Differentiation-Induced Remodelling of Store-Operated Calcium Entry Is Independent of Neuronal or Glial Phenotype but Modulated by Cellular Context.

Neurogenesis is a complex process leading to the generation of neuronal networks and glial cell types from stem cells or intermediate progenitors. Mapping subcellular and molecular changes accompanying the switch from proliferation to differentiation is vital for developing therapeutic targets for neurological diseases. Neuronal (N-type) and glial (S-type) phenotypes within the SH-SY5Y neuroblastoma cell line have distinct differentiation responses to 9-cis-retinoic acid (9cRA). In both cell phenotypes, these were accompanied at the single cell level by an uncoupling of Ca2+ store release from store-operated Ca2+ entry (SOCE), mediated by changes in the expression of calcium release-activated calcium pore proteins. This remodelling of calcium signalling was moderated by the predominant cell phenotype within the population. N- and S-type cells differed markedly in their phenotypic stability after withdrawal of the differentiation inducer, with the phenotypic stability of S-type cells, both morphologically and with respect to SOCE properties, in marked contrast to the lability of the N-type phenotype. Furthermore, the SOCE response of I-type cells, a presumed precursor to both N- and S-type cells, varied markedly in different cell environments. These results demonstrate the unique biology of neuronal and glial derivatives of common precursors and suggest that direct or indirect interactions between cell types are vital components of neurogenesis that need to be considered in experimental models.

Resculpting the binding pocket of APC superfamily LeuT-fold amino acid transporters.

Amino acid transporters are essential components of prokaryote and eukaryote cells, possess distinct physiological functions, and differ markedly in substrate specificity. Amino acid transporters can be both drug targets and drug transporters (bioavailability, targeting) with many monogenic disorders resulting from dysfunctional membrane transport. The largest collection of amino acid transporters (including the mammalian SLC6, SLC7, SLC32, SLC36, and SLC38 families), across all kingdoms of life, is within the Amino acid-Polyamine-organoCation (APC) superfamily. The LeuT-fold is a paradigm structure for APC superfamily amino acid transporters and carriers of sugars, neurotransmitters, electrolytes, osmolytes, vitamins, micronutrients, signalling molecules, and organic and fatty acids. Each transporter is specific for a unique sub-set of solutes, specificity being determined by how well a substrate fits into each binding pocket. However, the molecular basis of substrate selectivity remains, by and large, elusive. Using an integrated computational and experimental approach, we demonstrate that a single position within the LeuT-fold can play a crucial role in determining substrate specificity in mammalian and arthropod amino acid transporters within the APC superfamily. Systematic mutation of the amino acid residue occupying the equivalent position to LeuT V104 titrates binding pocket space resulting in dramatic changes in substrate selectivity in exemplar APC amino acid transporters including PAT2 (SLC36A2) and SNAT5 (SLC38A5). Our work demonstrates how a single residue/site within an archetypal structural motif can alter substrate affinity and selectivity within this important superfamily of diverse membrane transporters.

Store-operated Ca(2+) entry in proliferating and retinoic acid-differentiated N- and S-type neuroblastoma cells.

Neuroblastoma cell lines are heterogeneous, comprised of at least three distinct cell phenotypes; neuroblastic N-type cells, non-neuronal substrate-adherent S-type cells and intermediate I-type cells. N- and S-type cell populations were enriched from the parental SH-SY5Y neuroblastoma cell line and induced to differentiate by the addition of retinoic acid (RA), a drug used in the treatment of neuroblastoma. N- and S-type cells were identified based on their differential expression of ß-tubulin III, vimentin and Bcl-2. Store-operated Ca(2+) entry (SOCE) was then measured in proliferating and differentiated N- and S-type cell populations and the expression of STIM1, Orai1 and TRPC1, three proteins reported to play a key role in SOCE, was determined. In N-type cells the RA-induced switch from proliferation to differentiation was accompanied by a down-regulation in SOCE. STIM1 and Orai1 expression became down-regulated in differentiated cells, consistent with their respective roles as ER Ca(2+) sensor and store-operated Ca(2+) channel (SOC). TRPC1 became up-regulated suggesting that TRPC1 is not involved in SOCE, at least in differentiated N-type cells. In S-type cells SOCE remained active following the RA-induced switch from proliferation to differentiation and the expression of STIM1 and Orai1 remained unchanged. TRPC1 was not expressed in S-type cells. Our results indicate that differentiation of neuronal cells is associated with a remodelling of SOCE. Therapeutic targeting of SOCE proteins could potentially be a means of promoting neuronal differentiation in the treatment of neuroblastoma.

