Diisothiocyanostilbene-2,2'-disulfonate Research Articles

Effects of elevated pCO2 on physiological performance of marine microalgae Dunaliella salina (Chlorophyta, Chlorophyceae

The present study was conducted to determine the effects of elevated pCO2 on growth, photosynthesis, dark respiration and inorganic carbon acquisition in the marine microalga Dunaliella salina. To accomplish this, D. salina was incubated in semi-continuous cultures under present-day CO2 levels (390 μatm, pHNBS: 8.10), predicted year 2100 CO2 levels (1 000 μatm, pHNBS: 7.78) and predicted year 2300 CO2 levels (2 000 μatm, pHNBS: 7.49). Elevated pCO2 significantly enhanced photosynthesis (in terms of gross photosynthetic O2 evolution, effective quantum yield (ΔF/F' m ), photosynthetic efficiency (α), maximum relative electron transport rate (rETRmax) and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity) and dark respiration of D. salina, but had insignificant effects on growth. The photosynthetic O2 evolution of D. salina was significantly inhibited by the inhibitors acetazolamide (AZ), ethoxyzolamide (EZ) and 4,4’-diisothiocyanostilbene-2,2′-disulfonate (DIDS), indicating that D. salina is capable of acquiring HCOˉ 3 via extracellular carbonic anhydrase and anion-exchange proteins. Furthermore, the lower inhibition of the photosynthetic O2 evolution at high pCO2 levels by AZ, EZ and DIDS and the decreased carbonic anhydrase showed that carbon concentrating mechanisms were down-regulated at high pCO2. In conclusion, our results show that photosynthesis, dark respiration and CCMs will be affected by the increased pCO2/low pH conditions predicted for the future, but that the responses of D. salina to high pCO2/low pH might be modulated by other environmental factors such as light, nutrients and temperature. Therefore, further studies are needed to determine the interactive effects of pCO2, temperature, light and nutrients on marine microalgae.

Papain cleavage of the 38,000-dalton fragment inhibits the binding of 4, 4'-diisothiocyanostilbene-2, 2'-disulfonate to lys-539 on the 60,000-dalton fragment in human band 3.

Human band 3 is a 98-kDa transmembrane (TM) protein comprising 14 TM segments. Papain cleavages band 3 into 38- and 60-kDa fragments. Under vigorous conditions, the cleavage of the loop region between the TM 7 of gate domain and the TM 8 of core domain in the 38-kDa fragment produces 7- and 31-kDa fragments. Conformational changes of the TM 5 segment containing Lys-539 by cleavage of the 38-kDa fragment remain unclear. Pressure-induced haemolysis of erythrocytes was suppressed by binding of 4, 4'-diisothiocyanostilbene-2, 2'-disulfonate (DIDS) to Lys-539. Such effect of DIDS was not observed upon cleavage of the 38-kDa fragment, because of inhibition of DIDS binding to Lys-539. Using fluorescence of DIDS labelled to Lys-539, conformational changes of band 3 were examined. Fluorescence spectra demonstrated that the molecular motion of DIDS is more restricted upon digestion of the 38-kDa fragment. Interestingly, the quenching of DIDS fluorescence showed that Hg2+ is less accessible to DIDS upon digestion of the 38-kDa fragment. Taken together, we propose that the conformational changes of the TM 5 segment characterized by the sequestration and restricted motion of Lys-539 are induced by the cleavage of the loop region between the TM 7 and the TM 8.

Data supporting the involvement of the adenine nucleotide translocase conformation in opening the Tl(+)-induced permeability transition pore in Ca(2+)-loaded rat liver mitochondria.

