How do calcium channels transport calcium ions?

Calcium channel activity is crucial for many fundamental physiological processes ranging from the heart beat to synaptic transmission. The channel-forming protein, of about 2000 amino acids, comprises four domains internally homologous to each other. Voltage-dependent Ca2+ channels are the most selective ion channels known. Under physiological conditions, they prefer Ca2+ over Na+ by a ratio of about 1000:1. To explain at the same time the exquisite ion selectivity and the large Ca2+ ion turnover rate of Ca2+ channels (approximately 3 x 10(6) ions/s), two kind models have been proposed. In one, the conduction pathway possesses two high-affinity binding sites. When two Ca2+ ions are bound to each site, the mutual repulsion between them speeds the exit rate for the ions, causing greater ion permeation through the pore. The second model hypothesizes the existence of a single site having a charged structure able to attract multiple, interacting ions, simultaneously. Recent studies that combine mutagenesis and electrophysiology show that the high-affinity binding site is formed by a ring of glutamate residues located in the pore forming region of the Ca2+ channel. As proposed in the second class of models, the results suggest that four glutamate residues, one glutamate donated by each repeat, combine to form a single high-affinity site. In this review the different conduction models for Ca2+ channels are discussed and confronted with structural data.     

Fibroblast growth factor-23 abolishes 1,25-dihydroxyvitamin D₃-enhanced duodenal calcium transport in male mice

Despite being widely recognized as the important bone-derived phosphaturic hormone, whether fibroblast growth factor (FGF)-23 modulated intestinal calcium absorption remained elusive. Since FGF-23 could reduce the circulating level of 1,25-dihydroxyvitamin D? [1,25(OH)?D?], FGF-23 probably compromised the 1,25(OH)?D?-induced intestinal calcium absorption. FGF-23 may also exert an inhibitory action directly through FGF receptors (FGFR) in the intestinal cells. Herein, we demonstrated by Ussing chamber technique that male mice administered 1 μg/kg 1,25(OH)?D? sc daily for 3 days exhibited increased duodenal calcium absorption, which was abolished by concurrent intravenous injection of recombinant mouse FGF-23. This FGF-23 administration had no effect on the background epithelial electrical properties, i.e., short-circuit current, transepithelial potential difference, and resistance. Immunohistochemical evidence of protein expressions of FGFR isoforms 1-4 in mouse duodenal epithelial cells suggested a possible direct effect of FGF-23 on the intestine. This was supported by the findings that FGF-23 directly added to the serosal compartment of the Ussing chamber and completely abolished the 1,25(OH)?D?-induced calcium absorption in the duodenal tissues taken from the 1,25(OH)?D?-treated mice. However, direct FGF-23 exposure did not decrease the duodenal calcium absorption without 1,25(OH)?D? preinjection. The observed FGF-23 action was mediated by MAPK/ERK, p38 MAPK, and PKC. Quantitative real-time PCR further showed that FGF-23 diminished the 1,25(OH)?D?-induced upregulation of TRPV5, TRPV6, and calbindin-D(9k), but not PMCA(1b) expression in the duodenal epithelial cells. In conclusion, besides being a phosphatonin, FGF-23 was shown to be a novel calcium-regulating hormone that acted directly on the mouse intestine, thereby compromising the 1,25(OH)?D?-induced calcium absorption.     

The stimulus-secretion coupling of glucose-induced insulin release. Does glycolysis control calcium transport in the B-cell?

1. The metabolism of glucose and the exchangeable Ca2+ pool were measured in rat pancreatic islets, in order to assess the possible significance of glycolysis in the process of glucose-induced insulin release. 2. At high glucose concentration (16.7 mM), glucose was metabolized at the following rate (pmol of glucose residue/h per islet +/- S.E.M.): 131 +/- 11 for glucose uptake, 129+/-8 for glucose utilization, as judged by the conversion of [5-3H]glucose into 3H2O,60+/-2 for lactate output and 25+/-2 for glucose oxidation. 3. The secretory pattern usually correlated with the metabolic data. For instance, the ability of different sugars (glucose, mannose, fructose, galactose, D-glyceraldehyde) to stimulate lactate output closely paralleled their relative insulinotropic capacity. A disparity between metabolic and secretory responses was, however, encountered in the presence of dibutyryl cyclic AMP and theophylline. 4. Despite this contrasting behaviour, the size of the Ca2+- exchangeable pool (net uptake of 45Ca2+) was invariably proportional to the rate of lactate output under all experimental conditions examined. It is concluded that glycolysis usually exerts a tight control on the rate constant for Ca2+ transport across the B-cell membrane.     

