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Acid-sensing ion channels (ASICs) are proton-gated cation channels widely expressed in the nervous system. ASIC gating is modulated by divalent cations as well as small molecules; however, the molecular determinants of gating modulation by divalent cations are not well understood. Previously, we identified two small molecules that bind to ASIC1a at a novel site in the acidic pocket and modulate ASIC1 gating in a manner broadly resembling divalent cations, raising the possibility that these small molecules may help to illuminate the molecular determinants of gating modulation by divalent cations. Here, we examined how these two groups of modulators might interact as well as mutational effects on ASIC1a gating and its modulation by divalent cations. Our results indicate that binding of divalent cations to an acidic pocket site plays a key role in gating modulation of the channel., (© 2024. The Author(s).)

Swelling of epidermal cell walls decreases cell-to-cell adhesion and increases cracking susceptibility in sweet cherry. Ca is suggested to decrease cracking susceptibility by crosslinking of cell wall components and, possibly, by decreasing swelling. The objective is to test this hypothesis. The effect of Ca on swelling of anticlinal epidermal cell walls was quantified microscopically in vivo using excised skin sections and in vitro using extracted cell walls. After removal of turgor, cell wall thickness increased. Incubation in CaCl 2 decreased cell wall thickness up to 3 mM CaCl 2 . At higher concentrations thickness remained constant. Decreased cell wall swelling in vivo also occurred with other salts of divalent and trivalent cations, but not with those of monovalent cations. Decreased swelling was due to the Ca cation, the anions had no effect. Ca also decreased swelling of cell walls that were already swollen. CaCl 2 also decreased swelling of extracted cell walls in vitro. There was no effect on swelling pressure. The effect on swelling increased as the CaCl 2 concentration increased. Chlorides of divalent and trivalent cations, but not those of monovalent cations decreased swelling in vitro. The decrease in swelling among the divalent cations was linearly related to the radius of the cation. The results indicate that Ca decreases cracking susceptibility by decreasing swelling., (© 2022. The Author(s).)

The expression of the channels-enzymes TRPM6 and TRPM7 in the human heart remains poorly defined, and TRPM6 is generally considered not to be expressed in cardiomyocytes. We examined their expression at protein and mRNA levels using right atrial samples resected from patients (n = 72) with or without ischemic heart disease (IHD) and samples from all chamber walls of explanted human hearts (n = 9). TRPM6 and TRPM7 proteins were detected using immunofluorescence on isolated cardiomyocytes, ELISA on tissue homogenates, and immunostaining of cardiac tissue, whereas their mRNAs were detected by RT-qPCR. Both TRPM6 and TRPM7 were present in all chamber walls, with TRPM7 being more abundant. TRPM6 was co-expressed with TRPM7. The expression levels were dependent on cell incubation conditions (presence or absence of divalent cations, pH of the extracellular milieu, presence of TRP channel inhibitors 2-aminoethoxydiphenyl-borate and carvacrol). These drugs reduced TRPM7 immunofluorescence but increased that of TRPM6. TRPM6 and TRPM7 expression was increased in tissues from IHD patients. This is the first demonstration of the presence and co-expression of TRPM6 and TRPM7 in cardiomyocytes from all chamber walls of the human heart. The increased TRPM6 and TRPM7 expression in IHD suggests that the chanzymes are involved in the pathophysiology of the disease., (© 2021. The Author(s).)

We previously demonstrated that loss of Cdk5 in breast cancer cells promotes ROS-mediated cell death by inducing mitochondrial permeability transition pore (mPTP) opening (Oncogene 37, 1788-1804). However, the molecular mechanism by which Cdk5 loss causes mPTP opening remains to be investigated. Using primary mouse embryonic fibroblasts (MEFs) isolated from Cdk5 -/- mouse embryos, we show that absence of Cdk5 causes a significant increase in both mPTP opening and mitochondrial Ca 2+ level. Analysis of subcellular fractions of MEFs demonstrates that Cdk5 localizes in the mitochondria-associated endoplasmic reticulum (ER) membrane (MAM) and Cdk5 loss in MAMs causes increased ER-mitochondria tethering, a process required for Ca 2+ transfer from the ER to the mitochondria. Loss of Cdk5 also causes increased ATP-mediated mitochondrial Ca 2+ uptake from the ER. Inhibition of ER Ca 2+ release or mitochondrial Ca 2+ uptake in Cdk5 -/- MEFs prevents mPTP opening, indicating that mPTP opening in Cdk5 -/- MEFs is due to increased Ca 2+ transfer from the ER to the mitochondria. Altogether, our findings suggest that Cdk5 in MAMs regulates mitochondrial Ca 2+ homeostasis that is disturbed upon Cdk5 loss, which leads to mPTP opening.

Pannexin 3 (Panx3) is a regulator of bone formation. Panx3 forms three distinct functional channels: hemichannels, gap junctions, and endoplasmic reticulum (ER) Ca 2+ channels. However, the gating mechanisms of the Panx3 channels remain unclear. Here, we show that the Panx3 ER Ca 2+ channel is modulated by phosphorylation of the serine 68 residue (Ser68) to promote osteoblast differentiation. Among the 17 candidate phosphorylation sites identified, the mutation of Ser68 to Ala (Ser68Ala) was sufficient to inhibit Panx3-mediated osteoblast differentiation via reduction of Osterix and ALP expression. Using a Ser68 phospho-specific antibody (P-Panx3) revealed Panx3 was phosphorylated in prehypertrophic, hypertrophic chondrocytes, and bone areas of the newborn growth plate. In osteogenic C2C12 cells, P-Panx3 was located on the ER membranes. Importantly, the Ser68Ala mutation only affected Panx3 ER Ca 2+ channel function. Ser68 on Panx3 was phosphorylated by ATP stimulation and PI3K/Akt signaling. Finally, real-time FRET imaging and ratio analysis revealed that the Panx3 channel conformation was sensitive to ATP. Together, the phosphorylation of Panx3 at Ser68 is an essential step controlling the gating of the Panx3 ER Ca 2+ channel to promote osteogenesis.

Exploration of microbial-meteorite redox interactions highlights the possibility of bioprocessing of extraterrestrial metal resources and reveals specific microbial fingerprints left on extraterrestrial material. In the present study, we provide our observations on a microbial-meteorite nanoscale interface of the metal respiring thermoacidophile Metallosphaera sedula. M. sedula colonizes the stony meteorite Northwest Africa 1172 (NWA 1172; an H5 ordinary chondrite) and releases free soluble metals, with Ni ions as the most solubilized. We show the redox route of Ni ions, originating from the metallic Ni° of the meteorite grains and leading to released soluble Ni 2+ . Nanoscale resolution ultrastructural studies of meteorite grown M. sedula coupled to electron energy loss spectroscopy (EELS) points to the redox processing of Fe-bearing meteorite material. Our investigations validate the ability of M. sedula to perform the biotransformation of meteorite minerals, unravel microbial fingerprints left on meteorite material, and provide the next step towards an understanding of meteorite biogeochemistry. Our findings will serve in defining mineralogical and morphological criteria for the identification of metal-containing microfossils.

