VDAC closure increases calcium ion flux

VDAC is the major permeability pathway in the mitochondrial outer membrane and can control the flow of metabolites and ions. Therefore Ca²⁺ flux across the outer membrane occurs mainly through VDAC. Since both Ca²⁺ fluxes and VDAC are involved in apoptosis, we examined whether Ca²⁺ is required for channel formation by VDAC isolated from rat liver. The voltage gating of VDAC does not require Ca²⁺ and it functions normally with or without Ca²⁺. Additionally, VDAC generally shows a higher permeability to Ca²⁺ in the closed states (states with lower permeability to metabolites) than that in the open state. Thus VDAC closure, which induces apoptosis, also favors Ca²⁺ flux into mitochondria, which can also lead to permeability transition and cell death. These results are consistent with the view that VDAC closure is a pro-apoptotic signal.     

Molecular dynamics studies of ion permeation in VDAC

The voltage-dependent anion channel (VDAC) in the outer membrane of mitochondria serves an essential role in the transport of metabolites and electrolytes between the cell matrix and mitochondria. To examine its structure, dynamics, and the mechanisms underlying its electrophysiological properties, we performed a total of 1.77 μs molecular dynamics simulations of human VDAC isoform 1 in DOPE/DOPC mixed bilayers in 1 M KCl solution with transmembrane potentials of 0, ±25, ±50, ±75, and ±100 mV. The calculated conductance and ion selectivity are in good agreement with the experimental measurements. In addition, ion density distributions inside the channel reveal possible pathways for different ion species. Based on these observations, a mechanism underlying the anion selectivity is proposed; both ion species are transported across the channel, but the rate for K⁺ is smaller than that for Cl⁻ because of the attractive interactions between K⁺ and residues on the channel wall. This difference leads to the anion selectivity of VDAC.     

Hypoxia increases calcium flux through cortical neuron glutamate receptors via protein kinase C

The effects of 30 s to 10 min hypoxia (PO2-10 mmHg) on glutamate receptor activity were studied in murine cortical neurons. Receptor activity was assessed as a rise in intracellular calcium concentration ([Ca2+]i) following a 10 s application of 1 mm glutamate or 100 micro mN-methy-d-aspartate (NMDA) in the presence of 0.1 mm Mg2+ and 10 micro m glycine. Change in [Ca2+]i elicited by glutamate increased 26% (n = 192, p < 0.001) and that to NMDA by 74% (n = 9, p < 0.01) during a 100-s period of hypoxia. After 10 min hypoxia, responses to glutamate were 62% smaller than those in normoxia, with increased basal intracellular [Ca2+]i predicting reduced receptor activity. When neurons were exposed to NMDA after 10 min of hypoxia, [Ca2+]i increases were 12% smaller than after 100 s hypoxia, but still 53% larger than in oxygenated neurons (n = 9, p = 0.01). Neurons expressed relatively similar amounts of NR2A, -B, -C, and -D subunits. The phosphorylation of NMDA NR1 subunits increased during hypoxia. Pre-treatment of neurons with a protein kinase C (PKC) inhibitor (chelerythrine, 10 micro m) prevented increases in N-methy-d-aspartate receptor (NMDAR) activity during hypoxia and reduced the phosphorylation of NR1 subunits. These results suggest that enhancement of glutamate receptor activity during the first minutes of hypoxia is mediated by phosphorylation of NMDARs by PKC and that other mechanisms, possibly involving intracellular calcium, limit glutamate receptor-mediated calcium influx during longer periods of hypoxia.     

Brownian dynamics simulations of ion transport through the VDAC

It is important to gain a physical understanding of ion transport through the voltage-dependent anion channel (VDAC) because this channel provides primary permeation pathways for metabolites and electrolytes between the cytosol and mitochondria. We performed grand canonical Monte Carlo/Brownian dynamics (GCMC/BD) simulations to explore the ion transport properties of human VDAC isoform 1 (hVDAC1; PDB:2K4T) embedded in an implicit membrane. When the MD-derived, space-dependent diffusion constant was used in the GCMC/BD simulations, the current-voltage characteristics and ion number profiles inside the pore showed excellent agreement with those calculated from all-atom molecular-dynamics (MD) simulations, thereby validating the GCMC/BD approach. Of the 20 NMR models of hVDAC1 currently available, the third one (NMR03) best reproduces both experimental single-channel conductance and ion selectivity (i.e., the reversal potential). In addition, detailed analyses of the ion trajectories, one-dimensional multi-ion potential of mean force, and protein charge distribution reveal that electrostatic interactions play an important role in the channel structure and ion transport relationship. Finally, the GCMC/BD simulations of various mutants based on NMR03 show good agreement with experimental ion selectivity. The difference in ion selectivity between the wild-type and the mutants is the result of altered potential of mean force profiles that are dominated by the electrostatic interactions.     

