Artigo Acesso aberto Revisado por pares

Interaction of calmodulin with Sec61α limits Ca 2+ leakage from the endoplasmic reticulum

2010; Springer Nature; Volume: 30; Issue: 1 Linguagem: Inglês

10.1038/emboj.2010.284

ISSN

1460-2075

Autores

Frank Erdmann, Nico Schäuble, Sven Lang, Martin Jung, Alf Honigmann, Mazen Ahmad, Johanna Dudek, Julia Benedix, Anke Harsman, Annika Kopp, Volkhard Helms, Adolfo Cavalié, Richard Wagner, Richard Zimmermann,

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Transgenic Plants and Applications

Resumo

Article23 November 2010free access Interaction of calmodulin with Sec61α limits Ca2+ leakage from the endoplasmic reticulum Frank Erdmann Frank Erdmann Biophysics, Osnabrück University, Osnabrück, GermanyPresent address: Physiologie I, Universität Münster, Münster 48149, Germany Search for more papers by this author Nico Schäuble Nico Schäuble Medical Biochemistry and Molecular Biology, Saarland University, Homburg, Germany Search for more papers by this author Sven Lang Sven Lang Medical Biochemistry and Molecular Biology, Saarland University, Homburg, Germany Search for more papers by this author Martin Jung Martin Jung Medical Biochemistry and Molecular Biology, Saarland University, Homburg, Germany Search for more papers by this author Alf Honigmann Alf Honigmann Biophysics, Osnabrück University, Osnabrück, Germany Search for more papers by this author Mazen Ahmad Mazen Ahmad Computational Biology, Saarland University, Saarbrücken, Germany Search for more papers by this author Johanna Dudek Johanna Dudek Medical Biochemistry and Molecular Biology, Saarland University, Homburg, Germany Search for more papers by this author Julia Benedix Julia Benedix Medical Biochemistry and Molecular Biology, Saarland University, Homburg, Germany Search for more papers by this author Anke Harsman Anke Harsman Biophysics, Osnabrück University, Osnabrück, Germany Search for more papers by this author Annika Kopp Annika Kopp Biophysics, Osnabrück University, Osnabrück, Germany Search for more papers by this author Volkhard Helms Volkhard Helms Computational Biology, Saarland University, Saarbrücken, Germany Search for more papers by this author Adolfo Cavalié Corresponding Author Adolfo Cavalié Experimental and Clinical Pharmacology and Toxicology, Saarland University, Homburg, Germany Search for more papers by this author Richard Wagner Corresponding Author Richard Wagner Biophysics, Osnabrück University, Osnabrück, Germany Search for more papers by this author Richard Zimmermann Corresponding Author Richard Zimmermann Medical Biochemistry and Molecular Biology, Saarland University, Homburg, Germany Search for more papers by this author Frank Erdmann Frank Erdmann Biophysics, Osnabrück University, Osnabrück, GermanyPresent address: Physiologie I, Universität Münster, Münster 48149, Germany Search for more papers by this author Nico Schäuble Nico Schäuble Medical Biochemistry and Molecular Biology, Saarland University, Homburg, Germany Search for more papers by this author Sven Lang Sven Lang Medical Biochemistry and Molecular Biology, Saarland University, Homburg, Germany Search for more papers by this author Martin Jung Martin Jung Medical Biochemistry and Molecular Biology, Saarland University, Homburg, Germany Search for more papers by this author Alf Honigmann Alf Honigmann Biophysics, Osnabrück University, Osnabrück, Germany Search for more papers by this author Mazen Ahmad Mazen Ahmad Computational Biology, Saarland University, Saarbrücken, Germany Search for more papers by this author Johanna Dudek Johanna Dudek Medical Biochemistry and Molecular Biology, Saarland University, Homburg, Germany Search for more papers by this author Julia Benedix Julia Benedix Medical Biochemistry and Molecular Biology, Saarland University, Homburg, Germany Search for more papers by this author Anke Harsman Anke Harsman Biophysics, Osnabrück University, Osnabrück, Germany Search for more papers by this author Annika Kopp Annika Kopp Biophysics, Osnabrück University, Osnabrück, Germany Search for more papers by this author Volkhard Helms Volkhard Helms Computational Biology, Saarland University, Saarbrücken, Germany Search for more papers by this author Adolfo Cavalié Corresponding Author Adolfo