Phytic acid (PA) is a natural-occurring antioxidant, which plays an important role in many biological processes. PA is recognized as a potent inhibitor of lipid peroxidation because of its high affinity to multivalent cations, and it can play a role in osteogenic processes. However, its powerful chelating capacity is controversial because it can lead to a severe reduction of mineral availability in the organism. For this reason, compounds with beneficial biological properties of PA, but a modular ion binding capacity, are of high interest. In this work, we report the synthesis and physicochemical characterization of two hydroxylic derivatives of PA, named glycerylphytates (GPhy), through a condensation reaction of PA with glycerol (G). Both derivatives present antioxidant properties, measured by ferrozine/FeCl 2 method and chelating activity with calcium ions depending on the content of glyceryl groups incorporated. Besides, the hydroxylic modification not only modulates the ion binding affinity of derivatives but also improves their cytocompatibility in human bone marrow mesenchymal cells (MSCs). Furthermore, GPhy derivatives display osteogenic properties, confirmed by COL1A and ALPL expression depending on composition. These positive features convert GPhy compounds into potent alternatives for those skeletal diseases treatments where PA is tentatively applied.

Ca 2+ -transcription coupling controls gene expression patterns that define vascular smooth muscle cell (VSMC) phenotype. Although not well understood this allows normally contractile VSMCs to become proliferative following vessel injury, a process essential for repair but which also contributes to vascular remodelling, atherogenesis and restenosis. Here we show that the Ca 2+ /HCO 3 - -sensitive enzyme, soluble adenylyl cyclase (sAC), links Ca 2+ influx in human coronary artery smooth muscle cells (hCASMCs) to 3',5'-cyclic adenosine monophosphate (cAMP) generation and phosphorylation of the transcription factor Ca 2+ /cAMP response element binding protein (CREB). Store-operated Ca 2+ entry (SOCE) into hCASMCs expressing the FRET-based cAMP biosensor H187 induced a rise in cAMP that mirrored cytosolic [Ca 2+ ]. SOCE also activated the cAMP effector, protein kinase A (PKA), as determined by the PKA reporter, AKAR4-NES, and induced phosphorylation of vasodilator-stimulated phosphoprotein (VASP) and CREB. Transmembrane adenylyl cyclase inhibition had no effect on the SOCE-induced rise in cAMP, while sAC inhibition abolished SOCE-generated cAMP and significantly reduced SOCE-induced VASP and CREB phosphorylation. This suggests that SOCE in hCASMCs activates sAC which in turn activates the cAMP/PKA/CREB axis. sAC, which is insensitive to G-protein modulation but responsive to Ca 2+ , pH and ATP, may therefore act as an overlooked regulatory node in vascular Ca 2+ -transcription coupling.

In most species, fertilization induces Ca 2+ transients in the egg. In mammals, the Ca 2+ rises are triggered by phospholipase Cζ (PLCζ) released from the sperm; IP 3 generated by PLCζ induces Ca 2+ release from the intracellular Ca 2+ store through IP 3 receptor, termed IP 3 -induced Ca 2+ release. Here, we developed new fluorescent IP 3 sensors (IRIS-2s) with the wider dynamic range and higher sensitivity (Kd = 0.047-1.7 μM) than that we developed previously. IRIS-2s employed green fluorescent protein and Halo-protein conjugated with the tetramethylrhodamine ligand as fluorescence resonance energy transfer (FRET) donor and acceptor, respectively. For simultaneous imaging of Ca 2+ and IP 3 , using IRIS-2s as the IP 3 sensor, we developed a new single fluorophore Ca 2+ sensor protein, DYC3.60. With IRIS-2s and DYC3.60, we found that, right after fertilization, IP 3 concentration ([IP 3 ]) starts to increase before the onset of the first Ca 2+ wave. [IP 3 ] stayed at the elevated level with small peaks followed after Ca 2+ spikes through Ca 2+ oscillations. We detected delays in the peak of [IP 3 ] compared to the peak of each Ca 2+ spike, suggesting that Ca 2+ -induced regenerative IP 3 production through PLC produces small [IP 3 ] rises to maintain [IP 3 ] over the basal level, which results in long lasting Ca 2+ oscillations in fertilized eggs.

The prion protein (PrP) is a cell surface protein that in disease misfolds and becomes infectious causing Creutzfeldt-Jakob disease in humans, scrapie in sheep, and chronic wasting disease in deer and elk. Little is known regarding the dimerization of PrP and its role in disease. We developed a bioluminescent prion assay (BPA) to quantify PrP dimerization by bimolecular complementation of split Gaussia luciferase (GLuc) halves that are each fused to PrP. Fusion constructs between PrP and N- and C-terminal GLuc halves were expressed on the surface of RK13 cells (RK13-DC cells) and dimerized to yield a bioluminescent signal that was decreased in the presence of eight different antibodies to PrP. Dimerization of PrP was independent of divalent cations and was induced under stress. Challenge of RK13-DC cells with seven different prion strains did not lead to detectable infection but was measurable by bioluminescence. Finally, we used BPA to screen a compound library for compounds inhibiting PrP dimerization. One of the most potent compounds to inhibit PrP dimerization was JTC-801, which also inhibited prion replication in RML-infected ScN2a and SMB cells with an EC 50 of 370 nM and 220 nM, respectively. We show here that BPA is a versatile tool to study prion biology and to identify anti-prion compounds.

A synthetic derivative, GnRH [6-D-Phe], stable against enzymatic degradation, self-assembles and forms nanostructures and fibrils upon a pH shift in the presence of different concentrations of Zn 2+ in vitro. Attenuated Total Reflection Fourier Transform Infrared spectroscopy (ATR-FTIR) revealed the existence of higher order assembly of Zn 2+ : GnRH [6-D-Phe]. Nuclear Magnetic Resonance spectroscopy (NMR) indicated a weak interaction between Zn 2+ and GnRH [6-D-Phe]. Atomic Force Microscopy (AFM) showed the existence of GnRH [6-D-Phe] oligomers and fibrils. Molecular Dynamic (MD) simulation of the 10:1 Zn 2+ : GnRH [6-D-Phe] explored the interaction and dimerization processes. In contrast to already existing short peptide fibrils, GnRH [6-D-Phe] nanostructures and fibrils form in a Tris-buffered pH environment in a controlled manner through a temperature reduction and a pH shift. The lyophilized Zn 2+ : GnRH [6-D-Phe] assembly was tested as a platform for the sustained delivery of GnRH [6-D-Phe] and incorporated into two different oil vehicle matrices. The in vitro release was slow and continuous over 14 days and not influenced by the oil matrix.