Phenotypic modulation of human urinary tract stroma-derived fibroblasts by transforming growth factor beta3.

OBJECTIVES: Animal models have described critical roles for transforming growth factor beta (TGFbeta) isoforms in modulating urinary tract stroma phenotype. TGFbeta3 is of particular interest because it may regulate TGFbeta1 and TGFbeta2 expression, but its modulatory affect has not been so well characterized in human cells. In this study, we aim to determine whether TGFbeta3 treatment induced differentiation of human urinary tract stroma-derived fibroblasts to a smooth muscle-like phenotype. METHODS: We established cultures of human urinary tract stroma-derived fibroblasts and studied the effects of TGFbeta3 treatment using proliferation assays, cell cycle analysis, immunocytochemistry, and Western blotting for expression of differentiation marker and downstream regulators, and fura-2 fluorescence to study the effects on intracellular calcium. RESULTS: TGFbeta3 treatment induced proliferation that peaked at 72 hours, followed by enhanced expression of alpha-smooth muscle actin (alpha-SMA) with a maximal 3.4-fold increase at 168 hours. TGFbeta3 treatment decreased resting [Ca(2+)](i) by 70% and caused a 95% decrease in stimulated internal Ca(2+) release regulated by the sarcoplasmic/endoplasmic calcium-ATPase pump. These effects were associated with upregulation of nuclear activator of T cells -1 (NFAT), a known regulator of cell differentiation. CONCLUSIONS: TGFbeta3 treatment causes a time-specific response in the presence of serum, whereby fibroblasts initially proliferate and subsequently differentiate to a smooth muscle-like phenotype. This sequence was associated with stabilization of [Ca(2+)](i) stores, suggesting a role in the induction of hyperplasia and reduction of contractility; phenomena associated with a number of urinary tract pathologies.

Protein-folding location can regulate manganese-binding versus copper- or zinc-binding.

Metals are needed by at least one-quarter of all proteins. Although metallochaperones insert the correct metal into some proteins, they have not been found for the vast majority, and the view is that most metalloproteins acquire their metals directly from cellular pools. However, some metals form more stable complexes with proteins than do others. For instance, as described in the Irving-Williams series, Cu(2+) and Zn(2+) typically form more stable complexes than Mn(2+). Thus it is unclear what cellular mechanisms manage metal acquisition by most nascent proteins. To investigate this question, we identified the most abundant Cu(2+)-protein, CucA (Cu(2+)-cupin A), and the most abundant Mn(2+)-protein, MncA (Mn(2+)-cupin A), in the periplasm of the cyanobacterium Synechocystis PCC 6803. Each of these newly identified proteins binds its respective metal via identical ligands within a cupin fold. Consistent with the Irving-Williams series, MncA only binds Mn(2+) after folding in solutions containing at least a 10(4) times molar excess of Mn(2+) over Cu(2+) or Zn(2+). However once MncA has bound Mn(2+), the metal does not exchange with Cu(2+). MncA and CucA have signal peptides for different export pathways into the periplasm, Tat and Sec respectively. Export by the Tat pathway allows MncA to fold in the cytoplasm, which contains only tightly bound copper or Zn(2+) (refs 10-12) but micromolar Mn(2+) (ref. 13). In contrast, CucA folds in the periplasm to acquire Cu(2+). These results reveal a mechanism whereby the compartment in which a protein folds overrides its binding preference to control its metal content. They explain why the cytoplasm must contain only tightly bound and buffered copper and Zn(2+).