Cavalié Experimental and Clinical Pharmacology and Toxicology, Saarland University, Homburg, Germany Search for more papers by this author Richard Wagner Corresponding Author Richard Wagner Biophysics, Osnabrück University, Osnabrück, Germany Search for more papers by this author Richard Zimmermann Corresponding Author Richard Zimmermann Medical Biochemistry and Molecular Biology, Saarland University, Homburg, Germany Search for more papers by this author Author Information Frank Erdmann1,‡, Nico Schäuble2,‡, Sven Lang2, Martin Jung2, Alf Honigmann1, Mazen Ahmad3, Johanna Dudek2, Julia Benedix2, Anke Harsman1, Annika Kopp1, Volkhard Helms3, Adolfo Cavalié 4, Richard Wagner 1 and Richard Zimmermann 2 1Biophysics, Osnabrück University, Osnabrück, Germany 2Medical Biochemistry and Molecular Biology, Saarland University, Homburg, Germany 3Computational Biology, Saarland University, Saarbrücken, Germany 4Experimental and Clinical Pharmacology and Toxicology, Saarland University, Homburg, Germany ‡These authors contributed equally to this work *Corresponding authors: Experimental and Clinical Pharmacology and Toxicology, Saarland University, Homburg 66421, Germany. Tel.: +496 841 162 6151; Fax: +496 841 162 6402; E-mail: [email protected], Osnabrück University, Osnabrück 49076, Germany. Tel.: +495 419 692 851; Fax: +495 419 692 243; E-mail: [email protected] Biochemistry and Molecular Biology, Saarland University, Building 44, Homburg 66421, Germany. Tel.: +496 841 162 6510; Fax: +496 841 162 6288; E-mail: [email protected] The EMBO Journal (2011)30:17-31https://doi.org/10.1038/emboj.2010.284 Present address: Physiologie I, Universität Münster, Münster 48149, Germany PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info In eukaryotes, protein transport into the endoplasmic reticulum (ER) is facilitated by a protein-conducting channel, the Sec61 complex. The presence of large, water-filled pores with uncontrolled ion permeability, as formed by Sec61 complexes in the ER membrane, would seriously interfere with the regulated release of calcium from the ER lumen into the cytosol, an essential mechanism for intracellular signalling. We identified a calmodulin (CaM)-binding motif in the cytosolic N-terminus of mammalian Sec61α that bound CaM but not Ca2+-free apocalmodulin with nanomolar affinity and sequence specificity. In single-channel measurements, CaM potently mediated Sec61-channel closure in Ca2+-dependent manner. At the cellular level, two different CaM antagonists stimulated calcium release from the ER through Sec61 channels. However, protein transport into microsomes was not modulated by Ca2+-CaM. Molecular modelling of the ribosome/Sec61/CaM complexes supports the view that simultaneous ribosome and CaM binding to the Sec61 complex may be possible. Overall, CaM is involved in limiting Ca2+ leakage from the ER. Introduction In eukaryotes, the endoplasmic reticulum (ER) has central roles in the synthesis, folding, and sorting of proteins as well as in acting as a dynamic calcium reservoir, which is essential for cellular calcium signalling, a process tightly controlled by electrical and chemical stimuli (Berridge, 2002). How are these key processes coordinated to maintain the functional integrity of the ER? The products of almost a third of eukaryotic genes are integrated into the membrane or transported to the lumen of the ER (Blobel and Dobberstein, 1975), facilitated by a protein translocase with Sec61α, Sec61β, and Sec61γ as core components (Görlich and Rapoport, 1993; Hartmann et al, 1994). Channel pore diameters have been shown to range from 5–8 Å for the closed crystal structure of the archaean ortholog (van den Berg et al, 2004) to 26–60 Å for the mammalian Sec61 complex as deduced from fluorescence quenching (Hamman et al, 1997) and electrophysiological experiments (Wirth et al, 2003). During protein synthesis at the ER, the permeability of the translocon for small ions has to be tightly controlled. Accordingly, the ribosome–Sec61 complex is assumed to be impermeable to ions during nascent chain transport (Crowley et al, 1994). However, this view was recently challenged by 3D reconstructions after cryo-EM for Sec61/ribosome–complexes, which revealed a gap between Sec61 and the ribosome (Menetret et