Human KLK8/neuropsin, a kallikrein-related serine peptidase, is mostly expressed in skin and the hippocampus regions of the brain, where it regulates memory formation by synaptic remodeling. Substrate profiles of recombinant KLK8 were analyzed with positional scanning using fluorogenic tetrapeptides and the proteomic PICS approach, which revealed the prime side specificity. Enzyme kinetics with optimized substrates showed stimulation by Ca 2+ and inhibition by Zn 2+ , which are physiological regulators. Crystal structures of KLK8 with a ligand-free active site and with the inhibitor leupeptin explain the subsite specificity and display Ca 2+ bound to the 75-loop. The variants D70K and H99A confirmed the antagonistic role of the cation binding sites. Molecular docking and dynamics calculations provided insights in substrate binding and the dual regulation of activity by Ca 2+ and Zn 2+ , which are important in neuron and skin physiology. Both cations participate in the allosteric surface loop network present in related serine proteases. A comparison of the positional scanning data with substrates from brain suggests an adaptive recognition by KLK8, based on the tertiary structures of its targets. These combined findings provide a comprehensive picture of the molecular mechanisms underlying the enzyme activity of KLK8.

Zinc modulates the biological function of histidine-rich glycoprotein (HRG) through binding to its His-rich region (HRR). The Zn 2+ -binding properties of a 35 amino-acid biologically-active peptide mimic of the HRR, HRGP330, were investigated using dissociative mass spectrometry approaches in addition to travelling-wave ion mobility mass spectrometry (TWIM-MS). Native mass spectrometry confirmed zinc binding to HRGP330; however, broadening of the 1 H NMR resonances upon addition of Zn 2+ ions precluded the attainment of structural information. A complementary approach employing TWIM-MS indicated that HRGP330 has a more compact structure in the presence of Zn 2+ ions. Top-down MS/MS data supported a metal-binding-induced conformational change, as fewer fragments were observed for Zn 2+ -bound HRGP330. Zn 2+ -bound fragments of both N-terminal and C-terminal ends of the peptide were identified from collision-induced dissociation (CID) and electron transfer dissociation/proton transfer reaction (ETD/PTR) experiments, suggesting that multiple binding sites exist within this region of HRG. The combination of mass spectrometry and NMR approaches provides new insight into the highly dynamic interaction between zinc and this His-rich peptide.

The ryanodine receptor 1 is a large calcium ion channel found in mammalian skeletal muscle. The ion channel gained a lot of attention recently, after multiple independent authors published near-atomic cryo electron microscopy data. Taking advantage of the unprecedented quality of structural data, we performed molecular dynamics simulations on the entire ion channel as well as on a reduced model. We calculated potentials of mean force for Ba 2+ , Ca 2+ , Mg 2+ , K + , Na + and Cl - ions using umbrella sampling to identify the key residues involved in ion permeation. We found two main binding sites for the cations, whereas the channel is strongly repulsive for chloride ions. Furthermore, the data is consistent with the model that the receptor achieves its ion selectivity by over-affinity for divalent cations in a calcium-block-like fashion. We reproduced the experimental conductance for potassium ions in permeation simulations with applied voltage. The analysis of the permeation paths shows that ions exit the pore via multiple pathways, which we suggest to be related to the experimental observation of different subconducting states.

α-Synuclein (αSyn) is an intrinsically disordered protein, the aggregation of which is highly related to the pathology of diverse α-synucleinopathies. Various hard divalent metal cations have been shown to affect αSyn aggregation. Especially, Ca 2+ is suggested to be a crucial ion due to its physiological relevance to α-synucleinopathies. However, the molecular origin of αSyn aggregation mediated by the metal ions is not fully elucidated. In this study, we revealed that hard divalent metal ions had almost identical influences on αSyn aggregation. Based on these similarities, the molecular role of Ca 2+ was investigated as a representative metal ion. Herein, we demonstrated that binding of multiple Ca 2+ ions induces structural transition of αSyn monomers to extended conformations, which promotes rapid αSyn fibrillation. Additionally, we observed that Ca 2+ induced further interfibrillar aggregation via electrostatic and hydrophobic interactions. Our results from multiple biophysical methods, including ion mobility-mass spectrometry (IM-MS), synchrotron small-angle X-ray scattering (SAXS), transmission electron microscopy (TEM), provide detailed information on the structural change of αSyn and the aggregation process mediated by Ca 2+ . Overall, our study would be valuable for understanding the influence of Ca 2+ on the aggregation of αSyn during the pathogenesis of α-synucleinopathies.

Hypertension is a systemic disorder that affects numerous physiological processes throughout the body. Improper sodium transport is a common comorbidity of hypertension, and sodium transport is also critical for maintaining the secretion of submandibular glands, whether the function of submandibular glands is affected by hypertension remains unclear. To determine whether hypertension induces changes in the protein expression of submandibular glands, we compared the proteome of submandibular glands from 14-week-old spontaneously hypertensive rats (SHR) and Wistar Kyoto (WKY) rats using LC-MS/MS. The results revealed that 95 proteins displayed different levels of expression between the submandibular glands from the SHRs and WKYs. Among these, 35 proteins were more abundant, and 60 proteins were less abundant in the SHR compared with the WKY rats. Specifically, aquaporin 5 and parvalbumin, which are correlated with water transport and intracellular Ca 2+ signal transduction, were verified to exhibit differences in protein abundance. Impaired Ca 2+ response to carbachol was confirmed in the acinar cells from SHRs, and hyposecretion by the submandibular glands was further confirmed by in vivo saliva collection. In conclusion, the proteomic analysis of the submandibular glands of SHRs revealed novel changes in protein abundance that provides possible mechanisms connecting hypertension and hyposecretion in submandibular glands.

Loss of synapses or alteration of synaptic activity is associated with cognitive impairment observed in a number of psychiatric and neurological disorders, such as schizophrenia and Alzheimer's disease. Therefore successful development of in vitro methods that can investigate synaptic function in a high-throughput format could be highly impactful for neuroscience drug discovery. We present here the development, characterisation and validation of a novel high-throughput in vitro model for assessing neuronal function and synaptic transmission in primary rodent neurons. The novelty of our approach resides in the combination of the electrical field stimulation (EFS) with data acquisition in spatially separated areas of an interconnected neuronal network. We integrated our methodology with state of the art drug discovery instrumentation (FLIPR Tetra) and used selective tool compounds to perform a systematic pharmacological validation of the model. We investigated pharmacological modulators targeting pre- and post-synaptic receptors (AMPA, NMDA, GABA-A, mGluR2/3 receptors and Nav, Cav voltage-gated ion channels) and demonstrated the ability of our model to discriminate and measure synaptic transmission in cultured neuronal networks. Application of the model described here as an unbiased phenotypic screening approach will help with our long term goals of discovering novel therapeutic strategies for treating neurological disorders.