Dietary fructose inhibits lactation-induced adaptations in rat 1,25-(OH)?D? synthesis and calcium transport

We recently showed that excessive fructose consumption, already associated with numerous metabolic abnormalities, reduces rates of intestinal Ca(2+) transport. Using a rat lactation model with increased Ca(2+) requirements, we tested the hypothesis that mechanisms underlying these inhibitory effects of fructose involve reductions in renal synthesis of 1,25-(OH)(2)D(3). Pregnant and virgin (control) rats were fed isocaloric fructose or, as controls, glucose, and starch diets from d 2 of gestation to the end of lactation. Compared to virgins, lactating dams fed glucose or starch had higher rates of intestinal transcellular Ca(2+) transport, elevated intestinal and renal expression of Ca(2+) channels, Ca(2+)-binding proteins, and CaATPases, as well as increased levels of 25-(OH)D(3) and 1,25-(OH)(2)D(3). Fructose consumption prevented almost all of these lactation-induced increases, and reduced vitamin D receptor binding to promoter regions of Ca(2+) channels and binding proteins. Changes in 1,25-(OH)(2)D(3) level were tightly correlated with alterations in expression of 1α-hydroxylase but not with levels of parathyroid hormone and of 24-hydroxylase. Bone mineral density, content, and mechanical strength each decreased with lactation, but then fructose exacerbated these effects. When Ca(2+) requirements increase during lactation or similar physiologically challenging conditions, excessive fructose consumption may perturb Ca(2+) homeostasis because of fructose-induced reductions in synthesis of 1,25-(OH)(2)D(3).     

1α,25-dihydroxy-vitamin D-3 regulates ATP-dependent calcium transport in basolateral plasma membranes of rat enterocytes

Basolateral plasma membrane vesicles of rat small intestinal epithelium accumulate calcium through an ATP-dependent pumping system. The activity of this system is highest in duodenum and decreases towards the ileum. This distribution along the intestinal tract is similar as the active calcium absorption capacity of intact intestinal epithelial segments. ATP-dependent calcium uptake in basolateral membrane vesicles from duodenum and ileum increased significantly after repletion of young vitamin D-3-deficient rats with 1α,25-dihydroxy-vitamin D-3. Ca²⁺-ATPase activity in duodenal basolateral membranes increased to the same extend as ATP-dependent calcium transport, but (Na⁺ + K⁺)-ATPase activity remained unaltered.     

1,25-Dihydroxyvitamin D₃ contributes to regulating mammary calcium transport and modulates neonatal skeletal growth and turnover cooperatively with calcium

To assess the interaction of 1,25(OH)(2)D(3) and dietary calcium on mammary calcium transport in lactating dams and skeletal growth and turnover in the neonate, female lactating 1α(OH)ase(+/-) or 1α(OH)ase(-/-) mice were fed either a high-calcium diet containing 1.5% calcium in the drinking water or a "rescue diet." Dietary effects on the expression of molecules mediating mammary calcium transport were determined in the dams, and the effects of milk calcium content were assessed on skeletal growth and turnover in 2-wk-old 1,25(OH)(2)D(3)-deficient pups. Results showed that the reduction of milk calcium levels in the 1α(OH)ase(-/-) dams and the elevation of milk calcium levels in dams fed the rescue diet were associated with the down- or upregulation of calbindin D(9k) and plasma membrane Ca(2+) ATPase isoform 2b expression, respectively, in mammary epithelial cells. The action of ambient calcium in stimulating skeletal growth in the neonates appeared to supercede the direct action of 1,25(OH)(2)D(3), and the response of chondrocytes in the neonates to elevated calcium was more sensitive in hypocalcemic animals. Osteopenia was more apparent in pups nursed by dams with lower milk calcium than in 1,25(OH)(2)D(3)-deficient pups nursed by dams with higher milk calcium. Bone formation parameters were increased significantly in all pups fed by dams on the rescue diet but were still lower in 1α(OH)ase(-/-) pups than in 1α(OH)ase(+/-) pups. Consequently, there is an important contributory role of calcium in conjunction with 1,25(OH)(2)D(3) to mammary calcium transport in lactating dams and skeletal growth and turnover in the neonate.     