al, 2008; Becker et al, 2009). In the empty state after the translocation event, that is when the translocon is still ribosome bound but unoccupied by a polypeptide chain, and in the ribosome-depleted state the translocon complex seems to transiently allow the passage of small molecules and calcium ions (Ca2+) (Roy and Wonderlin, 2003; Flourakis et al, 2006; Giunti et al, 2007; Ong et al, 2007). Furthermore, the mammalian Sec61 complex shows characteristics of an ion channel under these conditions (Simon and Blobel, 1991; Wirth et al, 2003; Wonderlin, 2009). These results indicate that, at least in mammals, calcium ions could be lost from the ER during or after the termination of protein translocation. The controlled release of Ca2+ from the ER lumen to the cell cytosol is one of the key factors in the regulation of many physiological processes, including muscle contraction, exocytosis, and apoptosis. In the resting state, the Ca2+ concentration in the ER lumen is the result of a balance between Ca2+ uptake by sarcoplasmic ER calcium ATPases (SERCAs) (Wuytack et al, 2002) and Ca2+ leakage currents (Camello et al, 2002). Recent studies have proposed that the Sec61 complex may contribute to Ca2+ leakage (Lomax et al, 2002; van Coppenolle et al, 2004; Flourakis et al, 2006; Giunti et al, 2007). However, uncontrolled leakage of Ca2+ through the large, aqueous Sec61 pore would seriously interfere with the capacity of the ER membrane to maintain Ca2+ concentrations roughly three to four orders of magnitude higher than in cytosol (Yu and Hinkle, 2000; Smith et al, 2001; Alvarez, 2002). In addition, leakage would compromise the regulated release of calcium from the ER lumen in specific spatial and temporal patterns. Calmodulin (CaM) is a highly conserved protein with two pairs of EF-hand motifs (Babu et al, 1985). The binding of Ca2+ to these motifs induces a conformational change in CaM that, in turn, regulates the activity of CaM target proteins in a calcium-dependent manner. Ion channels are a major class of CaM-regulated proteins, particularly those committed to calcium homeostasis (Yamada et al, 1995; Zühlke et al, 1999; DeMaria et al, 2001; Yamaguchi et al, 2001; Bähler and Rhoads, 2002; Dick et al, 2008; Tadross et al, 2008; Wang et al, 2008). The IQ motif and the 1-8-14 motif are widely distributed, evolutionarily conserved motifs mediating the binding of CaM to interaction partners (Rhoads and Friedberg, 1997; Puntervoll et al, 2003). Here, we show that Ca2+-CaM can bind to an IQ motif that is present in the cytosolic N-terminus of the α subunit of the Sec61 complex. The binding of Ca2+-CaM to the Sec61 complex triggers closure of the Sec61 channel and is sensitive towards CaM antagonists that interfere with substrate binding of CaM. At the cellular level, the same CaM antagonists stimulate Ca2+ leakage from the ER via Sec61 complexes. Molecular modelling and docking analysis of Ca2+-CaM and the ribosome/Sec61 complex support the view that Ca2+-CaM and ribosomes simultaneously bind to the Sec61 complex. This finding is consistent with our observation that Ca2+-CaM does not interfere with cotranslational protein transport into the ER. Thus, CaM contributes to cellular calcium homeostasis by limiting Ca2+ leakage from the ER at the level of Sec61 complexes. Results CaM binds to a conserved motif of the Sec61α subunit in a calcium-dependent and sequence-specific manner Using computational prediction services (http://calcium.uhnres.utoronto.ca/ctdb/ctdb/sequence.html) and the eukaryotic linear motif server (http://www.elm.eu.org), we identified an IQ motif in the cytosolic N-terminus of mammalian Sec61α (Figure 1A and B). The motif 19IQKPERKIQFKEKV32 from the Sec61α subunit is highly conserved in higher eukaryotes and compares well with the IQ consensus motif ([IVL]QxxxRxxxx[RK]xx[FILVWY]) found in unconventional myosins, neuromodulin, and neurogranin (Yap et al, 2000; Puntervoll et al, 2003) and also conforms to the 1-8-14 motif (Table I). Figure 1.Sec61α IQ motif and its interactions with calmodulin. (A) The IQ motif (red), as proposed for unconventional myosins and others (Rhoads and Friedberg, 1997), that was identified in the sequence of the Sec61α subunit from Canis lupus familiaris. Predicted transmembrane