Store-operated Ca 2+ entry (SOCE), a ubiquitous mechanism that allows recovery of Ca 2+ ions from the extracellular space, has been proposed to limit fatigue during repetitive skeletal muscle activity. However, the subcellular location for SOCE in muscle fibers has not been unequivocally identified. Here we show that exercise drives a significant remodeling of the sarcotubular system to form previously unidentified junctions between the sarcoplasmic reticulum (SR) and transverse-tubules (TTs). We also demonstrate that these new SR-TT junctions contain the molecular machinery that mediate SOCE: stromal interaction molecule-1 (STIM1), which functions as the SR Ca 2+ sensor, and Orai1, the Ca 2+ -permeable channel in the TT. In addition, EDL muscles isolated from exercised mice exhibit an increased capability of maintaining contractile force during repetitive stimulation in the presence of 2.5 mM extracellular Ca 2+ , compared to muscles from control mice. This functional difference is significantly reduced by either replacement of extracellular Ca 2+ with Mg 2+ or the addition of SOCE inhibitors (BTP-2 and 2-APB). We propose that the new SR-TT junctions formed during exercise, and that contain STIM1 and Orai1, function as Ca 2+ Entry Units (CEUs), structures that provide a pathway to rapidly recover Ca 2+ ions from the extracellular space during repetitive muscle activity.

Anaphylactoid reactions are common clinical acute adverse drug reactions that can exacerbate a patient's condition and produce effects that may become life-threatening. Therefore, it is important to establish a novel method to evaluate drugs for anaphylactoid reactions. In this study, we developed a sensitive and rapid method to detect histamine release from LAD2 cells using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and constructed a relative release index based on various release curve parameters, including allergen release time and sudden change rate, to evaluate the potential and strength of allergen-induced anaphylactoid reactions. This LAD2 release model was used to evaluate anaphylactoid reactions induced by ciprofloxacin, norfloxacin, lomefloxacin, moxifloxacin, and baicalin. The results positively correlated with those obtained with an Evans blue ear test and negatively correlated with the Ca 2+ influx EC 50 . In summary, the current study established a novel in vitro method to analyze the properties of histamine release from LAD2 cells and characterize the sensitization and strength of sensitization of drugs or components that may induce anaphylactoid reactions.

Acidic microenvironment is commonly observed in tumour tissues, including glioblastoma (GBM), the most aggressive and lethal brain tumour in adults. Acid sensing ion channels (ASICs) are neuronal voltage-insensitive sodium channels, which are sensors of extracellular protons. Here we studied and functionally characterized ASICs in two primary glioblastoma stem cell lines as cell culture models. We detected transcripts of the ACCN2 and ACCN3 genes, coding for ASIC1 and ASIC3, respectively, but not transcripts of ACCN1 (coding for ASIC2). Available microarray data confirmed that ACCN1 is downregulated in glioma. Western blotting confirmed expression of ASIC1 and ASIC3, the most proton-sensitive ASICs, in both GBM cell lines. We characterized ASICs functionally using whole-cell patch clamp and detected different types of acid-sensitive currents. Some of these currents had kinetics typical for ASICs and were sensitive to specific toxin inhibitors of ASIC1a or ASIC3, demonstrating that the GBM cell lines express functional ASIC1a and ASIC3 that may enable GBM cells to sensitively detect extracellular pH in a tumour tissue. Microarray data revealed that expression of ACCN2 and ACCN3 is associated with improved survival of patients suffering from gliomas, suggesting that preserved susceptibility to extracellular pH may impair tumour growth.

The clinical significance of STIM proteins and Orai Ca 2+ channels in tumor progression has been demonstrated in different types of cancers. Podosomes are dynamic actin-rich cellular protrusions that facilitate cancer cell invasiveness by degrading extracellular matrix. Whether STIM1-dependent Ca 2+ signaling facilitates cancer cell invasion through affecting podosome formation remains unclear. Here we show that the invasive fronts of cancer tissues overexpress STIM1, accompanied by active store-operated Ca 2+ entry (SOCE). Interfering SOCE activity by SOCE inhibitors and STIM1 or Orai1 knockdown remarkably affects podosome rosettes formation. Mechanistically, STIM1-silencing significantly alters the podosome rosettes dynamics, shortens the maintenance phase of podosome rosettes and reduces cell invasiveness. The subsequently transient expression of STIM1 cDNA in STIM1-null (STIM1 -/- ) mouse embryo fibroblasts rescues the suppression of podosome formation, suggesting that STIM1-mediated SOCE activation directly regulates podosome formation. This study uncovers SOCE-mediated Ca 2+ microdomain that is the molecular basis for Ca 2+ sensitivity controlling podosome formation.

Comparison of kinetic and thermodynamic properties of IRP1 (iron regulatory protein1) binding to FRT (ferritin) and ACO2 (aconitase2) IRE-RNAs, with or without Mn 2+ , revealed differences specific to each IRE-RNA. Conserved among animal mRNAs, IRE-RNA structures are noncoding and bind Fe 2+ to regulate biosynthesis rates of the encoded, iron homeostatic proteins. IRP1 protein binds IRE-RNA, inhibiting mRNA activity; Fe 2+ decreases IRE-mRNA/IRP1 binding, increasing encoded protein synthesis. Here, we observed heat, 5 °C to 30 °C, increased IRP1 binding to IRE-RNA 4-fold (FRT IRE-RNA) or 3-fold (ACO2 IRE-RNA), which was enthalpy driven and entropy favorable. Mn 2+ (50 µM, 25 °C) increased IRE-RNA/IRP1 binding (K d ) 12-fold (FRT IRE-RNA) or 6-fold (ACO2 IRE-RNA); enthalpic contributions decreased ~61% (FRT) or ~32% (ACO2), and entropic contributions increased ~39% (FRT) or ~68% (ACO2). IRE-RNA/IRP1 binding changed activation energies: FRT IRE-RNA 47.0 ± 2.5 kJ/mol, ACO2 IRE-RNA 35.0 ± 2.0 kJ/mol. Mn 2+ (50 µM) decreased the activation energy of RNA-IRP1 binding for both IRE-RNAs. The observations suggest decreased RNA hydrogen bonding and changed RNA conformation upon IRP1 binding and illustrate how small, conserved, sequence differences among IRE-mRNAs selectively influence thermodynamic and kinetic selectivity of the protein/RNA interactions.

