Calcium-binding Protein Calmodulin Research Articles

Dual nuclear import mechanisms of sex determining factor SRY: intracellular Ca 2+ as a switch

The sex-determining region on the Y chromosome (SRY) has 2 nuclear localization signals (NLSs) that flank the DNA binding high mobility group (HMG) domain; the β-NLS and the CaM-NLS, which mediate nuclear transport through importin β1 (Impβ1) and the calcium-binding protein calmodulin (CaM), respectively. Here we reconstitute the nuclear import mediated by the 2 NLSs for the first time in vitro, establishing Ran independence of CaM-NLS-dependent transport. The β- and CaM-NLSs were found to be independently functional out of the context of the SRY HMG domain, dependent on Impβ1 and CaM binding, respectively. Intriguingly, direct protein binding assays also indicated competitive binding of Impβ1 and CaM to the SRY HMG domain. To assess the potential role of intracellular calcium in modulating SRY nuclear accumulation, Cos-7 cells expressing SRY and control constructs were treated with agents elevating or reducing intracellular Ca(2+) levels. The in vivo results, supported by experiments in vitro where transport was assessed with or without 2 μM Ca(2+), indicate a Ca(2+)-dependent mode of nuclear transport via the CaM-NLS/CaM, with inhibition of β-NLS/Impβ1-mediated nuclear import by intracellular Ca(2+). The results imply mutual exclusivity of nuclear transport via the 2 NLSs with intracellular Ca(2+) as the switch between the 2.

NO Way to Relax

The roles of nitric oxide (NO) in the heart have been studied intensely ever since the first report nearly 2 decades ago showing that endogenous NO modulates cardiac myocyte function.1 All 3 NO synthase (NOS) isoforms have been found in mammalian heart tissues (see reviews in Massion et al2 and Belge et al3). A complex array of myocyte and nonmyocyte cells in the heart express NOS isoforms, and the local generation of NO2,3 and reactive oxygen species (ROS)4 may exert both autocrine and paracrine effects on cellular function. Not only is the endothelial isoform of NOS (eNOS, or NOS3) robustly expressed in the endothelial cells of the cardiac vasculature, but eNOS is also present within cardiac myocytes, where the enzyme associates with the scaffolding/regulatory protein caveolin-3 in T tubules in plasmalemmal caveolae.5 The neuronal NOS (nNOS, or NOS1) is also expressed in cardiac myocytes, where the enzyme appears to be localized in the sarcoplasmic reticulum and modulates phospholamban phosphorylation.6 Although both eNOS and nNOS appear to be physiologically expressed in cardiac tissues, the inflammation-related NOS isoform (iNOS, or NOS2) only appears in the heart after immunoactivation; iNOS may modulate the decline in myocardial function seen in sepsis.7 There have been innumerable studies of the cardiac effects of various NOS inhibitors, NO-donating drugs, and NO itself.2 The cardiac phenotypes of NOS “knockout” mouse models have been characterized extensively, and mice lacking 1, 2, or all 3 NOS isoforms have been generated and characterized.8 In addition, the roles of NOS substrates, cofactors, and allosteric modulators have been explored exhaustively in diverse cardiac model systems. Emerging from this broad range of experimental evidence is an increasingly clear view that NO and NOS are key determinants of cardiac function. Yet the molecular mechanisms and …

Pro-apoptotic Par-4 and dopamine D2 receptor in temporal cortex in schizophrenia, bipolar disorder and major depression

Although the etiology of schizophrenia remains unknown, diverse neuropathological evidence suggests a disorder of synaptic connectivity. Apoptosis is a form of cell death that helps determine synaptic circuitry during neurodevelopment and altered regulation of apoptosis has been implicated in schizophrenia. Prostate apoptosis response-4 (Par-4) is an upstream regulator of apoptosis preferentially localized to synapses. Brain Par-4 levels are upregulated in response to pro-apoptotic stimuli in rodent models and in patients with classic neurodegenerative diseases. Recently, Par-4 was also found to form a complex with the dopamine D2 receptor (D2DR) in competition with the calcium-binding protein calmodulin, implicating Par-4 as an important regulatory component in normal dopamine signaling. Interestingly, mutant mice with disrupted Par-4/D2DR interaction demonstrated depressive-like behaviors, suggesting a potential role for Par-4 in both depression and schizophrenia. In this study, Par-4, D2DR and calmodulin protein levels were measured using semiquantitative Western blotting in postmortem temporal cortex in subjects with schizophrenia, major depression and bipolar disorder. Compared to normal controls, mean Par-4 levels appeared slightly lower in schizophrenia and bipolar disorder. However, in major depression, Par-4 was decreased by 67% compared to normal controls. No differences were found between any groups for calmodulin or for the D2DR 48kDa band. The D2DR 98kDa band was lower by 50% in the schizophrenia compared to control groups. Changes in the Par-4/D2DR signaling pathway represent a novel mechanism that may link apoptotic and dopamine signaling pathways in major depression and schizophrenia.