Insect ryanodine receptors: molecular targets for novel pest control chemicals.

Ryanodine receptors (RyRs) are a distinct class of ligand-gated calcium channels controlling the release of calcium from intracellular stores. They are located on the sarcoplasmic reticulum of muscle and the endoplasmic reticulum of neurons and many other cell types. Ryanodine, a plant alkaloid and an important ligand used to characterize and purify the receptor, has served as a natural botanical insecticide, but attempts to generate synthetic commercial analogues of ryanodine have proved unsuccessful. Recently two classes of synthetic chemicals have emerged resulting in commercial insecticides that target insect RyRs. The phthalic acid diamide class has yielded flubendiamide, the first synthetic ryanodine receptor insecticide to be commercialized. Shortly after the discovery of the phthalic diamides, the anthranilic diamides were discovered. This class has produced the insecticides Rynaxypyr and Cyazypyr. Here we review the structure and functions of insect RyRs and address the modes of action of phthalic acid diamides and anthranilic diamides on insect ryanodine receptors. Particularly intersting is the inherent selectivity both chemical classes exhibit for insect RyRs over their mammalian counterparts. The future prospects for RyRs as a commercially-validated target site for insect control chemicals are also considered.

Changes in functional properties of the caffeine-sensitive Ca2+ store during differentiation of human SH-SY5Y neuroblastoma cells.

We have used single cell fluorescence imaging techniques to examine how functional properties of the caffeine-sensitive Ca(2+) store change during differentiation of a sub-population of caffeine-sensitive SH-SY5Y cells. Application of caffeine (30 mM) 1-10.5 min after a 'priming' depolarisation pulse of 55 mM K(+) revealed that the caffeine-sensitive store in undifferentiated cells remained replete, whereas that in 9-cis retinoic acid (9cRA)-differentiated cells spontaneously dissipated with a t(1/2) of 2.8 min, and was essentially completely depleted approximately 10 min after priming. In 9cRA-differentiated cells that were stimulated with methacholine (10 microM) 1 min after priming, the amplitude, rate of rise and propagation velocity of the Ca(2+) wave in the neurites were all constant, whereas these kinetic parameters all progressively decreased as the wave travelled along the neurites in cells that were stimulated 10 min after priming. Use-dependent block with ryanodine inhibited the global Ca(2+) signal in 9cRA-differentiated cells stimulated with methacholine 1 min after priming (71+/-8%) but not 10 min after priming. Depolarisation was more effective at priming the caffeine-sensitive Ca(2+) store in 9cRA-differentiated cells, which lack a functional store-operated Ca(2+) entry pathway. We conclude that differentiation of caffeine-sensitive SH-SY5Y cells is accompanied by an increase in lability of the caffeine-sensitive Ca(2+) store, and that spontaneous dissipation of Ca(2+) from the store limits the time course of its molecular 'memory' during which it can amplify the hormone-induced Ca(2+) signal by Ca(2+)-induced Ca(2+) release.

A constitutively active nonselective cation conductance underlies resting Ca2+ influx and secretion in bovine adrenal chromaffin cells.