Lagging strand DNA synthesis by DNA polymerase requires RNA primers produced by DNA primase. The N-terminal primase domain of the gene 4 protein of phage T7 comprises a zinc-binding domain that recognizes a specific DNA sequence and an RNA polymerase domain that catalyzes RNA polymerization. Based on its crystal structure, the RNA polymerase domain contains two Mg(II) ions. Mn(II) substitution leads to elevated RNA primer synthesis by T7 DNA primase. NMR analysis revealed that upon binding Mn(II), T7 DNA primase undergoes conformational changes near the metal cofactor binding site that are not observed when the enzyme binds Mg(II). A machine-learning algorithm called linear discriminant analysis (LDA) was trained by using the large collection of Mn(II) and Mg(II) binding sites available in the protein data bank (PDB). Application of the model to DNA primase revealed a preference in the enzyme's second metal binding site for Mn(II) over Mg(II), suggesting that T7 DNA primase activity modulation when bound to Mn(II) is based on structural changes in the enzyme.

Vancomycin is known to bind to Zn(II) and can induce a zinc starvation response in bacteria. Here we identify a novel polymerization of vancomycin dimers by structural analysis of vancomycin-Zn(II) crystals and fibre X-ray diffraction. Bioassays indicate that this structure is associated with an increased antibiotic activity against bacterial strains possessing high level vancomycin resistance mediated by the reprogramming of peptidoglycan biosynthesis to use precursors terminating in D-Ala-D-Lac in place of D-Ala-D-Ala. Polymerization occurs via interaction of Zn(II) with the N-terminal methylleucine group of vancomycin, and we show that the activity of other glycopeptide antibiotics with this feature can also be similarly augmented by Zn(II). Construction and analysis of a model strain predominantly using D-Ala-D-Lac precursors for peptidoglycan biosynthesis during normal growth supports the hypothesis that Zn(II) mediated vancomycin polymerization enhances the binding affinity towards these precursors.

The Ca 2+ -activated K + channel, Slo1, has an unusually large conductance and contains a voltage sensor and multiple chemical sensors. Dual activation by membrane voltage and Ca 2+ renders Slo1 central to processes that couple electrical signalling to Ca 2+ -mediated events such as muscle contraction and neuronal excitability. Here we present the cryo-electron microscopy structure of a full-length Slo1 channel from Aplysia californica in the presence of Ca 2+ and Mg 2+ at a resolution of 3.5 Å. The channel adopts an open conformation. Its voltage-sensor domain adopts a non-domain-swapped attachment to the pore and contacts the cytoplasmic Ca 2+ -binding domain from a neighbouring subunit. Unique structural features of the Slo1 voltage sensor suggest that it undergoes different conformational changes than other known voltage sensors. The structure reveals the molecular details of three distinct divalent cation-binding sites identified through electrophysiological studies of mutant Slo1 channels.

Sildenafil relaxes vascular smooth muscle cells and is used to treat pulmonary artery hypertension as well as erectile dysfunction. However, the effectiveness of sildenafil on skeletal muscle and the benefit of its clinical use have been controversial, and most studies focus primarily on tissues and organs from disease models without cellular examination. Here, the effects of sildenafil on skeletal muscle at the cellular level were examined using mouse primary skeletal myoblasts (the proliferative form of skeletal muscle stem cells) and myotubes, along with single-cell Ca 2+ imaging experiments and cellular and biochemical studies. The proliferation of skeletal myoblasts was enhanced by sildenafil in a dose-independent manner. In skeletal myotubes, sildenafil enhanced the activity of ryanodine receptor 1, an internal Ca 2+ channel, and Ca 2+ movement that promotes skeletal muscle contraction, possibly due to an increase in the resting cytosolic Ca 2+ level and a unique microscopic shape in the myotube membranes. Therefore, these results suggest that the maintenance ability of skeletal muscle mass and the contractility of skeletal muscle could be improved by sildenafil by enhancing the proliferation of skeletal myoblasts and increasing the Ca 2+ availability of skeletal myotubes, respectively.

The pro-apoptotic Bcl-2 protein Bax can permeabilize the outer mitochondrial membrane and therefore commit human cells to apoptosis. Bax is regulated by constant translocation to the mitochondria and retrotranslocation back into the cytosol. Bax retrotranslocation depends on pro-survival Bcl-2 proteins and stabilizes inactive Bax. Here we show that Bax retrotranslocation shuttles membrane-associated and membrane-integral Bax from isolated mitochondria. We further discover the mitochondrial porin voltage-dependent anion channel 2 (VDAC2) as essential component and platform for Bax retrotranslocation. VDAC2 ensures mitochondria-specific membrane association of Bax and in the absence of VDAC2 Bax localizes towards other cell compartments. Bax retrotranslocation is also regulated by nucleotides and calcium ions, suggesting a potential role of the transport of these ions through VDAC2 in Bax retrotranslocation. Together, our results reveal the unanticipated bifunctional role of VDAC2 to target Bax specifically to the mitochondria and ensure Bax inhibition by retrotranslocation into the cytosol.

Precise regulation of calcium homeostasis is essential for many physiological functions. The Ca(2+)-selective transient receptor potential (TRP) channels TRPV5 and TRPV6 play vital roles in calcium homeostasis as Ca(2+) uptake channels in epithelial tissues. Detailed structural bases for their assembly and Ca(2+) permeation remain obscure. Here we report the crystal structure of rat TRPV6 at 3.25 Å resolution. The overall architecture of TRPV6 reveals shared and unique features compared with other TRP channels. Intracellular domains engage in extensive interactions to form an intracellular 'skirt' involved in allosteric modulation. In the K(+) channel-like transmembrane domain, Ca(2+) selectivity is determined by direct coordination of Ca(2+) by a ring of aspartate side chains in the selectivity filter. On the basis of crystallographically identified cation-binding sites at the pore axis and extracellular vestibule, we propose a Ca(2+) permeation mechanism. Our results provide a structural foundation for understanding the regulation of epithelial Ca(2+) uptake and its role in pathophysiology.

