Free concentrations of sodium, potassium and calcium in chromaffin granules.
Ontology highlight
ABSTRACT:
SUBMITTER: Haigh JR
PROVIDER: S-EPMC1138534 | biostudies-other | 1989 Apr
REPOSITORIES: biostudies-other
Similar Datasets
Project description:Synaptotagmin-7 (Syt-7) is one of two major calcium sensors for exocytosis in adrenal chromaffin cells, the other being synaptotagmin-1 (Syt-1). Despite a broad appreciation for the importance of Syt-7, questions remain as to its localization, function in mediating discharge of dense core granule cargos, and role in triggering release in response to physiological stimulation. These questions were addressed using two distinct experimental preparations-mouse chromaffin cells lacking endogenous Syt-7 (KO cells) and a reconstituted system employing cell-derived granules expressing either Syt-7 or Syt-1. First, using immunofluorescence imaging and subcellular fractionation, it is shown that Syt-7 is widely distributed in organelles, including dense core granules. Total internal reflection fluorescence (TIRF) imaging demonstrates that the kinetics and probability of granule fusion in Syt-7 KO cells stimulated by a native secretagogue, acetylcholine, are markedly lower than in WT cells. When fusion is observed, fluorescent cargo proteins are discharged more rapidly when only Syt-1 is available to facilitate release. To determine the extent to which the aforementioned results are attributable purely to Syt-7, granules expressing only Syt-7 or Syt-1 were triggered to fuse on planar supported bilayers bearing plasma membrane SNARE proteins. Here, as in cells, Syt-7 confers substantially greater calcium sensitivity to granule fusion than Syt-1 and slows the rate at which cargos are released. Overall, this study demonstrates that by virtue of its high affinity for calcium and effects on fusion pore expansion, Syt-7 plays a central role in regulating secretory output from adrenal chromaffin cells.
| S-EPMC7426247 | biostudies-literature
Project description:Transcellular calcium transport is an essential activity in mineralized tissue formation, including dental hard tissues. In many organ systems, this activity is regulated by membrane-bound sodium/calcium (Na(+)/Ca(2+)) exchangers, which include the NCX and NCKX [sodium/calcium-potassium (Na(+)/Ca(2+)-K(+)) exchanger] proteins. During enamel maturation, when crystals expand in thickness, Ca(2+) requirements vastly increase but exactly how Ca(2+) traffics through ameloblasts remains uncertain. Previous studies have shown that several NCX proteins are expressed in ameloblasts, although no significant shifts in expression were observed during maturation which pointed to the possible identification of other Ca(2+) membrane transporters. NCKX proteins are encoded by members of the solute carrier gene family, Slc24a, which include 6 different proteins (NCKX1-6). NCKX are bidirectional electrogenic transporters regulating Ca(2+) transport in and out of cells dependent on the transmembrane ion gradient. In this study we show that all NCKX mRNAs are expressed in dental tissues. Real-time PCR indicates that of all the members of the NCKX group, NCKX4 is the most highly expressed gene transcript during the late stages of amelogenesis. In situ hybridization and immunolocalization analyses clearly establish that in the enamel organ, NCKX4 is expressed primarily by ameloblasts during the maturation stage. Further, during the mid-late maturation stages of amelogenesis, the expression of NCKX4 in ameloblasts is most prominent at the apical poles and at the lateral membranes proximal to the apical ends. These data suggest that NCKX4 might be an important regulator of Ca(2+) transport during amelogenesis.
| S-EPMC3535175 | biostudies-literature
Project description:In the article a brief description of the biological basis of the regulation of human biological clocks was presented in order to introduce the role of circadian rhythms in physiology and specifically in the pharmacological translational tools based on the computational physiology models to motivate the need to provide models of circadian fluctuation in plasma cations. The main aim of the study was to develop statistical models of the circadian rhythm of potassium, sodium, and calcium concentrations in plasma. The developed ion models were further tested by assessing their influence on QT duration (cardiac endpoint) as simulated by the biophysically detailed models of human left ventricular cardiomyocyte. The main results are model equations along with an electronic supplement to the article that contains a fully functional implementation of all models.