Neutrophil cathepsin G increases calcium flux and inositol polyphosphate production in cultured endothelial cells

Exposure of endothelial cells (ENDO) to human neutrophil cathepsin G (CG) increases albumin flux across the endothelial monolayer. Since calcium influences cell shape and barrier function of ENDO monolayers, the current study was designed to determine if CG acted through alterations in Ca2+ homeostasis in ENDO. The role of Ca2+ in the increased permeability of ENDO monolayers to albumin after exposure to CG was studied by using ENDO monolayers cultured on polycarbonate filters. Exposure of ENDO monolayers to CG in the presence of the Ca2+-antagonist lanthanum partially prevented the increase in albumin flux, but exposure in the presence of agents that block voltage-regulated calcium channels did not block the increase in albumin flux. To monitor the effect of CG on Ca2+-flux, ENDO were labeled with 45Ca2+ and changes in Ca2+ flux were monitored by the release of 45Ca2+. From 1 to 15 minutes after exposure of ENDO to CG, there was increased release of 45Ca2+ compared with control cells. Calcium channel blocking agents did not inhibit the increased release of 45Ca2+, but lanthanum partially blocked the increase. The increased release of Ca2+ appeared to be due, at least in part, to activation of phospholipase C because there was an increase both in inositol polyphosphate species and in diglycerides after incubation of ENDO with CG. These studies support the hypothesis that CG increases the flux of calcium in ENDO, that this increase in Ca2+ flux may result from activation of phospholipase C, and that this system may be involved in the decreased barrier properties of the ENDO after CG exposure.     

Wolframin expression induces novel ion channel activity in endoplasmic reticulum membranes and increases intracellular calcium

Wolfram syndrome is an autosomal recessive neuro-degenerative disorder associated with juvenile onset non-autoimmune diabetes mellitus and progressive optic atrophy. The disease has been attributed to mutations in the WFS1 gene, which codes for a protein predicted to possess 9-10 transmembrane segments. Little is known concerning the function of the WFS1 protein (wolframin). Endoglycosidase H digestion, immunocytochemistry, and subcellular fractionation studies all indicated that wolframin is localized to the endoplasmic reticulum in rat brain hippocampus and rat pancreatic islet beta-cells, and after ectopic expression in Xenopus oocytes. Reconstitution of wolframin from oocyte membranes into planar lipid bilayers demonstrated that the protein induced a large cation-selective ion channel that was blocked by Mg2+ or Ca2+. Inositol triphosphate was capable of activating channels in the fused bilayers that were similar to channel components induced by wolframin expression. Expression of wolframin also increased cytosolic calcium levels in oocytes. Wolframin thus appears to be important in the regulation of intracellular Ca2+ homeostasis. Disruption of this function may place cells at risk to suffer inappropriate death decisions, thus accounting for the progressive beta-cell loss and neuronal degeneration associated with the disease.     

Concentration dependent ion selectivity in VDAC: a molecular dynamics simulation study

The voltage-dependent anion channel (VDAC) forms the major pore in the outer mitochondrial membrane. Its high conducting open state features a moderate anion selectivity. There is some evidence indicating that the electrophysiological properties of VDAC vary with the salt concentration. Using a theoretical approach the molecular basis for this concentration dependence was investigated. Molecular dynamics simulations and continuum electrostatic calculations performed on the mouse VDAC1 isoform clearly demonstrate that the distribution of fixed charges in the channel creates an electric field, which determines the anion preference of VDAC at low salt concentration. Increasing the salt concentration in the bulk results in a higher concentration of ions in the VDAC wide pore. This event induces a large electrostatic screening of the charged residues promoting a less anion selective channel. Residues that are responsible for the electrostatic pattern of the channel were identified using the molecular dynamics trajectories. Some of these residues are found to be conserved suggesting that ion permeation between different VDAC species occurs through a common mechanism. This inference is buttressed by electrophysiological experiments performed on bean VDAC32 protein akin to mouse VDAC.     