Calcium‐binding proteins in skeletal muscles of the mdx mice: potential role in the pathogenesis of Duchenne muscular dystrophy

Duchenne muscular dystrophy is one of the most common hereditary diseases. Abnormal ion handling renders dystrophic muscle fibers more susceptible to necrosis and a rise in intracellular calcium is an important initiating event in dystrophic muscle pathogenesis. In the mdx mice, muscles are affected with different intensities and some muscles are spared. We investigated the levels of the calcium-binding proteins calsequestrin and calmodulin in the non-spared axial (sternomastoid and diaphragm), limb (tibialis anterior and soleus), cardiac and in the spared extraocular muscles (EOM) of control and mdx mice. Immunoblotting analysis showed a significant increase of the proteins in the spared mdx EOM and a significant decrease in the most affected diaphragm. Both proteins were comparable to the cardiac muscle controls. In limb and sternomastoid muscles, calmodulin and calsequestrin were affected differently. These results suggest that differential levels of the calcium-handling proteins may be involved in the pathogenesis of myonecrosis in mdx muscles. Understanding the signaling mechanisms involving Ca(2+)-calmodulin activation and calsequestrin expression may be a valuable way to develop new therapeutic approaches to the dystrophinopaties.

A Double Tyrosine Motif in the Cardiac Sodium Channel Domain III-IV Linker Couples Calcium-dependent Calmodulin Binding to Inactivation Gating

Voltage-gated sodium channels maintain the electrical cadence and stability of neurons and muscle cells by selectively controlling the transmembrane passage of their namesake ion. The degree to which these channels contribute to cellular excitability can be managed therapeutically or fine-tuned by endogenous ligands. Intracellular calcium, for instance, modulates sodium channel inactivation, the process by which sodium conductance is negatively regulated. We explored the molecular basis for this effect by investigating the interaction between the ubiquitous calcium binding protein calmodulin (CaM) and the putative sodium channel inactivation gate composed of the cytosolic linker between homologous channel domains III and IV (DIII-IV). Experiments using isothermal titration calorimetry show that CaM binds to a novel double tyrosine motif in the center of the DIII-IV linker in a calcium-dependent manner, N-terminal to a region previously reported to be a CaM binding site. An alanine scan of aromatic residues in recombinant DIII-DIV linker peptides shows that whereas multiple side chains contribute to CaM binding, two tyrosines (Tyr(1494) and Tyr(1495)) play a crucial role in binding the CaM C-lobe. The functional relevance of these observations was then ascertained through electrophysiological measurement of sodium channel inactivation gating in the presence and absence of calcium. Experiments on patch-clamped transfected tsA201 cells show that only the Y1494A mutation of the five sites tested renders sodium channel steady-state inactivation insensitive to cytosolic calcium. The results demonstrate that calcium-dependent calmodulin binding to the sodium channel inactivation gate double tyrosine motif is required for calcium regulation of the cardiac sodium channel.

Thiol-Reactive Derivatives of the Solvatochromic 4-N,N-Dimethylamino-1,8-naphthalimide Fluorophore: A Highly Sensitive Toolset for the Detection of Biomolecular Interactions

The solvatochromic fluorophore 4-N,N-dimethylamino-1,8-naphthalimide (4-DMN) possesses extremely sensitive emission properties due largely to the low intrinsic fluorescence it exhibits in polar protic solvents such as water. This makes it well suited as a probe for the detection of a wide range of biomolecular interactions. Herein we report the development and evaluation of a new series of thiol-reactive agents derived from this fluorophore. The members of this series vary according to linker type and the electrophilic group required for the labeling of proteins and other biologically relevant molecules. Using the calcium-binding protein calmodulin as a model system, we compare the performance of the 4-DMN derivatives to that of several commercially available solvatochromic fluorophores identifying many key factors important to the successful application of such tools. This study also demonstrates the power of this new series of labeling agents by yielding a fluorescent calmodulin construct capable of producing a greater than 100-fold increase in emission intensity upon binding to calcium.