| S-EPMC3775438 | biostudies-literature
Project description:Small-conductance Ca2+-activated potassium channels (SK(Ca) channels) are heteromeric complexes of pore-forming main subunits and constitutively bound calmodulin. SK(Ca) channels in neuronal cells are activated by intracellular Ca2+ that increases during action potentials, and their ionic currents have been considered to underlie neuronal afterhyperpolarization. However, the ion selectivity of neuronal SK(Ca) channels has not been rigorously investigated. In this study, we determined the monovalent cation selectivity of a cloned rat SK(Ca) channel, rSK2, using heterologous expression and electrophysiological measurements. When extracellular K+ was replaced isotonically with Na+, ionic currents through rSK2 reversed at significantly more depolarized membrane potentials than the value expected for a Nernstian relationship for K+. We then determined the relative permeability of rSK2 for monovalent cations and compared them with those of the intermediate- and large-conductance Ca2+-activated K+ channels, IK(Ca) and BK(Ca) channels. The relative permeability of the rSK2 channel was determined as K+(1.0)>Rb+(0.80)>NH(4)+(0.19) approximately Cs+(0.19)>Li+(0.14)>Na+(0.12), indicating substantial permeability of small ions through the channel. Although a mutation near the selectivity filter mimicking other K+-selective channels influenced the size-selectivity for permeant ions, Na+ permeability of rSK2 channels was still retained. Since the reversal potential of endogenous SK(Ca) current is determined by Na+ permeability in a physiological ionic environment, the ion selectivity of native SK(Ca) channels should be reinvestigated and their in vivo roles may need to be restated.
| S-EPMC1366808 | biostudies-literature
Project description:The roles of nonmuscle myosin II and cortical actin filaments in chromaffin granule exocytosis were studied by confocal fluorescence microscopy, amperometry, and cell-attached capacitance measurements. Fluorescence imaging indicated decreased mobility of granules near the plasma membrane following inhibition of myosin II function with blebbistatin. Slower fusion pore expansion rates and longer fusion pore lifetimes were observed after inhibition of actin polymerization using cytochalasin D. Amperometric recordings revealed increased amperometric spike half-widths without change in quantal size after either myosin II inhibition or actin disruption. These results suggest that actin and myosin II facilitate release from individual chromaffin granules by accelerating dissociation of catecholamines from the intragranular matrix possibly through generation of mechanical forces.
| S-EPMC2768403 | biostudies-literature
Project description:Pore-spanning membranes (PSMs) composed of supported membrane parts as well as freestanding membrane parts are shown to be very versatile to investigate SNARE-mediated fusion on the single-particle level. They provide a planar geometry readily accessible by confocal fluorescence microscopy, which enabled us for the first time, to our knowledge, to investigate the fusion of individual natural secretory granules (i.e., chromaffin granules (CGs)) on the single-particle level by two-color fluorescence microscopy in a time-resolved manner. The t-SNARE acceptor complex ΔN49 was reconstituted into PSMs containing 2 mol % 1,2-dipalmitoyl-sn-glycero-3-phosphatidylinositol-4,5-bisphosphate and Atto488-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, and CGs were fluorescently labeled with 2-((1E,3E)-5-((Z)-3,3-dimethyl-1-octadecylindolin-2-ylidene)penta-1,3-dien-1-yl)-3,3-dimethyl-1-octadecyl-3H-indol-1-ium perchlorate. We compared the dynamics of docked and hemifused CGs as well as their fusion efficacy and kinetics with the results obtained for synthetic synaptobrevin 2-doped vesicles fusing with PSMs of the same composition. Whereas the synthetic vesicles were fully immobile on supported PSMs, docked as well as hemifused CGs were mobile on both PSM parts, which suggests that this system resembles more closely the natural situation. The fusion process of CGs proceeded through three-dimensional post-lipid-mixing structures, which were readily resolved on the gold-covered pore rims of the PSMs and which are discussed in the context of intermediate states observed in live cells.
| S-EPMC6350068 | biostudies-literature
Project description:Sodium/calcium(-potassium) exchangers (NCX and NCKX) are critical for the rapid extrusion of calcium, which follows the stimulation of a variety of excitable cells. To further understand the mechanisms of calcium regulation in signaling, we have cloned a Drosophila sodium/calcium-potassium exchanger, Nckx30C. The overall deduced protein topology for NCKX30C is similar to that of mammalian NCKX, having five membrane-spanning domains in the NH(2) terminus separated from six at the COOH-terminal end by a large intracellular loop. We show that NCKX30C functions as a potassium-dependent sodium/calcium exchanger, and is not only expressed in adult neurons as was expected, but is also expressed during ventral nerve cord development in the embryo and in larval imaginal discs. Nckx30C is expressed in a dorsal-ventral pattern in the eye-antennal disc in a pattern that is similar to, but broader than that of wingless, suggesting that large fluxes of calcium may be occurring during imaginal disc development. Nckx30C may not only function in the removal of calcium and maintenance of calcium homeostasis during signaling in the adult, but may also play a critical role in signaling during development.