Inhibition by aluminum hydroxide of the voltage-dependent closure of the mitochondrial channel, VDAC

Micromolar quantities of aluminum have been found (Dill et al. (1987) J. Membrane Biol. 99, 187-196) to reduce the voltage dependence of the mitochondrial outer membrane channel, VDAC, from Neurospora crassa. In the present study, various metallic and organic ions were tested for possible aluminum-like effect, and only the trivalent metals exhibited a similar ability to reduce the channels voltage dependence. However, trivalency alone was not sufficient because lanthanum (III) had no effect. Quantitative analyses with three group IIIA metals (A1, Ga, and In) showed that, of the structural characteristics examined, the ability to form sufficient M(OH)3 at experimental pH was the primary property shared by all the effective metals. While providing new insight into the nature of VDAC's sensor, these results also indicate that aluminum-cell interaction may result from the presence of AI(OH)3 in solution in addition to the widely accepted AI3+-mediated interactions. While the [AI3+] is vanishingly low at neutral pH, the trihydroxide is the major form and should be considered as an important candidate for aluminum-induced cellular effects.     

Molecular genetics of the VDAC ion channel: Structural model and sequence analysis

The voltage-dependent anion-selective channel of the outer mitochondrial membrane provides a unique system in which to study the molecular basis of voltage gating of ion flow. We have cloned and sequenced acDNA coding for this protein in yeast. From the derived amino acid sequence, we have generated a preliminary model for the secondary structure of the protein which suggests that the protein forms a -barrel type structure. Comparison of the VDAC amino acid sequence with that of the bacterial porins has indicated that the two classes of molecules appear to be unrelated.     

A soluble mitochondrial protein increases the voltage dependence of the mitochondrial channel,VDAC

A soluble protein isolated from mitochondria has been found to modulate the voltage-dependent properties of the mitochondrial outer membrane channel, VDAC. This protein, called the VDAC modulator, was first found inNeurospora crassa and then discovered in species from other eukaryotic kingdoms. The modulator-containing fraction (at a crude protein concentration of 20 µg/ml) increases the voltage dependence of VDAC channels over 2–3-fold. At higher protein concentrations (50–100 µg/ml), some channels seem to remain in a closed state or be blocked while others display the higher voltage dependence and are able to close at low membrane potentials. By increasing the steepness of the voltage-dependent properties of VDAC channels, this modulator may serve as an amplifierin vivo to increase the sensitivity of the channels in response to changes in the cell's microenvironment, and consequently, regulate the metabolic flux across the outer mitochondrial membrane by controlling the gating of VDAC channels.     

Imaging ion flux and ion homeostasis in blood stage malaria parasites

The steady-state regulation of intracellular levels of essential ions and ionic gradients is critical for almost all functions within a cell. Thus, it is not surprising to find that ions have been shown to play an important role in numerous parasitic processes, such as invasion, development and possibly drug resistance mechanisms. Live cell imaging has become a widespread technique to visualize and quantify several of these processes, including pH and Ca²⁺ homeostasis, in an effort to better understand the biology and physiology of cells. This is now also the case for many human pathogens. The aim of this review is to emphasize the importance of this technique and provide an overview of what we have learned so far, using the malaria parasite Plasmodium falciparum as a paradigm.     

Plant cell growth and ion flux responses to the streptomycete phytotoxin thaxtomin A: calcium and hydrogen flux patterns revealed by the non-invasive MIFE technique

Thaxtomin A, a key phytotoxin produced by plant pathogenic Streptomyces sp., is implicit in common scab disease expression in potato. Primary targets and modes of action of thaxtomin A toxicity in plant cells are not well understood. In this work, early signalling events associated with thaxtomin A toxicity were studied using the ion-selective microelectrode ion flux estimation (MIFE) technique. Thaxtomin A-induced changes in net ion fluxes were measured across the plasma membrane (PM) of root and pollen tube tissue in Arabidopsis thaliana and tomato. Within a minute after toxin application, a rapid and short-lived Ca2+ influx was observed. Well ahead of the marked inhibition of root growth, a significant shift towards net H+ efflux across the PM occurred in all tissues. Similar to root tissues, thaxtomin A significantly modified ion flux profiles from growing pollen tubes. Thaxtomin A was more effective in young, physiologically active tissues (root elongation zone or pollen tube apex), suggesting a higher density of thaxtomin A-binding sites in these regions. Overall, our data provide the first evidence that thaxtomin A triggers an early signalling cascade, which may be crucial in plant-pathogen interactions. It also suggests a possible interaction between thaxtomin A and PM auxin receptors, as revealed from experiments on the auxin-sensitive ucu2-2/gi2 A. thaliana mutant.     