There is growing interest in anxiolytic and pro-social effects of the neuropeptide oxytocin (OXT), but the underlying intraneuronal mechanisms are largely unknown. Here we examined OXT-mediated anxiolysis in the hypothalamic paraventricular nucleus (PVN) of rats and effects of OXT administration on signaling events in hypothalamic primary and immortalized cells. In vivo, the application of SKF96365 prevented the anxiolytic activity of OXT in the PVN, suggesting that changes in intracellular Ca(2+) mediate the acute OXT behavioral effects. In vitro, mainly in the neurons with autonomous Ca(2+) oscillations, OXT increased intracellular Ca(2+) concentration and oscillation amplitude. Pharmacological intervention revealed OXT-dependent changes in Ca(2+) signaling that required activation of transient receptor potential vanilloid type-2 channel (TRPV2), mediated by phosphoinositide 3-kinase. TRPV2 induced the activation of the anxiolytic mitogen-activated protein kinase kinase (MEK1/2). In situ, immunohistochemistry revealed co-localization of TRPV2 and OXT in the PVN. Thus, functional and pharmacological analyses identified TRPV2 as a mediator of anxiolytic effects of OXT, conveying the OXT signal to MEK1/2 via modulation of intracellular Ca(2+).

Enzymes used for passaging human pluripotent stem cells (hPSCs) digest cell surface proteins, resulting in cell damage. Moreover, cell dissociation using divalent cation-free solutions causes apoptosis. Here we report that Mg(2+) and Ca(2+) control cell-fibronectin and cell-cell binding of hPSCs, respectively, under feeder- and serum-free culture conditions without enzyme. The hPSCs were detached from fibronectin-, vitronectin- or laminin-coated dishes in low concentrations of Mg(2+) and remained as large colonies in high concentrations of Ca(2+). Using enzyme-free solutions containing Ca(2+) without Mg(2+), we successfully passaged hPSCs as large cell clumps that showed less damage than cells passaged using a divalent cation-free solution or dispase. Under the same conditions, the undifferentiated and early-differentiated cells could also be harvested as a cell sheet without being split off. Our enzyme-free passage of hPSCs under a serum- and feeder-free culture condition reduces cell damage and facilitates easier and safer cultures of hPSCs.

Voltage-gated calcium (CaV) channels catalyse rapid, highly selective influx of Ca(2+) into cells despite a 70-fold higher extracellular concentration of Na(+). How CaV channels solve this fundamental biophysical problem remains unclear. Here we report physiological and crystallographic analyses of a calcium selectivity filter constructed in the homotetrameric bacterial NaV channel NaVAb. Our results reveal interactions of hydrated Ca(2+) with two high-affinity Ca(2+)-binding sites followed by a third lower-affinity site that would coordinate Ca(2+) as it moves inward. At the selectivity filter entry, Site 1 is formed by four carboxyl side chains, which have a critical role in determining Ca(2+) selectivity. Four carboxyls plus four backbone carbonyls form Site 2, which is targeted by the blocking cations Cd(2+) and Mn(2+), with single occupancy. The lower-affinity Site 3 is formed by four backbone carbonyls alone, which mediate exit into the central cavity. This pore architecture suggests a conduction pathway involving transitions between two main states with one or two hydrated Ca(2+) ions bound in the selectivity filter and supports a 'knock-off' mechanism of ion permeation through a stepwise-binding process. The multi-ion selectivity filter of our CaVAb model establishes a structural framework for understanding the mechanisms of ion selectivity and conductance by vertebrate CaV channels.

A series of olivomycin A derivatives containing different combinations of the acyl residues in the carbohydrate chains was obtained. The formation of complexes of Mg(2+)-coordinated dimers of these compounds with double-stranded DNA was studied using spectral methods such as absorption, fluorescence and circular dichroism (CD) spectral analyses. There was a good correlation of the values of binding constants of complexes (antibiotic)2Mg(2+)-DNA, the quantum yields of fluorescence and changes of the induced CD spectra with topoisomerase I inhibition and cytotoxicity. We demonstrate that the presence of the acyl groups in the saccharide residues of olivomycin A derivatives is absolutely necessary for a high cytotoxic potency of these antibiotics. On the basis of the experimental results and quantum chemical calculations, we presume that the acyl residue in the 4-O-position in the A-sugar residue is involved, to the most part, in the antibiotic-antibiotic interactions in the (olivomycin)2Mg(2+) dimers, whereas the O-acyl group in E-olivomicose residue largely participates in the formation of the (olivomycin)2Mg(2+)-DNA complexes.

Aim: The insulin-like growth factor-1 receptor (IGF1R) is over-expressed in a wide variety of tumors and contributes to tumor cell proliferation, metastasis and drug resistance. The aim of this study was to establish a sensitive screening platform to identify novel IGF1R inhibitors., Methods: The catalytic domain of IGF1R was expressed using the Bac-to-Bac baculovirus expression system. The screening platform for IGF1R inhibitors was established based on ELISA. The binding profile of IGF1R with the inhibitors was predicted with molecular docking and then subjected to the surface plasmon resonance (SPR) approach. The growth inhibition of cancer cells by the inhibitors was assessed with MTT assay. Apoptosis was analyzed using flow cytometry and Western blotting., Results: A naturally occurring small molecule compound hematoxylin was identified as the most potent inhibitor (IC₅₀ value=1.8±0.1 μmol/L) within a library of more than 200 compounds tested. Molecular simulation predicted the possible binding mode of hematoxylin with IGF1R. An SPR assay further confirmed that hematoxylin bound directly to IGF1R with high binding affinity (Kd=4.2 × 10⁻⁶ mol/L). In HL-60 cancer cells, hematoxylin inactivated the phosphorylation of IGF1R and downstream signaling and therefore suppressed cell proliferation. Mechanistic studies revealed that hematoxylin induced apoptosis in HL-60 cells via both extrinsic and intrinsic pathways., Conclusion: A simple, sensitive ELISA-based screening platform for identifying IGF1R inhibitors was established. Hematoxylin was identified as a promising IGF1R inhibitor with effective antitumor activity that deserves further investigation.

The slicer activity of the RNA-induced silencing complex resides within its Argonaute (Ago) component, in which the PIWI domain provides the catalytic residues governing guide-strand mediated site-specific cleavage of target RNA. Here we report on structures of ternary complexes of Thermus thermophilus Ago catalytic mutants with 5'-phosphorylated 21-nucleotide guide DNA and complementary target RNAs of 12, 15 and 19 nucleotides in length, which define the molecular basis for Mg(2+)-facilitated site-specific cleavage of the target. We observe pivot-like domain movements within the Ago scaffold on proceeding from nucleation to propagation steps of guide-target duplex formation, with duplex zippering beyond one turn of the helix requiring the release of the 3'-end of the guide from the PAZ pocket. Cleavage assays on targets of various lengths supported this model, and sugar-phosphate-backbone-modified target strands showed the importance of structural and catalytic divalent metal ions observed in the crystal structures.