There we made available information about the effects of the adenine nucleotide translocase (ANT) ‘c’ conformation fixers (phenylarsine oxide (PAO), tert-butylhydroperoxide (tBHP), and carboxyatractyloside) as well as thiol reagent (4,4′-diisothiocyanostilbene-2,2′-disulfonate (DIDS)) on isolated rat liver mitochondria. We observed a decrease in A540 (mitochondrial swelling) and respiratory control rates (RCRADP [state 3/state 4] and RCRDNP [2,4-dinitrophenol-uncoupled state/basal state or state 4]), as well as an increase in Ca2+-induced safranin fluorescence (F485/590, arbitrary units), showed a dissipation in the inner membrane potential (ΔΨmito), in experiments with energized rat liver mitochondria, injected into the buffer containing 25–75 mM TlNO3, 125 mM KNO3, and 100 µM Ca2+. The fixers and DIDS, in comparison to Ca2+ alone, greatly increased A540 decline and the rate of Ca2+-induced ΔΨmito dissipation. These reagents also markedly decreased RCRADP and RCRDNP. The MPTP inhibitors (ADP, cyclosporin A, bongkrekic acid, and N-ethylmaleimide) fixing the ANT in ‘m’ conformation significantly hindered the above-mentioned effects of the fixers and DIDS. A more complete scientific analysis of these findings may be obtained from the manuscript “To involvement the conformation of the adenine nucleotide translocase in opening the Tl+-induced permeability transition pore in Ca2+-loaded rat liver mitochondria” (Korotkov et al., 2016 [1]).

To involvement the conformation of the adenine nucleotide translocase in opening the Tl+-induced permeability transition pore in Ca2+-loaded rat liver mitochondria

The conformation of adenine nucleotide translocase (ANT) has a profound impact in opening the mitochondrial permeability transition pore (MPTP) in the inner membrane. Fixing the ANT in ‘c’ conformation by phenylarsine oxide (PAO), tert-butylhydroperoxide (tBHP), and carboxyatractyloside as well as the interaction of 4,4′-diisothiocyanostilbene-2,2′-disulfonate (DIDS) with mitochondrial thiols markedly attenuated the ability of ADP to inhibit the MPTP opening. We earlier found (Korotkov and Saris, 2011) that calcium load of rat liver mitochondria in medium containing TlNO3 and KNO3 stimulated the Tl+-induced MPTP opening in the inner mitochondrial membrane. The MPTP opening as well as followed increase in swelling, a drop in membrane potential (ΔΨmito), and a decrease in state 3, state 4, and 2,4-dinitrophenol-uncoupled respiration were visibly enhanced in the presence of PAO, tBHP, DIDS, and carboxyatractyloside. However, these effects were markedly inhibited by ADP and membrane-penetrant hydrophobic thiol reagent, N-ethylmaleimide (NEM) which fix the ANT in ‘m’ conformation. Cyclosporine A additionally potentiated these effects of ADP and NEM. Our data suggest that conformational changes of the ANT may be directly involved in the opening of the Tl+-induced MPTP in the inner membrane of Ca2+-loaded rat liver mitochondria. Using the Tl+-induced MPTP model is discussed in terms finding new transition pore inhibitors and inducers among different chemical and natural compounds.

Comparative research on inorganic carbon acquisition by the macroalgae Ulva prolifera (Chlorophyta) and Pyropia yezoensis (Rhodophyta)

The red macroalga Pyropia/Porphyra is one of the most important marine crops in the world; Pyropia cultivation is severely affected by the green macroalga Ulva spp., a fouling organism. In this study, growth competition and acquisition of dissolved inorganic carbon (Ci) by Ulva prolifera and Pyropia yezoensis were investigated to understand the physiological characteristics of the two species. Competition experimental results showed that the relative growth rate of U. prolifera was not affected, whereas the growth rate of P. yezoensis was significantly inhibited. U. prolifera exhibited a competitive advantage when these two species were cultured together. The two algal species displayed high pH compensation points, suggesting that U. prolifera and P. yezoensis can photosynthesize by using HCO3 −; the utilization ability of U. prolifera is stronger than that of P. yezoensis. The net photosynthetic rates of P. yezoensis and U. prolifera were significantly inhibited by the carbonic anhydrase (CA) inhibitors acetazolamide and 6-ethoxyzolamide indicating that CA is implicated in carbon-concentrating mechanisms (CCM). 4,4′-diisothiocyano-stilbene-2,2′-disulfonate (DIDS), another inhibitor that prevents direct HCO3 − uptake, did not significantly affect U. prolifera. Conversely, DIDS can sharply decrease the photosynthetic rate of P. yezoensis, particularly at high pH. U. prolifera and P. yezoensis also showed CO2-limited photosynthesis by which the half-saturating concentration of Ci exceeds that of seawater. U. prolifera did not show active HCO3 − uptake; instead, U. prolifera used HCO3 − via extracellular CA for photosynthetic carbon fixation. U. prolifera also utilizes extracellular CA-mediated HCO3 − absorption to a higher extent than P. yezoensis. By contrast, P. yezoensis shows active HCO3 − uptake and extracellular CCMs for photosynthetic carbon fixation. Therefore, these two algal species can survive in changing environments with high pH and low Ci concentration because of the action of CCMs.