We have combined fluorimetric measurements of the intracellular free Ca(2+) concentration ([Ca(2+)](i)) with the patch clamp technique, to investigate resting Ca(2+) entry in bovine adrenal chromaffin cells. Perfusion with nominally Ca(2+)-free medium resulted in a rapid, reversible decrease in [Ca(2+)](i), indicating a resting Ca(2+) permeability across the plasma membrane. Simultaneous whole-cell voltage-clamp showed a resting inward current that increased when extracellular Ca(2+) (Ca(2+)(o)) was lowered. This current had a reversal potential of around 0 mV and was carried by monovalent or divalent cations. In Na(+)-free extracellular medium there was a reduction in current amplitude upon removal of Ca(2+)(o), indicating the current can carry Ca(2+). The current was constitutively active and not enhanced by agents that promote Ca(2+)-store depletion such as thapsigargin. Extracellular La(3+) abolished the resting current, reduced resting [Ca(2+)](i) and inhibited basal secretion. Abolishment of resting Ca(2+) influx depleted the inositol 1,4,5-trisphosphate-sensitive Ca(2+) store without affecting the caffeine-sensitive Ca(2+) store. The results indicate the presence of a constitutively active nonselective cation conductance, permeable to both monovalent and divalent cations, that can regulate [Ca(2+)](i), the repletion state of the intracellular Ca(2+) store and the secretory response in resting cells.

Release and sequestration of Ca2+ by a caffeine- and ryanodine-sensitive store in a sub-population of human SH-SY5Y neuroblastoma cells.

We have used single cell fluorescence imaging techniques to examine the role that ryanodine receptors play in the stimulus-induced Ca(2+) responses of SH-SY5Y cells. The muscarinic agonist methacholine (1mM) resulted in a Ca(2+) signal in 95% of all cells. Caffeine (30 mM) however stimulated a Ca(2+) signal in only 1-7% of N-type (neuroblastic) cells within any given field. The caffeine response was independent of extracellular Ca(2+), regenerative in nature, and abolished in a use-dependent fashion by ryanodine. In caffeine-responsive cells, the magnitude of the methacholine-induced Ca(2+) signal was inhibited by 75.07 +/- 5.51% by pretreatment with caffeine and ryanodine, suggesting that the caffeine-sensitive store may act as a Ca(2+) source after muscarinic stimulation. When these data were combined with equivalent data from non-caffeine-responsive cells, the degree of apparent inhibition was significantly reduced. In contrast, after store depletion by caffeine, the Ca(2+) signal induced by 55 mM K(+) was potentiated 2.5-fold in the presence of ryanodine, suggesting that the store may act a Ca(2+) sink after depolarisation. We conclude that a caffeine- and ryanodine-sensitive store can act as a Ca(2+) source and sink in SH-SY5Y cells, and that effects of the store can become obscured if data from caffeine-insensitive cells are not excluded.

Mechanistic and functional changes in Ca2+ entry after retinoic acid-induced differentiation of neuroblastoma cells.

We have investigated effects of neuronal differentiation on hormone-induced Ca2+ entry. Fura-2 fluorescence measurements of undifferentiated SH-SY5Y neuroblastoma cells, stimulated with methacholine, revealed the presence of voltage-operated Ca2+-permeable, Mn2+-impermeable entry pathways, and at least two voltage-independent Ca2+- and Mn2+-permeable entry pathways, all of which apparently contribute to both peak and plateau phases of the Ca2+ signal. Similar experiments using 9-cis retinoic acid-differentiated cells, however, revealed voltage-operated Ca2+-permeable, Mn2+-impermeable channels, and, more significantly, the absence or down-regulation of the most predominant of the voltage-independent entry pathways. This down-regulated pathway is probably due to CCE (capacitative Ca2+ entry), since thapsigargin also stimulated Ca2+ and Mn2+ entry in undifferentiated but not differentiated cells. The Ca2+ entry components remaining in methacholine-stimulated differentiated cells contributed to only the plateau phase of the Ca2+ signal. We conclude that differentiation of SH-SY5Y cells results in a mechanistic and functional change in hormone-stimulated Ca2+ entry. In undifferentiated cells, voltage-operated Ca2+ channels, CCE and NCCE (non-CCE) pathways are present. Of the voltage-independent pathways, the predominant one appears to be CCE. These pathways contribute to both peak and plateau phases of the Ca2+ signal. In differentiated cells, CCE is either absent or down-regulated, whereas voltage-operated entry and NCCE remain active and contribute to only the plateau phase of the Ca2+ signal.