helices are underlined. (B) Predicted membrane topology of the mammalian Sec61α subunit. The IQ motif is indicated in red, the two cytosolic loops that most intimately contact with the ribosomal exit tunnel according to cryo-EM (Menetret et al, 2008; Becker et al, 2009) are indicated in green. Predicted transmembrane helices are shown in blue. N, N-terminus; C, C-terminus. (C) Fitted autocorrelation functions and residuals for the IQ488 peptide in the presence of 10 μM CaCl2 with the addition of 1 μM CaM or 4 μM GST-CaM. (D, E) Formation of the IQ488–CaM complex with increasing concentrations of CaM (D) or GST-CaM (E) in the presence of 4 mM CaCl2 or 10 mM EGTA. Error bars represent s.d. (F) IQ-peptide spots bound by 14C-labelled GST-CaM and 14C-labelled GST in the presence of 1 mM CaCl2 or 4 mM EGTA. Spots 1–7 and 10 correspond to the Sec61α IQ peptide (Homo sapiens). Amino acids were exchanged as indicated. Spots 8 and 9 correspond to the respective region of SecY (E. coli) and TRAM (H. sapiens) proteins, respectively. We note that the analysis of the various peptides was carried out three times. Furthermore, we note that similar results were obtained when unlabelled GST-CaM and anti-GST antibodies in combination with POD-coupled anti-rabbit antibodies, ECL™, and luminescence imaging were employed (data not shown). Download figure Download PowerPoint Table 1. Sequence alignments for Sec61α and SecY Species ID IQ motif Homo sapiens P61619 −19IQKPERKIQFKEKV32− Canis lupus familiaris NP_001000315.1 −19IQKPERKIQFKEKV32− Mus musculus P61620 −19IQKPERKIQFKEKV32− Gallus gallus XP_414364.2 −120IQKPERKIQFKEKV133− Danio rerio NP_963871.1 −19IQKPERKIQFKEKV32− Oncorhynchus mykiss Q98SN9 −19IQKPERKIQFKEKV32− Xenopus laevis Q7ZX87 −19IQKPERKIQFKEKV32− Drosophila melanogaster Q8STG9 −19IAKPERKIQFREKV32− Arabidopsis thaliana NP_174225.2 −21VQSADRKIPFREKV34− Oryza sativa NP_001049359.1 −21VQSADRKIPFREKV34− Plasmodium falciparum XP_001350175.1 −18VQSPDRKLPFKEKL31− Saccharomyces cerevisiae P32915 −20VIAPERKVPYNQKL33− Escherichia coli P0AGA3 −10QSAKGGLGELKRRL23− Thermus thermophilus Q72I24 −5FWSALQIPELRQRV18− Thermotoga maritima Q9X1I9 −5FKNAFKIPELRDRI18− Salmonella choleraesuis Q57J52 −10QSAKGGLGELKRRL23− Bacillus subtilis P16336 −5ISNFMRVSDIRNKI18− Methanococcus jannaschii Q60175 −14VELPVKEITFKEKL27− Methanobacterium thermoautotrophicum O26134 −18VKSPGYRVPFREKL31− Methanosarcina acetivorans Q8TRS4 −17VASPEKHVHFKDKL30− Halobacterium salinarium Q9HPB1 −17VERPEGHVPFRRKM30− Representative sequences were selected for ClustalW2 multiple sequence alignments. The IQ motif and homolog sequences are shown for eukaryotes, eubacteria, and archaea. To determine whether CaM binds to this Sec61α IQ motif, we synthesized the corresponding peptide, added a terminal cysteine, and labelled it with a fluorescent dye (Atto488). In fluorescence correlation spectroscopy (FCS) experiments (according to Petrasek and Schwille, 2008), the CaM–IQ488 complex formation was detected by an increase in the diffusion time (τD) for the IQ488 peptide. After the addition of 1 μM CaM in the presence of 10 μM CaCl2, τD increased from 148±5 μs to 199±4 μs (Figure 1C). To amplify the signal, we increased the molecular mass of CaM by glutathione-S-transferase (GST) fusion. With the addition of 4 μM GST-CaM, τD further increased to 297±8 μs (Figure 1C). We found that CaM binding to the IQ488 peptide in the presence of saturating calcium concentrations (4 mM) occurred via a single-site binding mechanism (Hill slope: 1.1±0.19; KD 121±28 nM; Figure 1D) and GST-CaM bound to the IQ488 peptide with a Hill slope of 0.88±0.07 and a KD of 115±12 nM (Figure 1E). In the absence of Ca2+, CaM did not interact with the IQ488 peptide at comparable concentrations (Figure 1D and E). We concluded that CaM bound to the Sec61α IQ peptide in a calcium-dependent manner and with high affinity. Next, we tested the sequence specificity of calcium-dependent CaM interactions with the Sec61α IQ motif. We synthesized IQ peptides with alanines in place of conserved amino acids and these were spotted on a cellulose membrane (Hilpert et al, 2007). Binding assays were carried out in the presence and absence of Ca2+ (Figure 1F). Consistent with the FCS results, the wild-type Sec61α IQ motif bound 