Three decades in transport business: studies of metabolite transport in chloroplasts – a personal perspective

This article gives an historical overview of our group's research on various metabolite translocators of chloroplasts, such as the translocators for phosphorylated intermediates of the Calvin–Benson cycle and of glycolysis, of ADP and ATP, of dicarboxylates, of pyruvate and of hexoses; how it began and where it led to. Wherever appropriate, references will be made to research in other laboratories. This revised version was published online in June 2006 with corrections to the Cover Date.     

δ-Aminolevulinic acid transport in Saccharomyces cerevisiae

• 1.1. This work represents the first approach to characterize the transport system of haem pathway precursors, such as δ-aminolevulinic acid (ALA), in two strains of Saccharomyces cerevisiae, a wild type, D27, and a HEM R⁺ mutant.
• 2.2. ALA transport occurs unidirectionally by a sole active system with an apparent K_M of 0.10 mM, at the optimum pH of 5.0. ALA uptake is influenced by both the carbon and nitrogen source; this suggests a rather complex regulation mechanism.
• 3.3. This transport is not mediated by the general amino acid permease (GAP).
• 4.4. ALA uptake is strongly inhibited by compounds harboring a methyl-amine terminus suggesting that this group is essential for ALA transport; however, the electric environment of the carboxylic group may be also important for the interaction between ALA and its transporter active site.
• 5.5. We have found differences in ALA transport which would indicate a different regulation mechanism for this system in both strain cells.

     

Cl− transport in basolateral renal medullary vesicles: I. Cl− transport in intact vesicles

Summary This paper provides the results of studies which characterized conductive³⁶Cl^– flux in basolaterally enriched membrane vesicles prepared from rabbit renal outer medulla. Conductive³⁶Cl^– uptake was studied under two different experimental conditions. In the first,³⁶Cl^– flux was driven by an inside positive voltage created with oppositely directed Cl^– and gluconate gradients. In the second, an inwardly direct K⁺ gradient was used to drive³⁶Cl^– uptake. By these two methods, voltage-sensitive³⁶Cl^– uptake was shown to comprise about 45 and 65%, respectively, of the initial rates of total³⁶Cl^– flux. Separate paired studies demonstrated that the conductive³⁶Cl^– uptake was inhibited by the Cl^– channel blocker diphenylamine-2-carboxylate (DPC) with an IC₅₀ for DPC of 154 m. The voltagedependent³⁶Cl^– uptake had an activation energy of 6.4 kcal/mole. This³⁶Cl^– conductance had an anion selectivity sequence of I^–>Cl^–NO ₃ ^– gluconate.     

Is the intrasomal phase of fast axonal transport driven by oscillations of intracellular calcium?

An hypothesis is presented suggesting that the delivery of vesicle-packaged protein from the neuronal soma to the axonal transport system is physiologically coupled to spontaneous fluctuations of intracellular calcium (Ca_i). Evidence is reviewed that oscillations of Ca_i, commonly detected as agonist-or voltage-triggered waves and spikes propagating through the cytosol, also occur as spontaneous events. Endogenously-generated oscillations are examined since intrasomal transport persists in the absence of extracellular signals or nerve impulse activity. Vesicle budding from the endoplasmic reticulum (ER) may be a key step at which anterograde transport is regulated by events related to the release and reuptake of ER stores of Ca²⁺.Special-issue dedicated to Dr. Sidney Ochs.     

Modelling sediment transport in shallow lakes — interactions between sediment transport and sediment composition

In shallow, wind exposed lakes, the light conditions, the cycling of nutrients, heavy metals and organic micro-pollutants and changes in the local composition of the sediment top layer can be dominated by resuspension/erosion of bottom sediment and sedimentation of suspended solids. A 2 dimensional model for Sediment Transport, Resuspension and Sedimentation in Shallow lakes (STRESS-2d), based on an existing transport model, is discussed. In the model, mass balance equations for the water compartment and the bottom sediment are solved numerically. Up to 7 sediment fractions can be taken into account, each having a specific set of resuspension/erosion and sedimentation parameter values. Several options for modelling the changes in the bottom sediment composition are available.A simulation experiment for Lake Veluwe (The Netherlands), in which model options with and without the distinction of sediment fractions were used, showed that using sediment fractions to account for the variability in the sediment composition leads to an improvement of the model results, particularly the simulated phosphorus sediment-water exchange fluxes. For Lake Ketel (The Netherlands) two options for modelling changes in the bottom sediment composition are compared. It is shown that an option in which a thin water-sediment layer on top of the more consolidated bottom sediment is simulated provides an improvement in the simulation of the suspended solids concentration.     