Human SLC4A11-C functions as a DIDS-stimulatable H⁺(OH⁻) permeation pathway: partial correction of R109H mutant transport.

The SLC4A11 gene mutations cause a variety of genetic corneal diseases, including congenital hereditary endothelial dystrophy 2 (CHED2), Harboyan syndrome, some cases of Fuchs' endothelial dystrophy (FECD), and possibly familial keratoconus. Three NH2-terminal variants of the human SLC4A11 gene, named SLC4A11-A, -B, and -C are known. The SLC4A11-B variant has been the focus of previous studies. Both the expression of the SLC4A11-C variant in the cornea and its functional properties have not been characterized, and therefore its potential pathophysiological role in corneal diseases remains to be explored. In the present study, we demonstrate that SLC4A11-C is the predominant SLC4A11 variant expressed in human corneal endothelial mRNA and that the transporter functions as an electrogenic H(+)(OH(-)) permeation pathway. Disulfonic stilbenes, including 4,4'-diisothiocyano-2,2'-stilbenedisulfonate (DIDS), 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonate (H2DIDS), and 4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulfonate (SITS), which are known to bind covalently, increased SLC4A11-C-mediated H(+)(OH(-)) flux by 150-200% without having a significant effect in mock-transfected cells. Noncovalently interacting 4,4'-diaminostilbene-2,2'-disulfonate (DADS) was without effect. We tested the efficacy of DIDS on the functionally impaired R109H mutant (SLC4A11-C numbering) that causes CHED2. DIDS (1 mM) increased H(+)(OH(-)) flux through the mutant transporter by ∼40-90%. These studies provide a basis for future testing of more specific chemically modified dilsulfonic stilbenes as potential therapeutic agents to improve the functional impairment of specific SLC4A11 mutant transporters.

Dye-permeable, voltage-gated channel on mouse fungiform taste bud cells

We show here the expression, permeability and pharmacology of a voltage-gated channel in certain taste bud cells (TBCs) which is known to be permeable to Lucifer Yellow CH (LY) and known to release ATP as a neurotransmitter in response to taste substances. LY dissolved in a 200mM K+-containing solution label TBCs immunoreactive to PLCβ2, a phospholipase subtype, but not the TBC subtype immunoreactive to SNAP-25, a SNARE protein. In addition to these subtypes, LY also labelled a few of the non-immunoreactive TBCs. Monovalent and divalent anion probes with molar mass less than 1200 also label PLCβ2-immunoreactive TBCs and a few non-immunoreactive TBCs, whereas a cation probe, rhodamine B, labels the cell membrane of TBCs nonselectively and K+ independently. The number of LY-labelled TBCs is decreased by 5μM DIDS (4,4′-diisothiocyanostilbene-2-2′disulfonate), 1mM octanol and 10−5M H+, but not by 10μM carbenoxolone, 2mM probenecid, 10mM TEA, or 30μM flufenamic acid. PLCβ2-immunoreactive TBCs and a few non-immunoreactive TBCs generate a TEA-insensitive outwardly rectifying current. DIDS decreases this current in magnitude with IC50 of ~0.4μM in a voltage-independent manner. Also 10−5M H+ and 1mM octanol decreases the current magnitude, but 10μM carbenoxolone and 2mM probenecid do not. These results show that the LY-permeable channel preferably permeates anions and occurs not only on PLCβ2-immunoreactive TBCs but also on certain non-immunoreactive TBCs. Also the results show that the pharmacology of the LY-permeable channel is different from hemichannels reported. The discussion focuses on the pharmacology and the role of the LY-permeable channel.

