells

The ceramide transport protein CERT mediates the inter-organelle transport of ceramide for the synthesis of sphingomyelin, presumably through endoplasmic reticulum (ER)-Golgi membrane contact sites. CERT has a short peptide motif named FFAT, which associates with the ER-resident membrane protein VAP. We show that the phosphorylation of CERT at serine 315, which is adjacent to the FFAT motif, markedly enhanced the interaction of CERT with VAP. The phosphomimetic CERT S315E mutant exhibited higher activity to support the ER-to-Golgi transport of ceramide than the wild-type control in a semi-intact cell system, and this enhanced activity was abrogated when its FFAT motif was deleted. The level of phosphorylation of CERT at Ser-315 increased in HeLa cells treated with a sphingolipid biosynthesis inhibitor or exogenous sphingomyelinase. Expression of CERT S315E induced intracellular punctate structures, to which CERT and VAP were co-localized, and the occurrence of the structure was dependent on both phosphatidylinositol 4-monophosphate binding and VAP binding activities of CERT. Phosphorylation of another region (named a serine-rich motif) in CERT is known to down-regulate the activity of CERT. Analysis of various CERT mutant constructs showed that the de-phosphorylation of the serine-rich motif and the phosphorylation of Ser-315 likely have the additive contribution to enhance the activity of CERT. These results demonstrate that the phosphorylation of CERT at the FFAT motif-adjacent serine affected its affinity for VAP, which may regulate the inter-organelle trafficking of ceramide in response to the perturbation of cellular sphingomyelin and/or other sphingolipids.     

Post-translational N-glycosylation of type I transmembrane KCNE1 peptides: implications for membrane protein biogenesis and disease

N-Glycosylation of membrane proteins is critical for their proper folding, co-assembly and subsequent matriculation through the secretory pathway. Here, we examine the kinetics of N-glycan addition to type I transmembrane KCNE1 K(+) channel β-subunits, where point mutations that prevent N-glycosylation at one consensus site give rise to disorders of the cardiac rhythm and congenital deafness. We show that KCNE1 has two distinct N-glycosylation sites: a typical co-translational site and a consensus site ～20 residues away that unexpectedly acquires N-glycans after protein synthesis (post-translational). Mutations that ablate the co-translational site concomitantly reduce glycosylation at the post-translational site, resulting in unglycosylated KCNE1 subunits that cannot reach the cell surface with their cognate K(+) channel. This long range inhibition is highly specific for post-translational N-glycosylation because mutagenic conversion of the KCNE1 post-translational site into a co-translational site restored both monoglycosylation and anterograde trafficking. These results directly explain how a single point mutation can prevent N-glycan attachment at multiple sites, providing a new biogenic mechanism for human disease.     

STIM1 and STIM2 Proteins Differently Regulate Endogenous Store-operated Channels in HEK293 Cells

The endoplasmic reticulum calcium sensors stromal interaction molecules 1 and 2 (STIM1 and STIM2) are key modulators of store-operated calcium entry. Both these sensors play a major role in physiological functions in normal tissue and in pathology, but available data on native STIM2-regulated plasma membrane channels are scarce. Only a few studies have recorded STIM2-induced CRAC (calcium release-activated calcium) currents. On the other hand, many cell types display store-operated currents different from CRAC. The STIM1 protein regulates not only CRAC but also transient receptor potential canonical (TRPC) channels, but it has remained unclear whether STIM2 is capable of regulating store-operated non-CRAC channels. Here we present for the first time experimental evidence for the existence of endogenous non-CRAC STIM2-regulated channels. As shown in single-channel patch clamp experiments on HEK293 cells, selective activation of native STIM2 proteins or STIM2 overexpression results in store-operated activation of I_min channels, whereas STIM1 activation blocks this process. Changes in the ratio between active STIM2 and STIM1 proteins can switch the regulation of I_min channels between store-operated and store-independent modes. We have previously characterized electrophysiological properties of different Ca²⁺ influx channels coexisting in HEK293 cells. The results of this study show that STIM1 and STIM2 differ in the ability to activate these store-operated channels; I_min channels are regulated by STIM2, TRPC3-containing I_NS channels are induced by STIM1, and TRPC1-composed I_max channels are activated by both STIM1 and STIM2. These new data about cross-talk between STIM1 and STIM2 and their different roles in store-operated channel activation are indicative of an additional level in the regulation of store-operated calcium entry pathways.     

Silencing of the Tandem Pore Domain Halothane-inhibited K+ Channel 2 (THIK2) Relies on Combined Intracellular Retention and Low Intrinsic Activity at the Plasma Membrane

The tandem pore domain halothane-inhibited K⁺ channel 1 (THIK1) produces background K⁺ currents. Despite 62% amino acid identity with THIK1, THIK2 is not active upon heterologous expression. Here, we show that this apparent lack of activity is due to a unique combination of retention in the endoplasmic reticulum and low intrinsic channel activity at the plasma membrane. A THIK2 mutant containing a proline residue (THIK2-A155P) in its second inner helix (M2) produces K⁺-selective currents with properties similar to THIK1, including inhibition by halothane and insensitivity to extracellular pH variations. Another mutation in the M2 helix (I158D) further increases channel activity and affects current kinetics. We also show that the cytoplasmic amino-terminal region of THIK2 (Nt-THIK2) contains an arginine-rich motif (RRSRRR) that acts as a retention/retrieval signal. Mutation of this motif in THIK2 induces a relocation of the channel to the plasma membrane, resulting in measurable currents, even in the absence of mutations in the M2 helix. Cell surface delivery of a Nt-THIK2-CD161 chimera is increased by mutating the arginines of the retention motif but also by converting the serine embedded in this motif to aspartate, suggesting a phosphorylation-dependent regulation of THIK2 trafficking.