The cytoskeleton in plasmodesmata: a role in intercellular transport?

Actin and myosin are components of the plant cell cytoskeleton that extend from cell to cell through plasmodesmata (PD), but it is unclear how they are organized within the cytoplasmic sleeve or how they might behave as regulatory elements. Early work used antibodies to locate actin and myosin to PD, at the electron microscope level, or to pitfields (aggregations of PD in the cell wall), using immunofluorescence techniques. More recently, a green fluorescent protein (GFP)-tagged plant myosin VIII was located specifically at PD-rich pitfields in cell walls. Application of actin or myosin disrupters may modify the conformation of PD and alter rates of cell-cell transport, providing evidence for a role in regulating PD permeability. Intriguingly, there is now evidence of differentiation between types of PD, some of which open in response to both actin and myosin disrupters, and others which are unaffected by actin disrupters or which close in response to myosin inhibitors. Viruses also interact with elements of the cytoskeleton for both intracellular and intercellular transport. The precise function of the cytoskeleton in PD may change during cell development, and may not be identical in all tissue types, or even in all PD within a single cell. Nevertheless, it is likely that actin- and myosin-associated proteins play a key role in regulating cell-cell transport, by interacting with cargo and loading it into PD, and may underlie the capacity for one-way transport across particular cell and tissue boundaries.     

Models of long-distance transport: how is carrier-dependent auxin transport regulated in the stem?

? This paper presents two models of carrier-dependent long-distance auxin transport in stems that represent the process at different scales. ? A simple compartment model using a single constant auxin transfer rate produced similar data to those observed in biological experiments. The effects of different underlying biological assumptions were tested in a more detailed model representing cellular and intracellular processes that enabled discussion of different patterns of carrier-dependent auxin transport and signalling. ? The output that best fits the biological data is produced by a model where polar auxin transport is not limited by the number of transporters/carriers and hence supports biological data showing that stems have considerable excess capacity to transport auxin. ? All results support the conclusion that auxin depletion following apical decapitation in pea (Pisum sativum) occurs too slowly to be the initial cause of bud outgrowth. Consequently, changes in auxin content in the main stem and changes in polar auxin transport/carrier abundance in the main stem are not correlated with axillary bud outgrowth.     

Mathematical models of α-synuclein transport in axons

To investigate possible effects of diffusion on α-synuclein (α-syn) transport in axons, we developed two models of α-syn transport, one that assumes that α-syn is transported only by active transport, as part of multiprotein complexes, and a second that assumes an interplay between motor-driven and diffusion-driven α-syn transport. By comparing predictions of the two models, we were able to investigate how diffusion could influence axonal transport of α-syn. The predictions obtained could be useful for future experimental work aimed at elucidating the mechanisms of axonal transport of α-syn. We also attempted to simulate possible defects in α-syn transport early in Parkinson's disease (PD). We assumed that in healthy axons α-syn localizes in the axon terminal while in diseased axons α-syn does not localize in the terminal (this was simulated by postulating a zero α-syn flux into the terminal). We found that our model of a diseased axon predicts the build-up of α-syn close to the axon terminal. This build-up could cause α-syn accumulation in Lewy bodies and the subsequent axonal death pattern observed in PD (‘dying back’ of axons).     

A role for voltage gated T-type calcium channels in mediating "capacitative" calcium entry?

Calcium entry through plasma membrane calcium channels is one of the most important cell signaling mechanism involved in such diverse functions as secretion, contraction and cell growth by regulating gene expression, proliferation and apoptosis. The identity of plasma membrane calcium channels, the main regulators of calcium entry, involved in cell proliferation has been thus extensively sought. Among these, a calcium entry pathway called capacitative calcium entry (CCE), activated by calcium store depletion, is particularly important in non-excitable cells. Though this capacitative calcium entry is generally supposed to occur through TRP channels there is some evidence that voltage-dependent T-type calcium channels may contribute to calcium entry after store depletion. Here we show that though mibefradil, a T-type calcium channel blocker, is able to reduce capacitative calcium entry induced by either thapsigargin or ATP, this was not mimicked by any other T-type calcium channel inhibitors even in cells overexpressing alpha(1H) T-type calcium channels, leading us to conclude that T-type calcium channels are not responsible for the capacitative calcium entry observed in different cancer cell lines. On the contrary, we show that the action of mibefradil on capacitative calcium entry is due to an action on store-operated calcium channels.