Mitochondrial calcium flux in temperature-sensitive mutants of

The mechanisms of mitochondrial calcium flux in normal and temperature-sensitive mutants of were investigated. Adult mitochondria from all stocks, when analysed with an oxygen electrode, gave respiration rates which exhibited normal responses to adenosine diphosphate and uncoupling agents but no stimulation by calcium. In contrast, calcium stimulates the respiration rate of normal larval mitochondria particularly those of the second instar. This is not evident in second instar mitochondria from the temperature sensitive mutants. There is a rapid accumulation of mitochondrial calcium during normal larval ontogenesis. The levels in temperature-sensitive second instar mitochondria are higher than those of any of the normal larval stages. Adult mitochondria in all cases contain very low levels of calcium. The amount of calcium taken up by mitochondria of second instar temperature-sensitive mutants is lower than that of normal. This may reflect the higher endogenous levels already present in the former. The implications of these variations in calcium metabolism for the behavioural defects of the temperature sensitive mutants is discussed.     

Physiological elevations in cytoplasmic free calcium by cold or ion injection result in transient closure of higher plant plasmodesmata

The concentration of cytoplasmic free calcium ([Ca²⁺]_cyt) required to close higher plant plasmodesmata was investigated using corn (Zea mays L. cv. Black Mexican Sweet) suspension-culture cells. Physiological elevations of [Ca²⁺]_cyt were applied by cold treatment, and ion injection was also used to increase [Ca²⁺]_cyt, by diffusion (for small increases) or by iontophoresis (for larger increases). The impact of such treatments on [Ca²⁺]_cyt was measured by ratiometric ion imaging. Intercellular communication during treatments was monitored using our recently developed electrophysiological technique that allows the electrical resistance of plasmodesmata and the plasma membranes of a sister-cell pair to be measured. A 4-fold increase in the calculated resistance of single plasmodesmata was observed in response to cold treatment that caused a 2-fold increase in average [Ca²⁺]_cyt (from 107 to 210 nM). In response to iontophoresis of Ca²⁺, plasmodesmata were observed to go from “open” (low resistance) to “shut” (high resistance) and then back “open” within 10 s. Our results thus indicate that higher plant plasmodesmata respond quickly to physiological changes in [Ca²⁺]_cyt. Received: 2 June 1999 / Accepted: 16 July 1999     

Pollen tube growth is coupled to the extracellular calcium ion flux and the intracellular calcium gradient: effect of BAPTA-type buffers and hypertonic media.

Lily pollen tubes possess a steep, tip-focused intracellular Ca2+ gradient and a tip-directed extracellular Ca2+ influx. Ratiometric ion imaging revealed that the gradient extends from above 3.0 microM at the apex to approximately 0.2 microM within 20 microns from the tip, while application of the Ca(2+)-specific vibrating electrode indicated that the extracellular influx measured between 1.4 and 14 pmol cm-2 sec-1. We examined the relationship between these phenomena and their role in tube growth by using different 1,2-bis(o-aminophenoxy)ethane N,N,N',N'-tetraacetic acid (BAPTA)-type buffers and hypertonic media. Injection of active BAPTA-type buffers or application of elevated levels of sucrose reversibly inhibited growth, destroyed tip zonation of organelles, and modified normal patterns of cytoplasmic streaming. Simultaneously, these treatments dissipated both the intracellular tip-focused gradient and the extracellular Ca2+ flux. Of the BAPTA-type buffers, 5,5'-dibromo-BAPTA (dissociation constant [Kd] is 1.5 microM) and 4,4'-difluoro-BAPTA (Kd of 1.7 microM) exhibited greater activity than those buffers with either a higher affinity (5,5'-dimethyl-BAPTA, Kd of 0.15 microM; BAPTA, Kd of 0.21 microM; 5,5'-difluoro-BAPTA, Kd of 0.25 microM) or lower affinity (5-methyl, 5'-nitro-BAPTA, Kd of 22 microM) for Ca2+. Our findings provide evidence that growing pollen tubes have open Ca2+ channels in their tip and that these channels become inactivated in nongrowing tubes. The studies with elevated sucrose support the view that stretching of the apical plasma membrane contributes to the maintenance of the Ca2+ signal.     

Monovalent ion and calcium ion fluxes in sarcoplasmic reticulum

Summary The ion permeability of sarcoplasmic reticulum vesicles from skeletal and heart muscle has been characterized by radioisotope flux, osmotic and membrane potential measurements, and by incorporating vesicles into planar phospholipid bilayers. The sarcoplasmic reticulum membrane is uniquely permeable to various biologically relevant monovalent ions. At least two and possibly three separate passive permeation systems for monovalent ions have been identified: 1) a K⁺, Na⁺ channel, 2) an anion channel, and 3) a H⁺ (OH^–) permeable pathway which may or may not be synonymous with the anion channel. A possible physiological function of these monovalent ion permeation systems is to permit rapid movement of K⁺, Na⁺, H⁺ and Cl across the membrane to counter electrogenic Ca²⁺ fluxes during Ca²⁺ release and uptake by sacroplasmic reticulum.