ATP storage and uptake by isolated pancreatic zymogen granules

ATP is released from pancreatic acini in response to cholinergic and hormonal stimulation. The same stimuli cause exocytosis of ZG (zymogen granules) and release of digestive enzymes. The aim of the present study was to determine whether ZG stored ATP and to characterize the uptake mechanism for ATP transport into the ZG. ZG were isolated and the ATP content was measured using luciferin/luciferase assays and was related to protein in the sample. The estimate of ATP concentration in freshly isolated granules was 40-120 microM. The ATP uptake had an apparent Km value of 4.9+/-2.1 mM when granules were incubated without Mg2+ and a Km value of 0.47+/-0.05 mM in the presence of Mg2+, both in pH 6.0 buffers. The uptake of ATP was significantly higher at pH 7.2 compared with pH 6.0 solutions. The anion transport blockers DIDS (4,4'-di-isothiocyanostilbene-2,2'-disulfonate) and Evans Blue inhibited ATP transport. Western blot analysis on the ZG showed the presence of VNUT (vesicular nucleotide transporter). Together, these findings indicate that VNUT may be responsible for the ATP uptake into ZG. Furthermore, the present study shows the presence of ATP together with digestive enzymes in ZG. This indicates that co-released ATP would regulate P2 receptors in pancreatic ducts and, thus, ductal secretion, and this would aid delivery of enzymes to the duodenum.

Electrophysiological characterization of ATPases in native synaptic vesicles and synaptic plasma membranes

Vesicular V-ATPase (V-type H+-ATPase) and the plasma membrane-bound Na+/K+-ATPase are essential for the cycling of neurotransmitters at the synapse, but direct functional studies on their action in native surroundings are limited due to the poor accessibility via standard electrophysiological equipment. We performed SSM (solid supported membrane)-based electrophysiological analyses of synaptic vesicles and plasma membranes prepared from rat brains by sucrose-gradient fractionation. Acidification experiments revealed V-ATPase activity in fractions containing the vesicles but not in the plasma membrane fractions. For the SSM-based electrical measurements, the ATPases were activated by ATP concentration jumps. In vesicles, ATP-induced currents were inhibited by the V-ATPase-specific inhibitor BafA1 (bafilomycin A1) and by DIDS (4,4'-di-isothiocyanostilbene-2,2'-disulfonate). In plasma membranes, the currents were inhibited by the Na+/K+-ATPase inhibitor digitoxigenin. The distribution of the V-ATPase- and Na+/K+-ATPase-specific currents correlated with the distribution of vesicles and plasma membranes in the sucrose gradient. V-ATPase-specific currents depended on ATP with a K0.5 of 51+/-7 microM and were inhibited by ADP in a negatively co-operative manner with an IC50 of 1.2+/-0.6 microM. Activation of V-ATPase had stimulating effects on the chloride conductance in the vesicles. Low micromolar concentrations of DIDS fully inhibited the V-ATPase activity, whereas the chloride conductance was only partially affected. In contrast, NPPB [5-nitro-2-(3-phenylpropylamino)-benzoic acid] inhibited the chloride conductance but not the V-ATPase. The results presented describe electrical characteristics of synaptic V-ATPase and Na+/K+-ATPase in their native surroundings, and demonstrate the feasibility of the method for electrophysiological studies of transport proteins in native intracellular compartments and plasma membranes.