14C-labelled GST-CaM in the presence of free calcium (Figure 1F, spots 1, 7, and 10; defined as 100% binding). When Ca2+ was chelated with EGTA, GST-CaM binding was nearly abolished (spots 1, 7, and 10; 17±5% binding). The exchange of the conserved residues to alanine at positions 7 and 11 drastically reduced CaM binding (spots 5 and 6; 24±4.8% binding). However, modifying the residues at positions 2 and 3 did not alter CaM binding (spots 2, 3, and 4; 80±16% binding). As negative controls, the N-terminus of the Sec61α ortholog in Escherichia coli, SecY, and the N-terminus of the human TRAM protein were analysed. As expected, the peptides from the SecY and TRAM proteins did not bind CaM (spots 8 and 9; 12±7% and 2±0.2% binding, respectively). When 14C-labelled GST was added to the IQ constructs as a control, no binding was observed (Figure 1F). We concluded that CaM bound to the Sec61α IQ peptide in a sequence-specific and calcium-dependent manner. CaM interacts with the native Sec61 complex We studied the binding of CaM to native Sec61 complexes, present in rough microsomes (RM), by flotation in sucrose gradients. GST-CaM was incubated with RM, or RM that had been pretreated with puromycin and high salt in order to remove ribosomes (termed PKRM; Görlich and Rapoport, 1993), or purified and reconstituted Sec61 complex (Görlich and Rapoport, 1993), in the presence or absence of Ca2+. Subsequently, the samples were adjusted for a high sucrose concentration, placed on the bottom of centrifuge tubes and covered with the sucrose gradient. After centrifugation, fractions were taken and analysed by SDS–PAGE, western blot, and immunodetection of GST-CaM and Sec61β. GST-CaM floated with RM in the presence, and perhaps to a lesser extent in the absence of Ca2+ (Figure 2B and C; 3.47±0.84% and 1.47±0.59%, respectively, flotation). In addition, we observed that a CaM antagonist that interferes with substrate binding, such as trifluoperazine (Johnson and Fugman, 1983; Vandonselaar et al, 1994), interferes with binding of GST-CaM to RM in the presence of Ca2+ (Figure 2D; 0.51±0.11%). GST-CaM also floated with PKRM and reconstituted Sec61 complex in the presence of Ca2+ (Figure 2E and G; 3.57±0.68% and 29.37±4.56%, respectively, flotation), but not without any membranes or with protein-free liposomes (Figure 2A and F; 0.22% and 0.75±0.47%, respectively, flotation). We concluded that the CaM-binding site in the membrane resident Sec61 complex was active and accessible to CaM even in the presence of ribosomes and that the interaction of CaM with the Sec61 complex involved CaM's substrate-binding site. Figure 2.Sec61 complex interactions with calmodulin. (A) Flotation analysis of GST-CaM in the presence of calcium in sucrose gradients. (B, C) Coflotation experiments in sucrose gradients of canine pancreatic rough microsomes (RM) with GST-CaM in the presence (B) or absence (C) of calcium. (D) Coflotation experiments in sucrose gradients of RM with GST-CaM in the simultaneous presence of calcium and trifluoperazine (TFP). (E, G) Coflotation experiments in sucrose gradients of PKRM (E) and Sec61 proteoliposmes (G) with GST-CaM in the presence of calcium. (F) Coflotation experiments of protein-free liposomes with GST-CaM in the presence of calcium. We note that the analysis was carried out between three (buffer, liposomes) and five times (RM, PKRM, Sec61 proteoliposomes) with similar results. (H) GST-CaM and GST were derivatized with FeBABE according to the manufacturer's protocol (indicated with asterisk) or were left underivatized, and incubated with Sec61 proteoliposomes for 30 min at room temperature in the presence or absence of calcium as indicated. Activation with ascorbate and peroxide was carried out for 30 s. We note that the analysis was carried out three times with similar results. All samples were analysed by SDS–PAGE, western blotting, and immunodetection of GST-CaM, Sec61α, or Sec61β in combination with POD-coupled anti-rabbit antibodies, ECL™, and luminescence imaging as indicated. In all panels, the areas of interest for single western blots and gels are shown, in panels B through E and G the two parts that are divided by a white space represent two consecutive immunodetections for the same blots. Download figure