The magnesium ion, Mg2+, is essential for myriad biochemical processes and remains the only major biological ion whose transport mechanisms remain unknown. The CorA family of magnesium transporters is the primary Mg2+ uptake system of most prokaryotes and a functional homologue of the eukaryotic mitochondrial magnesium transporter. Here we determine crystal structures of the full-length Thermotoga maritima CorA in an apparent closed state and its isolated cytoplasmic domain at 3.9 A and 1.85 A resolution, respectively. The transporter is a funnel-shaped homopentamer with two transmembrane helices per monomer. The channel is formed by an inner group of five helices and putatively gated by bulky hydrophobic residues. The large cytoplasmic domain forms a funnel whose wide mouth points into the cell and whose walls are formed by five long helices that are extensions of the transmembrane helices. The cytoplasmic neck of the pore is surrounded, on the outside of the funnel, by a ring of highly conserved positively charged residues. Two negatively charged helices in the cytoplasmic domain extend back towards the membrane on the outside of the funnel and abut the ring of positive charge. An apparent Mg2+ ion was bound between monomers at a conserved site in the cytoplasmic domain, suggesting a mechanism to link gating of the pore to the intracellular concentration of Mg2+.

HutP regulates the expression of the hut structural genes of Bacillus subtilis by an anti-termination mechanism and requires two components, Mg2+ ions and L-histidine. HutP recognizes three UAG triplet units, separated by four non-conserved nucleotides on the terminator region. Here we report the 1.60-A resolution crystal structure of the quaternary complex (HutP-L-histidine-Mg2+-21-base single-stranded RNA). In the complex, the RNA adopts a novel triangular fold on the hexameric surface of HutP, without any base-pairing, and binds to the protein mostly by specific protein-base interactions. The structure explains how the HutP and RNA interactions are regulated critically by the l-histidine and Mg2+ ion through the structural rearrangement. To gain insights into these structural rearrangements, we solved two additional crystal structures (uncomplexed HutP and HutP-L-histidine-Mg2+) that revealed the intermediate structures of HutP (before forming an active structure) and the importance of the Mg2+ ion interactions in the complexes.

Ribozymes enhance chemical reaction rates using many of the same catalytic strategies as protein enzymes. In the hepatitis delta virus (HDV) ribozyme, site-specific self-cleavage of the viral RNA phosphodiester backbone requires both divalent cations and a cytidine nucleotide. General acid-base catalysis, substrate destabilization and global and local conformational changes have all been proposed to contribute to the ribozyme catalytic mechanism. Here we report ten crystal structures of the HDV ribozyme in its pre-cleaved state, showing that cytidine is positioned to activate the 2'-OH nucleophile in the precursor structure. This observation supports its proposed role as a general base in the reaction mechanism. Comparison of crystal structures of the ribozyme in the pre- and post-cleavage states reveals a significant conformational change in the RNA after cleavage and that a catalytically critical divalent metal ion from the active site is ejected. The HDV ribozyme has remarkable chemical similarity to protein ribonucleases and to zymogens for which conformational dynamics are integral to biological activity. This finding implies that RNA structural rearrangements control the reactivity of ribozymes and ribonucleoprotein enzymes.

During intracellular Ca2+ signalling mitochondria accumulate significant amounts of Ca2+ from the cytosol. Mitochondrial Ca2+ uptake controls the rate of energy production, shapes the amplitude and spatio-temporal patterns of intracellular Ca2+ signals, and is instrumental to cell death. This Ca2+ uptake is undertaken by the mitochondrial Ca2+ uniporter (MCU) located in the organelle's inner membrane. The uniporter passes Ca2+ down the electrochemical gradient maintained across this membrane without direct coupling to ATP hydrolysis or transport of other ions. Carriers are characterized by turnover numbers that are typically 1,000-fold lower than ion channels, and until now it has been unclear whether the MCU is a carrier or a channel. By patch-clamping the inner mitochondrial membrane, we identified a previously unknown Ca2+-selective ion channel sensitive to inhibitors of mitochondrial Ca2+ uptake. Our data indicate that this unique channel binds Ca2+ with extremely high affinity (dissociation constant < or =2 nM), enabling high Ca2+ selectivity despite relatively low cytoplasmic Ca2+ concentrations. The channel is inwardly rectifying, making it especially effective for Ca2+ uptake into energized mitochondria. Thus, we conclude that the properties of the current mediated by this novel channel are those of the MCU.

Transient receptor potential (TRP) proteins are cation-selective channels that function in processes as diverse as sensation and vasoregulation. Mammalian TRP channels that are gated by heat and capsaicin (>43 degrees C; TRPV1 (ref. 1)), noxious heat (>52 degrees C; TRPV2 (ref. 2)), and cooling (< 22 degrees C; TRPM8 (refs 3, 4)) have been cloned; however, little is known about the molecular determinants of temperature sensing in the range between approximately 22 degrees C and 40 degrees C. Here we have identified a member of the vanilloid channel family, human TRPV3 (hTRPV3) that is expressed in skin, tongue, dorsal root ganglion, trigeminal ganglion, spinal cord and brain. Increasing temperature from 22 degrees C to 40 degrees C in mammalian cells transfected with hTRPV3 elevated intracellular calcium by activating a nonselective cationic conductance. As in published recordings from sensory neurons, the current was steeply dependent on temperature, sensitized with repeated heating, and displayed a marked hysteresis on heating and cooling. On the basis of these properties, we propose that hTRPV3 is thermosensitive in the physiological range of temperatures between TRPM8 and TRPV1.

Interactions with divalent metal ions are essential for the folding and function of the catalytic RNA component of the tRNA processing enzyme ribonuclease P (RNase P RNA). However, the number and location of specific metal ion interactions in this large, highly structured RNA are poorly understood. Using atomic mutagenesis and quantitative analysis of thiophilic metal ion rescue we provide evidence for metal ion interactions at the pro-R(P) and pro-S(P) non-bridging phosphate oxygens at nucleotide A67 in the universally conserved helix P4. Moreover, second-site modifications within helix P4 and the adjacent single stranded region (J3/4) provide the first evidence for metal ion interactions with nucleotide base functional groups in RNase P RNA and reveal the presence of an additional metal ion important for catalytic function. Together, these data are consistent with a cluster of metal ion interactions in the P1-P4 multi-helix junction that defines the catalytic core of the RNase P ribozyme.