| S-EPMC2151195 | biostudies-literature
Project description:The potassium sodium niobate, K0.5Na0.5NbO₃, solid solution (KNN) is considered as one of the most promising, environment-friendly, lead-free candidates to replace highly efficient, lead-based piezoelectrics. Since the first reports of KNN, it has been recognized that obtaining phase-pure materials with a high density and a uniform, fine-grained microstructure is a major challenge. For this reason the present paper reviews the different methods for consolidating KNN ceramics. The difficulties involved in the solid-state synthesis of KNN powder, i.e., obtaining phase purity, the stoichiometry of the perovskite phase, and the chemical homogeneity, are discussed. The solid-state sintering of stoichiometric KNN is characterized by poor densification and an extremely narrow sintering-temperature range, which is close to the solidus temperature. A study of the initial sintering stage revealed that coarsening of the microstructure without densification contributes to a reduction of the driving force for sintering. The influences of the (K + Na)/Nb molar ratio, the presence of a liquid phase, chemical modifications (doping, complex solid solutions) and different atmospheres (i.e., defect chemistry) on the sintering are discussed. Special sintering techniques, such as pressure-assisted sintering and spark-plasma sintering, can be effective methods for enhancing the density of KNN ceramics. The sintering behavior of KNN is compared to that of a representative piezoelectric lead zirconate titanate (PZT).
| S-EPMC5458861 | biostudies-other
Project description:Adrenomedullary chromaffin cells respond to sympathetic nervous system activation by secreting a cocktail of potent neuropeptides and hormones into the circulation. The distinct phases of the chromaffin cell secretory response have been attributed to the progressive fusion of distinct populations of dense core granules with different activation kinetics. However, it has been difficult to define what distinguishes these populations at the molecular level. Functional segregation of granule pools may depend on selective sorting of synaptotagmin-1 (Syt-1) and synaptotagmin-7 (Syt-7), which our previous work showed are rarely cosorted to the same granule. Here we assess the consequences of selective sorting of Syt isoforms in chromaffin cells, particularly with respect to granule dynamics and activation kinetics. Upon depolarization of cells expressing fluorescent Syt isoforms using elevated K+, we find that Syt-7 granules fuse with faster kinetics than Syt-1 granules, irrespective of stimulation strength. Pharmacological blockade of Ca2+ channels reveals differential dependence of Syt-1 versus Syt-7 granule exocytosis on Ca2+ channel subtypes. Syt-7 granules also show a greater tendency to fuse in clusters than Syt-1 granules, and granules harboring Syt-1 travel a greater distance before fusion than those with Syt-7, suggesting that there is spatial and fusion-site heterogeneity among the two granule populations. However, the greatest functional difference between granule populations is their responsiveness to Ca2+ Upon introduction of Ca2+ into permeabilized cells, Syt-7 granules fuse with fast kinetics and high efficacy, even at low Ca2+ levels (e.g., when cells are weakly stimulated). Conversely, Syt-1 granules require a comparatively larger increase in intracellular Ca2+ for activation. At Ca2+ concentrations above 30 µM, activation kinetics are faster for Syt-1 granules than for Syt-7 granules. Our study provides evidence for functional specialization of chromaffin cell granules via selective expression of Syt isoforms with different Ca2+ sensitivities.
| S-EPMC5560776 | biostudies-literature
Project description:Soluble N-ethylmaleimide-sensitive factor activating protein receptor (SNARE) proteins are the main catalysts for membrane fusion in the secretory pathway of eukaryotic cells. In vitro, SNAREs are sufficient to mediate effective fusion of both native and artificial membranes. Here we have established, to our knowledge, a new platform for monitoring SNARE-mediated docking and fusion between giant unilamellar vesicles (GUVs) and smaller liposomes or purified secretory granules with high temporal and spatial resolution. Analysis of fusion is restricted to the free-standing part of the GUV-membrane exhibiting low curvature and a lack of surface contact, thus avoiding adhesion-mediated interference with the fusion reaction as in fusion with supported bilayers or surface-immobilized small vesicles. Our results show that liposomes and chromaffin granules fuse with GUVs containing activated SNAREs with only few milliseconds delay between docking and fusion. We conclude that after initial contact in trans, SNAREs alone can complete fusion at a rate close to fast neuronal exocytosis.
| S-EPMC5607038 | biostudies-literature