Download PowerPoint Alternatively, interaction of CaM with native Sec61 complex was analysed by employing the reagent FeBABE that can be attached to a bait protein and used to artificially hydrolyze nearby peptide bonds in a prey protein (Ghaim et al, 1995). GST-CaM was derivatized with FeBABE, incubated with reconstituted Sec61 complex, and activated with ascorbate and peroxide (i.e. converted to active protease). Subsequently, the samples were analysed by SDS–PAGE, western blot, and immunodetection of Sec61α with an antibody that is directed against the C-terminus of Sec61α. Derivatized and activated GST-CaM cleaved Sec61α in the presence and absence of Ca2+ to the indicated C-terminal 20 kDa fragment plus two less abundant fragments of larger molecular mass (Figure 2H, lanes 3 and 5). Without FeBABE attachment, there was no proteolytic cleavage observed (Figure 2H, lane 2). Furthermore, there was no proteolytic cleavage observed when FeBABE modified and activated GST was employed (Figure 2H, lane 4). Thus, the artificial protease approach confirmed the conclusion that the CaM-binding site in the native Sec61 complex is accessible to CaM. However, we note that evidence for a direct interaction between CaM and Sec61 in its native context remains to be fully explored. CaM binding induces calcium-dependent Sec61-channel closure We also characterized the effect of CaM on Sec61-channel activity using electrophysiology. Vesicles containing native or reconstituted Sec61 complexes were osmotically fused to lipid bilayers as previously described (Wirth et al, 2003). In transport-competent vesicles that were derived from RM, single-channel activities were observed after treatment with puromycin, which caused the termination of translocation and the dissociation of ribosomes (Figure 3A, middle trace; Supplementary Figures S1 and S2). The addition of 500 nM CaM to RM channels led to closure of the pore in the presence of calcium (Figure 3A, lower trace). In agreement with the FCS results, we found that a Ca2+ concentration of 10 μM was sufficient to induce CaM-mediated channel closure (Figure 3B). In the absence of calcium, no channel closure was observed with the addition of CaM (Figure 3C). A statistic evaluation of the CaM effect on the channel open probability is shown in Figure 3D. Comparing the open probability before and after the addition of CaM showed an explicit closure of the channel for all membrane potentials examined. Again, the closure of the Sec61 complex by CaM was strictly dependent on free Ca2+; in the presence of 10 mM EGTA, we observed no reduction in the open probability with the addition of 500 nM CaM (Figure 3D). An additional experiment demonstrated that the observed effects involved a CaM/Sec61 interaction. With proteoliposomes that contained only purified Sec61 complexes, the observed effect was indistinguishable from the results with RM preparations (Figure 3E). Figure 3.Electrophysiological properties of Sec61 in the presence of calmodulin. (A) Current traces from rough microsomes (RM) preparations before (middle trace) and after (lower trace) the addition of 500 nM calmodulin to both compartments (cis/trans), at a membrane potential of −30 mV and in the presence of 13 mM CaCl2 and 200 μM puromycin. The upper trace shows a control experiment in the absence of puromycin. The frequency histograms (right panels) depict the main current levels observed in the recordings shown in the left panels. Baseline and the high conductance state of the Sec61 channel are indicated with dashed lines. (B) Voltage ramp recording of a RM channel before (grey) and after (black) the addition of 500 nM calmodulin cis/trans in the presence of 10 μM CaCl2. Measurements were performed in 100 mM KCl, 10 mM Mops/Tris (pH 7.0) cis/trans and 200 μM puromycin. (C) Voltage ramp recording of a bilayer containing a single-Sec61 channel from RM vesicles before (grey) and after (black) the addition of 500 nM calmodulin cis/trans in the absence of Ca2+ (10 mM EGTA). Measurements were performed in 100 mM KCl, 10 mM Mops/Tris (pH 7.0) cis/trans and 200 μM puromycin. (D) Open probability of the Sec61 channel from RM vesicles before and after the addition of 500 nM calmodulin cis/trans in the presence (13 mM CaCl2) or absence (10 