Importins and Beyond: Non‐Conventional Nuclear Transport Mechanisms

The movement of proteins between the cytoplasm and the nucleus conventionally involves the recognition of nuclear targeting signals by members of the importin (Imp) superfamily of nuclear transporters, followed by translocation through the nuclear envelope-embedded nuclear pore complexes (NPCs). It is becoming increasingly apparent, however, that distinct alternative pathways for nuclear transport exist and are relatively abundant. This review examines several of these novel pathways, including facilitation of Imp-dependent transport by microtubule motors, and Imp-independent pathways involving either other transport molecules such as the calcium-binding protein calmodulin or through direct binding to the components of the NPC. The existence of these pathways and the fact that many proteins appear to possess separate Imp-dependent and -independent nuclear import mechanisms ensure that the cell can function under conditions in which Imp-dependent transport is inhibited and/or modulate the efficiency of Imp-dependent transport itself, according to the need.

Heterobifunctional isotope‐labeled amine‐reactive photo‐cross‐linker for structural investigation of proteins by matrix‐assisted laser desorption/ionization tandem time‐of‐flight and electrospray ionization LTQ‐Orbitrap mass spectrometry

Chemical cross-linking combined with a subsequent enzymatic digestion and mass spectrometric analysis of the created cross-linked products presents an alternative approach to assess low-resolution protein structures. By covalently connecting pairs of functional groups within a protein or a protein complex a set of structurally defined interactions is built up. We synthesized the heterobifunctional amine-reactive photo-cross-linker N-succinimidyl p-benzoyldihydrocinnamate as a non-deuterated (SBC) and doubly deuterated derivative (SBDC). Applying a 1:1 mixture of SBC and SBDC for cross-linking experiments aided the identification of cross-linked amino acids in the mass spectra based on the characteristic isotope patterns of fragment ions. The cross-linker was applied to the calcium-binding protein calmodulin with a subsequent analysis of cross-linked products by nano-high-performance liquid chromatography matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (nano-HPLC/MALDI-TOF/TOF-MS) and nano-HPLC/nano-electrospray ionization (ESI)-LTQ-Orbitrap-MS.

Charge density-dependent modifications of hydration shell waters by Hofmeister ions.

Gadolinium (Gd(3+)) vibronic sideband luminescence spectroscopy (GVSBLS) is used to probe, as a function of added Hofmeister series salts, changes in the OH stretching frequency derived from first-shell waters of aqueous Gd(3+) and of Gd(3+) coordinated to three different types of molecules: (i) a chelate (EDTA), (ii) structured peptides (mSE3/SE2) of the lanthanide-binding tags (LBTs) family with a single high-affinity binding site, and (iii) a calcium-binding protein (calmodulin) with four binding sites. The vibronic sideband (VSB) corresponding to the OH stretching mode of waters coordinated to Gd(3+), whose frequency is inversely correlated with the strength of the hydrogen bonding to neighboring waters, exhibits an increase in frequency when Gd(3+) becomes coordinated to either EDTA, calmodulin, or mSE3 peptide. In all of these cases, the addition of cation chloride or acetate salts to the solution increases the frequency of the vibronic band originating from the OH stretching mode of the coordinated waters in a cation- and concentration-dependent fashion. The cation dependence of the frequency increase scales with charge density of the cations, giving rise to an ordering consistent with the Hofmeister ordering. On the other hand, water Raman spectroscopy shows no significant change upon addition of these salts. Additionally, it is shown that the cation effect is modulated by the specific anion used. The results indicate a mechanism of action for Hofmeister series ions in which hydrogen bonding among hydration shell waters is modulated by several factors. High charge density cations sequester waters in a configuration that precludes strong hydrogen bonding to neighboring waters. Under such conditions, anion effects emerge as anions compete for hydrogen-bonding sites with the remaining free waters on the surface of the hydration shell. The magnitude of the anion effect is both cation and Gd(3+)-binding site specific.