The N-methyl-D-aspartate subtype of glutamate receptor (NMDAR) serves critical functions in physiological and pathological processes in the central nervous system, including neuronal development, plasticity and neurodegeneration. Conventional heteromeric NMDARs composed of NR1 and NR2A-D subunits require dual agonists, glutamate and glycine, for activation. They are also highly permeable to Ca2+, and exhibit voltage-dependent inhibition by Mg2+. Coexpression of NR3A with NR1 and NR2 subunits modulates NMDAR activity. Here we report the cloning and characterization of the final member of the NMDAR family, NR3B, which shares high sequence homology with NR3A. From in situ and immunocytochemical analyses, NR3B is expressed predominantly in motor neurons, whereas NR3A is more widely distributed. Remarkably, when co-expressed in Xenopus oocytes, NR3A or NR3B co-assembles with NR1 to form excitatory glycine receptors that are unaffected by glutamate or NMDA, and inhibited by D-serine, a co-activator of conventional NMDARs. Moreover, NR1/NR3A or -3B receptors form relatively Ca2+-impermeable cation channels that are resistant to Mg2+, MK-801, memantine and competitive antagonists. In cerebrocortical neurons containing NR3 family members, glycine triggers a burst of firing, and membrane patches manifest glycine-responsive single channels that are suppressible by D-serine. By itself, glycine is normally thought of as an inhibitory neurotransmitter. In contrast, these NR1/NR3A or -3B 'NMDARs' constitute a type of excitatory glycine receptor.

The tumor suppressor p53 is a transcription factor which binds DNA through a structurally complex domain stabilized by a zinc atom. Zinc chelation disrupts the architecture of this domain, inducing the protein to adopt an immunological phenotype identical to that of many mutant forms of p53. In this report, we used 65Zn to show that incorporation of zinc within the protein was required for folding in the 'wild-type' conformation capable of specific DNA-binding. Using a cellular assay, we show that addition of extracellular zinc at concentrations within the physiological range (5 microM) was required for renaturation and reactivation of wild-type p53. Among other divalent metals tested (Cd2+, Cu2+, Co2+, Fe2+ and Ni2+), only Co2+ at 125 microM had a similar effect. Recombinant metallothionein (MT), a metal chelator protein, was found to modulate p53 conformation in vitro. In cultured cells, overexpression of MT by transfection could modulate p53 transcriptional activity. Taken together, these results suggest that zinc binding plays a regulatory role in the control of p53 folding and DNA-binding activity.

Polycystic kidney diseases are genetic disorders in which the renal parenchyma is progressively replaced by fluid-filled cysts. Two members of the polycystin family (polycystin-1 and -2) are mutated in autosomal dominant polycystic kidney disease (ADPKD), and polycystin-L is deleted in mice with renal and retinal defects. Polycystins are membrane proteins that share significant sequence homology, especially polycystin-2 and -L (50% identity and 71% similarity). The functions of the polycystins remain unknown. Here we show that polycystin-L is a calcium-modulated nonselective cation channel that is permeable to sodium, potassium and calcium ions. Patch-clamp experiments revealed single-channel activity with a unitary conductance of 137 pS. Channel activity was substantially increased when either the extracellular or intracellular calcium-ion concentration was raised, indicating that polycystin-L may act as a transducer of calcium-mediated signalling in vivo. Its large single-channel conductance and regulation by calcium ions distinguish it from other structurally related cation channels.

Electrolyte balances during acute renal failure treated with continuous convective techniques, such as continuous arteriovenous hemofiltration (CAVH) and its pumped variants, are largely dependent on the eloctrolyte plasma concentration available for ultrafiltration, the ultrafiltration rate and the composition of the replacement solution. As blood sodium concentrations measured by potentiometry (Na +P) and the total ultrafiltrate sodium concentration are very similar, Na +P can be taken as the value of ultrafilterable sodium when choosing the correct sodium concentration in the substitution fluid. In CAVH, the ultrafiltrate contains about 3 m Eq/liter of calcium and 1 m Eq/liter of magnesium that must be replaced by the substitution fluid in order to prevent hypocalcemia and hypomagnesemia. In addition, if plasma potassium levels are normal, 3 to 4 mEq/liter of potassium should be added to the replacement fluid to avoid hypokalemia. Although convection and diffusion are combined in continuous hemodialysis, solute transport is largely mediated by convection; however, the net removal of sodium and calcium is significantly influenced by their concentrations in the dialysate, and the risk of hypomagnesemia and hypokalemia can be attenuated by adjusting magnesium and potassium concentrations in the dialysis solution to levels near to the plasma water values. Since critically ill patients are prone to developing dialysis-induced hypophosphatemia, phosphorous must be monitored and supplemented if necessary, Since CRRT works continuously, serious derangement in fluid and electrolyte homeostasis may occur in the absence of careful prescription and extremely vigilant monitoring.

Rods and cones of the two vertebrate lateral eyes hyperpolarize when illuminated, a response generated by a cyclic GMP cascade leading to cGMP hydrolysis and consequently the closure of cGMP-gated, non-selective cation channels that are open in darkness. Lizards and other lower vertebrates also have a parietal (third) eye, which contains ciliary photoreceptors that under dark-adapted conditions depolarize to light instead. Depolarizing light responses are characteristic of most invertebrate rhabdomeric photoreceptors, and are thought to involve a phosphoinositide signalling pathway (see, for example, refs 7-9). Surprisingly, we have found in excised membrane patches a cGMP-gated channel that is selectively present at high density on the outer segment (the presumptive light-sensitive part) of the parietal eye photoreceptor. Like the light-activated channel of the cell, it is non-selective among cations. Inositol trisphosphate (InsP3) had no effect on the same membrane patches. These findings suggest that the photoreceptors of the parietal eye, like rods and cones, use a cGMP cascade and not an InsP3-mediated pathway for phototransduction, but in this case light increases cGMP. A unifying principle of evolutionary significance emerges: that phototransductions in various ciliary photoreceptors, whether hyperpolarizing or depolarizing, uniformly use a cGMP cascade and a cGMP-gated channel to generate the light response, although there are rich variations in the details.

In non-excitable cells, release of Ca2+ from the inositol 1,4,5-trisphosphate (InsP3)-sensitive store can activate Ca2+ entry. Very little is known about the signal mechanism relating store emptying to plasma membrane Ca2+ influx. It has been suggested that the signal may be either a diffusible messenger like an inositol phosphate, or the InsP3 receptor itself, which, by physically coupling to some component of Ca2+ entry in the plasma membrane, may link store release to Ca2+ entry. The nature of the Ca2+ entry pathway is also unclear. Only in mast cells has a very selective Ca2+ current been observed after store emptying. Activation of exogenous 5-hydroxytryptamine (5-HT) receptors expressed in Xenopus oocytes or direct injection of InsP3 evokes Ca2+ entry activated by InsP3 pool depletion. Here we investigate the nature of this influx pathway and find a current activated by pool depletion. This has an unusual selectivity in that it is more permeable to Ca2+ ions than to other divalent cations (Ba2+, Sr2+ or Mn2+). Moreover, a K+ permeability is also stimulated after pool depletion. The activation of this store depletion current involves both a phosphatase and an unidentified diffusible messenger. Both the Ca2+ entry pathway and the activating factors found here may be relevant to pool-depleted Ca2+ entry in a variety of non-excitable cells.