mM EGTA) of Ca2+ (n=6–9 bilayers). Error bars represent s.d. Measurement conditions: 100 mM KCl, 10 mM Mops/Tris (pH 7.0), 200 μM puromycin. (E) Voltage ramp recording of the purified Sec61 complex before (grey) and after (black) the addition of 1 μM calmodulin cis/trans in the presence of 13 mM CaCl2. The experiment was performed under symmetrical buffer conditions of 100 mM KCl, 10 mM EGTA, 10 mM Mops/Tris (pH 7.0), and 200 μM puromycin. Download figure Download PowerPoint Therefore, both the binding of free Ca2+ to CaM and the binding of CaM to Sec61 are essential for the regulation of the open probability. In contrast, the presence of calcium-free apocalmodulin had no effect on the Sec61 channel, consistent with the IQ motif-binding studies. CaM antagonists enhance calcium leakage from the ER in intact cells Under resting conditions of the cell, the cytosolic-free Ca2+ concentration is low due to the action of plasma membrane pumps and exchangers (50–100 nM). Simultaneously, the free Ca2+ concentration in the ER lumen is high, basically because of the action of SERCA (100–800 μM). This Ca2+ distribution is constantly challenged by the so-called calcium leak, a passive calcium efflux from the ER. The drug thapsigargin irreversibly inhibits SERCA, thereby unmasking the leak pathway. The leakage of Ca2+ from the ER can be visualized as an increase in the cytosolic calcium concentration by utilizing a Ca2+ indicator, such as FURA-2, in intact cells in the absence of extracellular calcium. Indirect evidence from various laboratories suggests that the Sec61 complex contributes to the leak after termination of protein translocation (Lomax et al, 2002; van Coppenolle et al, 2004; Flourakis et al, 2006; Giunti et al, 2007). Based on the above in vitro experiments, we expected CaM to contribute to limiting the Sec61-mediated calcium efflux under these conditions and for CaM antagonists to enhance the leak. Therefore, we investigated whether the presence of CaM antagonists that interfere with substrate binding by CaM, ophiobolin A (Leung et al, 1984; Au et al, 2000) and trifluoperazine (Johnson and Fugman, 1983; Vandonselaar et al, 1994), enhance Ca2+ efflux from the ER in the presence of thapsigargin. In Ca2+ imaging experiments with FURA-2, HeLa cells were treated with one of the two CaM antagonists for 10 min and, subsequently, Ca2+ was released by applying thapsigargin in the absence of external Ca2+. The two CaM antagonists ophiobolin A and trifluoperazine had similar enhancing and significant effects on the thapsigargin-induced calcium efflux (Figure 4A, B, and H). Thus, CaM contributes to reducing Ca2+ leakage from the ER under normal conditions. Figure 4.Live cell calcium imaging of HeLa cells in the presence of SEC61A1 siRNA and CaM antagonists. HeLa cells were loaded with the calcium indicator FURA-2 AM and experiments were carried out with a Ca2+-free buffer containing 0.5 mM EGTA. (A, B) Naive HeLa cells were treated with the indicated CaM antagonist or with the Ca2+-free buffer. Ca2+ release was initiated by applying thapsigargin in the absence of external Ca2+. Error bars represent standard errors of the means (s.e.m.). (C–F) HeLa cells were treated with one of the two siRNAs directed against SEC61A1 (E, F) or a negative control siRNA (C, D) for 96 h as indicated and calcium-imaging experiments were carried out as above. (G) Silencing was evaluated by western blot analysis using antibodies that were directed against Sec61α and β-actin (loading control). The primary antibodies were visualized by using ECL™ Plex secondary antibodies and fluorescence imaging. Average values are given, error bars represent standard errors of the means (s.e.m.). The numbers of experiments that were analysed are indicated. (H) Statistical analysis of the changes in the cytosolic Ca2+ concentration after the addition of thapsigargin (as indicated by the insert) in the experiments presented in A through F, plus the experiments with the second SEC61A1 siRNA. P-values <0.001 were defined as significant by unpaired t-test and are indicated by three asterisks (***), NS, not significant. The numbers of cells that were analysed are indicated. The experiments were carried out f

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