Gas-phase metalloprotein complexes interrogated by ion mobility-mass spectrometry

Gas-phase biomolecular structure may be explored through a number of analytical techniques. Ion mobility-mass spectrometry (IM-MS) continues to prove itself as a sensitive and reliable bioanalytical tool for gas-phase structure determination due to intense study and development over the past 15 years. A vast amount of research interest, especially in protein and peptide conformational studies has generated a wealth of structural information for biological systems from small peptides to megadalton-sized biomolecules. In this work, linear low field IM-MS has been used to study gas-phase conformations and determine rotationally averaged collision cross-sections of three metalloproteins—cytochrome c, haemoglobin and calmodulin. Measurements have been performed on the MoQToF, a modified QToF 1 instrument (Micromass UK Ltd., Manchester, UK) modified in house. Gas-phase conformations and cross-sections of multimeric cytochrome c ions of the form [ xM + nH +] n+ for x = 1–3 (monomer to trimer) have been successfully characterised and measured. We believe these to be the first reported collision cross-sections of higher order multimeric cytochrome c. Haemoglobin is investigated to obtain structural information on the associative mechanism of tetramer formation. Haemoglobin molecules, comprising apo- and holo-monomer chains, dimer and tetramer are transferred to the gas phase under a range of solution conditions. Structural information on the proposed critical intermediate, semi-haemoglobin, is reported. Cross-sections of the calcium binding protein calmodulin have been obtained under a range of calcium-bound conditions. Metalloprotein collision cross-sections from ion mobility measurements are compared with computationally derived values from published NMR and X-ray crystallography structural data. Finally we consider the change in the density of the experimentally measured rotationally averaged collision cross-section for compact geometries of the electrosprayed proteins.

The role of disordered ribosomal protein extensions in the early steps of eubacterial 50 S ribosomal subunit assembly.

Although during the past decade research has shown the functional importance of disorder in proteins, many of the structural and dynamics properties of intrinsically unstructured proteins (IUPs) remain to be elucidated. This review is focused on the role of the extensions of the ribosomal proteins in the early steps of the assembly of the eubacterial 50 S subunit. The recent crystallographic structures of the ribosomal particles have revealed the picture of a complex assembly pathway that condenses the rRNA and the ribosomal proteins into active ribosomes. However, little is know about the molecular mechanisms of this process. It is thought that the long basic r-protein extensions that penetrate deeply into the subunit cores play a key role through disorder-order transitions and/or co-folding mechanisms. A current view is that such structural transitions may facilitate the proper rRNA folding. In this paper, the structures of the proteins L3, L4, L13, L20, L22 and L24 that have been experimentally found to be essential for the first steps of ribosome assembly have been compared. On the basis of their structural and dynamics properties, three categories of extensions have been identified. Each of them seems to play a distinct function. Among them, only the coil-helix transition that occurs in a phylogenetically conserved cluster of basic residues of the L20 extension appears to be strictly required for the large subunit assembly in eubacteria. The role of α helix-coil transitions in 23 S RNA folding is discussed in the light of the calcium binding protein calmodulin that shares many structural and dynamics properties with L20.

Morphological and biochemical analyses of otoliths of the ice‐fish Chionodraco hamatus confirm a common origin with red‐blooded species

The morphology and composition of the three otoliths of the Antarctic ice-fish Chionodraco hamatus were studied by scanning electron microscopy and X-ray diffraction. The composition of the sagitta, lapillus and asteriscus protein matrices was also analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, western blots and confocal laser scanning microscopy to reveal the presence of and to localize the calcium-binding proteins calmodulin, calbindin and S-100. Morphological results indicated that the otoliths in this ice-fish were similar to those of Trematomus bernacchii, a red-blooded Antarctic species [B. Avallone et al. (2003) J. Submicrosc. Cytol. Pathol. 35, 69-76], but rather different from those of other teleosts. These two Antarctic species possessed a completely vateritic asteriscus, whereas their sagitta and lapillus were made mostly of aragonite. Parallel analysis of protein patterns in C. hamatus and T. bernacchii revealed that the sagitta significantly differed from the lapillus and asteriscus in both species. The sagitta did not contain the S-100 protein and showed calmodulin and calbindin located in discontinuous or incremental zones, respectively. These results demonstrate that the otoliths of C. hamatus and T. bernacchii share more resemblances than differences and support the idea of a common origin of these species.