Studies on the DIDS-binding Site of Monocarboxylate Transporter 1 Suggest a Homology Model of the Open Conformation and a Plausible Translocation Cycle

Site-directed mutagenesis of MCT1 was performed on exofacial lysines Lys38, Lys45, Lys282, and Lys413. K38Q-MCT1 and K38R-MCT1 were inactive when expressed at the plasma membrane of Xenopus laevis oocytes, whereas K45R/K282R/K413R-MCT1 and K45Q/K282Q/K413Q-MCT1 were active. The former exhibited normal reversible and irreversible inhibition by DIDS, whereas the latter showed less reversible and no irreversible inhibition. K45Q/K413Q-MCT1 retained some irreversible inhibition, whereas K45Q/K282Q-MCT1 and K282Q/K413Q-MCT1 did not. These data suggest that the two DIDS SO3− groups interact with positively charged Lys282 together with Lys45 and/or Lys413. This positions one DIDS isothiocyanate group close to Lys38, leading to its covalent modification and irreversible inhibition. Additional mutagenesis revealed that DIDS cross-links MCT1 to its ancillary protein embigin using either Lys38 or Lys290 of MCT1 and Lys160 or Lys164 of embigin. We have modeled a possible structure for the outward facing (open) conformation of MCT1 by employing modest rotations of the C-terminal domain of the inner facing conformation modeled previously. The resulting model structure has a DIDS-binding site consistent with experimental data and locates Lys38 in a hydrophobic environment at the bottom of a substrate-binding channel. Our model suggests a translocation cycle in which Lys38 accepts a proton before binding lactate. Both the lactate and proton are then passed through the channel via Asp302− and Asp306+, an ion pair already identified as important for transport and located adjacent to Phe360, which controls channel selectivity. The cross-linking data have also been used to model a structure of MCT1 bound to embigin that is consistent with published data.

Imaging Of The Diffusion Of Individual Band 3 Molecules On Whole Erythrocytes From Patients With Hereditary Hemolytic Disorders

The plasma membrane of the human erythrocyte is a composite structure that consists of a fluid lipid bilayer (including membrane-embedded and membrane-associated proteins) and a sub-surface scaffolding consisting of a six-fold network of the tetrameric flexible structural protein spectrin. The spectrin network is pinned to the fluid bilayer through a series of transmembrane and membrane associated proteins both at the six-fold junctions and near the mid-points along the spectrin tetramers between junctions. Common to both of these pinning points is the presence of the transmembrane anion exchange protein band 3 (AE1). Band 3 ties the six-fold junction to the bilayer through interaction with the protein adducin and ties the midpoint to the bilayer through interaction with ankyrin. Much of the remarkable mechanical characteristics of the red cell have been attributed to this membrane architecture. Further, red cells become fragile in pathologies known to disrupt the spectrin network or its pinning points to the membrane. To assess changes in the structure of the plasma membrane of pathologic red cells at the single molecule level, the mobility of individual band 3 molecules was observed on normal red cells as well as those from patients with several types of hereditary diseases including spherocytosis, elliptocytosis, and pyropokilocytosis. Specifically, individual band 3 molecules on whole red cells were labeled by quantum dots through the band 3 inhibitor 4,4′-diisothiocyanostilbene-2,2′-disulfonate (DIDS). We will present data on the mobility of band 3 in each of these cell types recorded at video imaging rates of 120 fps. The observation that membrane pathologies can be distinguished by the mobility of individual membrane molecules suggests that single particle tracking might constitute a useful tool for characterizing the “health” of a membrane.

Role of Fyn and PI3K in H2O2-induced inhibition of apical Cl−/OH− exchange activity in human intestinal epithelial cells