Crystallization and preliminary X-ray characterization of the genetically encoded fluorescent calcium indicator protein GCaMP2.

Fluorescent proteins and their engineered variants have played an important role in the study of biology. The genetically encoded calcium-indicator protein GCaMP2 comprises a circularly permuted fluorescent protein coupled to the calcium-binding protein calmodulin and a calmodulin target peptide, M13, derived from the intracellular calmodulin target myosin light-chain kinase and has been used to image calcium transients in vivo. To aid rational efforts to engineer improved variants of GCaMP2, this protein was crystallized in the calcium-saturated form. X-ray diffraction data were collected to 2.0 A resolution. The crystals belong to space group C2, with unit-cell parameters a = 126.1, b = 47.1, c = 68.8 A, beta = 100.5 degrees and one GCaMP2 molecule in the asymmetric unit. The structure was phased by molecular replacement and refinement is currently under way.

Transcriptional Analysis of Normal Human Fibroblast Responses to Microgravity Stress

To understand the molecular mechanism(s) of how spaceflight affects cellular signaling pathways, quiescent normal human WI-38 fibroblasts were flown on the STS-93 space shuttle mission. Subsequently, RNA samples from the space-flown and ground-control cells were used to construct two cDNA libraries, which were then processed for suppression subtractive hybridization (SSH) to identify spaceflight-specific gene expression. The SSH data show that key genes related to oxidative stress, DNA repair, and fatty acid oxidation are activated by spaceflight, suggesting the induction of cellular oxidative stress. This is further substantiated by the up-regulation of neuregulin 1 and the calcium-binding protein calmodulin 2. Another obvious stress sign is that spaceflight evokes the Ras/mitogen-activated protein kinase and phosphatidylinositol-3 kinase signaling pathways, along with up-regulating several G1-phase cell cycle traverse genes. Other genes showing up-regulation of expression are involved in protein synthesis and pro-apoptosis, as well as pro-survival. Interactome analysis of functionally related genes shows that c-Myc is the “hub” for those genes showing significant changes. Hence, our results suggest that microgravity travel may impact changes in gene expression mostly associated with cellular stress signaling, directing cells to either apoptotic death or premature senescence.

A New Role for Ankyrin Repeats

Transient receptor potential (TRP) channels are nonselective cation channels that act as sensors of heat and noxious chemicals and thus are important in pain perception. One member of this family, TRPV1, responds to such stimuli as heat, low pH, and capsaicin, the ingredient of chili peppers that makes them "hot." Two mechanisms are known to reduce the activity of TRP channels. The first, desensitization, occurs after prolonged exposure to a single stimulus. The second, tachyphylaxis, occurs after sequential exposures to the same stimulus. Increased intracellular Ca 2+ causes the desensitization of TRPV1 currents, and this may be mediated by the calcium-binding protein calmodulin (CaM). Lishko et al . solved the crystal structure of the ankyrin repeat domain (ARD) found in the N-terminal region of TRPV1. Ankyrin repeats are motifs containing 33 amino acid residues, and they are important for protein-protein interactions. As well as determining the protein structure of the ARD, the authors also discovered that ATP, which was present in the crystallization solution, was bound to the ARD. This interaction was not an artifact of the crystallization process as ATP-agarose formed a complex with purified TRPV1-ARD recombinant protein, and free ATP inhibited this interaction. The addition of either Mg 2+ or Ca 2+ reduced the binding of TRPV1-ARD to ATP-agarose. As discussed in the accompanying commentary by Myers and Julius, these findings are interesting because the N terminus of TRPV1 does not contain the "Walker box" motif often found in other proteins that bind ATP, and divalent cations usually stabilize the association between binding proteins and ATP, rather than disrupt them. Whole-cell patch clamp assays were performed on TRPV1-expressing insect cells to assess the effect of ATP on capsaicin-stimulated changes in TRPV1 current. The authors found (consistent with previous studies) that the addition of ATP sensitized TRPV1 and reduced tachyphylaxis after repeated exposure to capsaicin. Surprisingly, mutation of residues in the ATP-binding site resulted in mutant TRPV1 channels that showed a reduction in tachyphylaxis, even in the absence of ATP. The authors suggested that another factor that promotes tachyphylaxis must bind to the same site on TRPV1, and so mutations of this site block the binding of both ATP and this other factor, resulting in a net decrease in tachyphylaxis. Exclusion chromatography analysis showed that CaM could form a complex with TRPV1-ARD that was Ca 2+ -dependent and inhibitable by ATP. Patch clamp experiments with an antibody that sequesters CaM and blocks its activity showed that CaM promoted tachyphylaxis. ATP-binding site mutants of TRPV1-ARD did not bind CaM. Together, these data not only increase our understanding of the mechanisms that regulate TRPV1 channel activity but also present a new role for ankyrin repeats that may have consequences for the function of other ankyrin repeat-containing proteins. P. V. Lishko, E. Procko, X. Jin, C. B. Phelps, R. Gaudet, The ankyrin repeats of TRPV1 bind multiple ligands and modulate channel sensitivity. Neuron 54 , 905-918 (2007). [PubMed] B. R. Myers, D. Julius, TRP channel structural biology: New roles for an old fold. Neuron 54 , 847-850 (2007). [PubMed]

Calcium-Dependent Association of Calmodulin with the C-Terminal Domain of the Tetraspanin Protein Peripherin/rds

Peripherin/rds (p/rds), an integral membrane protein from the transmembrane 4 (TMF4) superfamily, possesses a multi-functional C-terminal domain that plays crucial roles in rod outer segment (ROS) disk renewal and structure. Here, we report that the calcium binding protein calmodulin (CaM) binds to the C-terminal domain of p/rds. Fluorescence spectroscopy reveals Ca2+-dependent association of CaM with a polypeptide corresponding to the C-terminal domain of p/rds. The fluorescence anisotropy of the polypeptide upon CaM titration yields a dissociation constant (KD) of 320 +/- 150 nM. The results of the fluorescence experiments were confirmed by GST-pull down analyses in which a GST-p/rds C-terminal domain fusion protein was shown to pull down CaM in a calcium-dependent manner. Moreover, molecular modeling and sequence predictions suggest that the CaM binding domain resides in a p/rds functional hot spot, between residues E314 and G329. Predictions were confirmed by peptide competition studies and a GST-p/rds C-terminal domain construct in which the putative Ca2+/CaM binding site was scrambled. This GST-polypeptide did not associate with Ca2+/CaM. This putative calmodulin domain is highly conserved between human, mouse, rat, and bovine p/rds. Finally, the binding of Ca2+/CaM inhibited fusion between ROS disk and ROS plasma membranes as well as p/rds C-terminal-domain-induced fusion in model membrane studies. These results offer a new mechanism for the modulation of p/rds function.

Calmodulin, conformational states, and calcium signaling. A single-molecule perspective.

Single-molecule fluorescence measurements can provide a new perspective on the conformations, dynamics, and interactions of proteins. Recent examples are described illustrating the application of single-molecule fluorescence spectroscopy to calcium signaling proteins with an emphasis on the new information available in single-molecule fluorescence burst measurements, resonance energy transfer, and polarization modulation methods. Calcium signaling pathways are crucial in many cellular processes. The calcium binding protein calmodulin (CaM) serves as a molecular switch to regulate a network of calcium signaling pathways. Single-molecule spectroscopic methods can yield insights into conformations and dynamics of CaM and CaM-regulated proteins. Examples include studies of the conformations and dynamics of CaM, binding of target peptides, and interaction with the plasma-membrane Ca2+ pump. Single-molecule resonance energy transfer measurements revealed conformational substates of CaM, and single-molecule polarization modulation spectroscopy was used to probe interactions between CaM and the plasma-membrane Ca2+-ATPase.