H(2)O(2) is a highly reactive oxygen metabolite that has been implicated as an important mediator of inflammation-induced intestinal injury associated with ischaemia/reperfusion, radiation and inflammatory bowel disease. Previous studies have shown that H(2)O(2) inhibits NaCl absorption and activates Cl(-) secretion in the rat and rabbit colon. To date, however, almost no information is available with respect to its effect on the human intestinal apical anion exchanger Cl(-)/OH(-) (HCO(3)(-)). The present studies were, therefore, undertaken to examine the direct effects of H(2)O(2) on OH(-) gradient-driven DIDS (4,4'-di-isothiocyanostilbene-2,2'-disulfonate)-sensitive (36)Cl(-) uptake utilizing a post-confluent transformed human intestinal epithelial cell line, Caco-2. Our results demonstrate that H(2)O(2) (1 mM for 60 min) significantly inhibited (approx. 60%; P<0.05) Cl(-)/OH(-) exchange activity in Caco-2 cells. H(2)O(2)-mediated inhibition of Cl(-)/OH(-) exchange activity involved the Src kinase Fyn and PI3K (phosphoinositide 3-kinase)-dependent pathways. H(2)O(2) also induced phosphorylation of Fyn and p85 (the regulatory subunit of PI3K) in Caco-2 cells. Moreover, an increased association of Fyn and p85 was observed in response to H(2)O(2), resulting in the activation of the downstream target PLCgamma1 (phospholipase Cgamma1). Elevated intracellular Ca(2+) levels and PKCalpha (protein kinase Calpha) functioned as downstream effectors of H(2)O(2)-induced PLCgamma1 activation. Our results, for the first time, provide evidence for H(2)O(2)-induced Src kinase Fyn/PI3K complex association. This complex association resulted in the subsequent activation of PLCgamma1 and Ca(2+)-dependent PKCalpha, resulting in the inhibition of Cl(-)/OH(-) exchange activity. These findings suggest that H(2)O(2)-induced inhibition of the Cl(-)/OH(-) exchange process may play an important role in the pathophysiology of diarrhoea associated with inflammatory disorders, where the amount of reactive oxygen species is markedly elevated.

Role of Calcium in Volume-Activated Chloride Currents in a Mouse Cholangiocyte Cell Line

Volume-activated Cl(-) channels (VACCs) play vital roles in many cells including cholangiocytes. Previously, we characterized the VACCs in mouse cholangiocytes. Since calcium plays an important role in VACC regulation in many cells, we have studied the effect of calcium modulation on the regulatory volume decrease (RVD) and VACC currents in mouse bile duct cells (MBDCs). Cell volume measurements were assessed by a Coulter counter with cell sizer, and conventional whole-cell patch-clamp techniques were used to study the role of calcium on RVD and VACC currents. Cell volume study indicated that MBDCs exhibited RVD, which was inhibited by 5-nitro-2'-(3-phenylpropylamino)-benzoate (NPPB), 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) and 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra-acetoxymethyl ester (BAPTA-AM) but not by removal of extracellular calcium. During hypotonic challenge, MBDCs exhibited an outwardly rectified current, which was significantly inhibited by administration of classical chloride channel inhibitors such as NPPB and tamoxifen. Chelation of the intracellular calcium with BAPTA-AM or removal of extracellular calcium and calcium channel blocker had no significant effect on VACC currents during hypotonic challenge. In addition to VACC, MBDC had a calcium-activated chloride channel, which was inhibited by NPPB. The present study is the first to systemically study the role of calcium on the VACC and RVD in mouse cholangiocytes and demonstrates that a certain level of intracellular calcium is necessary for RVD but the activation of VACC during RVD does not require calcium. These findings suggest that calcium does not have a direct regulatory role on VACC but has a permissive role on RVD in cholangiocytes.

Aldolase potentiates DIDS activation of the ryanodine receptor in rabbit skeletal sarcoplasmic reticulum

DIDS (4,4'-di-isothiocyanostilbene-2,2'-disulfonate), an anion channel blocker, triggers Ca2+ release from skeletal muscle SR (sarcoplasmic reticulum). The present study characterized the effects of DIDS on rabbit skeletal single Ca2+-release channel/RyR1 (ryanodine receptor type 1) incorporated into a planar lipid bilayer. When junctional SR vesicles were used for channel incorporation (native RyR1), DIDS increased the mean P(o) (open probability) of RyR1 without affecting unitary conductance when Cs+ was used as the charge carrier. Lifetime analysis of single RyR1 activities showed that 10 microM DIDS induced reversible long-lived open events (P(o)=0.451+/-0.038) in the presence of 10 microM Ca2+, due mainly to a new third component for both open and closed time constants. However, when purified RyR1 was examined in the same condition, 10 microM DIDS became considerably less potent (P(o)=0.206+/-0.025), although the caffeine response was similar between native and purified RyR1. Hence we postulated that a DIDS-binding protein, essential for the DIDS sensitivity of RyR1, was lost during RyR1 purification. DIDS-affinity column chromatography of solubilized junctional SR, and MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS analysis of the affinity-column-associated proteins, identified four major DIDS-binding proteins in the SR fraction. Among them, aldolase was the only protein that greatly potentiated DIDS sensitivity. The association between RyR1 and aldolase was further confirmed by co-immunoprecipitation and aldolase-affinity batch-column chromatography. Taken together, we conclude that aldolase is physically associated with RyR1 and could confer a considerable potentiation of the DIDS effect on RyR1.