GM‐CSF Expression in the Lung During Pneumocystis Pneumonia

GRANULOCYTE macrophage-colony stimulating factor (GM-CSF) is an important growth factor that promotes proliferation and maturation of macrophages and monocytes (Shibata et al. 2001). Granulocyte macrophage-colony stimulating factor also mediates cytokine production (Kremlev et al. 1998), stimulates expression of FcgR1 receptors and actin polymerization to increase phagocyte function (Berclaz et al. 2002), inhibits neutrophil apoptosis (Kobayashi et al. 2005), and enhances clearance of surfactant protein by alveolar macrophages in the lung (Ikegami et al. 1996). Granulocyte macrophage-colony stimulating factor present in the alveolar spaces of the lung is produced by alveolar macrophages, T lymphocytes, fibroblasts, and endothelial cells. The expression level of GM-CSF in some cell types is controlled by the ubiquitous calcium binding protein calmodulin through calmodulin-dependent protein kinase II (Liu and Grundstrom 2002). In Pneumocystis pneumonia (PcP), there is a reduction in alveolar macrophage-mediated phagocytosis (Lasbury et al. 2003), a build-up of surfactant protein A (Atochina et al. 2001; Phelps and Rose 1991), and a loss of alveolar macrophages by increased apoptosis (Lasbury et al. 2006). The present study sought to measure the levels of GM-CSF in alveolar macrophages and bronchoalveolar lavage (BAL) fluids from rats with PcP. We also examined the role of calmodulin in controlling GM-CSF expression in alveolar macrophages.

Ion Regulation of Homotypic Vacuole Fusion in Saccharomyces cerevisiae

Biological membrane fusion employs divalent cations as protein cofactors or as signaling ligands. For example, Mg2+ is a cofactor for the N-ethylmaleimide-sensitive factor (NSF) ATPase, and the Ca2+ signal from neuronal membrane depolarization is required for synaptotagmin activation. Divalent cations also regulate liposome fusion, but the role of such ion interactions with lipid bilayers in Rab- and soluble NSF attachment protein receptor (SNARE)-dependent biological membrane fusion is less clear. Yeast vacuole fusion requires Mg2+ for Sec18p ATPase activity, and vacuole docking triggers an efflux of luminal Ca2+. We now report distinct reaction conditions where divalent or monovalent ions interchangeably regulate Rab- and SNARE-dependent vacuole fusion. In reactions with 5 mm Mg2+, other free divalent ions are not needed. Reactions containing low Mg2+ concentrations are strongly inhibited by the rapid Ca2+ chelator BAPTA. However, addition of the soluble SNARE Vam7p relieves BAPTA inhibition as effectively as Ca2+ or Mg2+, suggesting that Ca2+ does not perform a unique signaling function. When the need for Mg2+, ATP, and Sec18p for fusion is bypassed through the addition of Vam7p, vacuole fusion does not require any appreciable free divalent cations and can even be stimulated by their chelators. The similarity of these findings to those with liposomes, and the higher ion specificity of the regulation of proteins, suggests a working model in which ion interactions with bilayer lipids permit Rab- and SNARE-dependent membrane fusion.

Regulation of the voltage-gated potassium channel KCNQ4 in the auditory pathway

The potassium channel KCNQ4, expressed in the mammalian cochlea, has been associated tentatively with an outer hair cell (OHC) potassium current, I(K,n), a current distinguished by an activation curve shifted to exceptionally negative potentials. Using CHO cells as a mammalian expression system, we have examined the properties of KCNQ4 channels under different phosphorylation conditions. The expressed current showed the typical KCNQ4 voltage-dependence, with a voltage for half-maximal activation (V(1/2)) of -25 mV, and was blocked almost completely by 200 microM linopirdine. Application of 8-bromo-cAMP or the catalytic sub-unit of PKA shifted V(1/2) by approximately -10 and -20 mV, respectively. Co-expression of KCNQ4 and prestin, the OHC motor protein, altered the voltage activation by a further -15 mV. Currents recorded with less than 1 nM Ca(2+) in the pipette ran down slowly (12% over 5 min). Buffering the pipette Ca(2+) to 100 nM increased the run-down rate sevenfold. Exogenous PKA in the pipette prevented the effect of elevated [Ca(2+)](i) on run-down. Inhibition of the calcium binding proteins calmodulin or calcineurin by W-7 or cyclosporin A, respectively, also prevented the calcium-dependent rapid run-down. We suggest that KCNQ4 phosphorylation via PKA and coupling to a complex that may include prestin can lead to the negative activation and the negative resting potential found in adult OHCs.