A novel systems biology/engineering approach solves fundamental molecular mechanistic problems in bioenergetics and motility

A large number of new insights and further intricate details of the molecular mechanisms of ATP synthesis and muscle contraction have been offered from the perspective of the torsional mechanism of energy transduction and ATP synthesis and the rotation-uncoiling-tilt (RUT) energy storage mechanism of muscle contraction. In this paper, a new systems thermodynamics analysis of oxidative- and photo-phosphorylation has been performed. New experimental data has been reported on the inhibition of ATP synthesis by known specific anion channel blockers: the triorganotin compound, tributyltin chloride (TBTCl), and the stilbene compound 4,4′-diisothiocyanostilbene-2,2′-disulfonate (DIDS), and interpreted as supporting the new framework. A bioinformatic analysis of the interacting a–c regions in F O has been carried out to locate the anion binding pocket in the anion–proton symsequenceporter and insights into the coordination chemistry of the bound chloride in its internal cavity at the lipid–water interface have been obtained. The need to look at ATP synthesis in F O as a multisubstrate reaction has been emphasized and a detailed microscopic explanation of the mechanism of inhibition by these blockers and its relationship to the conformational cycle within F O has been postulated. Such detailed explanations of the role of membrane elements in a lipophilic region have been shown to lead to deeper understanding and to offer a more realistic and complete picture of biological energy transduction than considering the bilayer as “mere insulation”, as in chemiosmotic dogma. Details of the elementary force production events by myosin II have been provided within the novel molecular systems framework of the RUT mechanism in which the S-1, the S-1–S-2 hinge, and the S-2 coiled coil all have essential roles in the in vivo contractile process. The new paradigm is shown to be consistent with the great body of experiments usually considered to support the swinging crossbridge/lever arm models of muscle contraction, because these events also occur during the cycle but as events preliminary to the occurrence of the power stroke. The crucial role of energy storage in mechanically strained nonequilibrium conformational states of myosin, specifically as a high-energy state of S-2 with uncoiled first few N-terminal heptads is highlighted. The propensity of these heptads to recoil and regain the resting state of the coiled coil is postulated to be a primary driving force of the power stroke. The new paradigms of ATP synthesis/muscle contraction have been shown to remove the inconsistencies present in previous theories and to have the sound backing of the first and second laws of thermodynamics, the principle of electrical neutrality, the laws of Newtonian mechanics, and the great conservation laws of mechanics. A systems integration of muscle contraction has been successfully made and a systems electrical analog constructed. The unifying laws and principles in the new theories have been further applied to understand the functioning of other protein molecular machines such as kinesin, ncd and unconventional myosins. Finally, the major physical, chemical, biological and technological implications arising as a result of this research have been discussed. Taken together, the new paradigms have been shown to solve fundamental molecular mechanistic problems in bioenergetics and motility and to offer a most detailed, unified and appealing picture of energy generation, transduction, storage and utilization processes in systems of biological molecular machines.

Photosynthetic use of exogenous inorganic carbon in the agarophyte Gracilaria lemaneiformis (Rhodophyta)

Oxygen evolution characteristics of the agarophyte Gracilaria lemaneiformis (Bory) Weber-van Bosse (Rhodophyta) were examined to establish the mechanism of photosynthetic use of exogenous inorganic carbon (Ci). Oxygen evolution in seawater of pH 8.0 was sharply reduced with the increase of pH. However, the rates of O 2 evolution at pH 8.0 and above exceeded the theoretical calculated maximum rates supported solely by the CO 2 supply from the spontaneous dehydration of HCO 3 − in the bulk seawater. In addition, G. lemaneiformis exhibited a high pH compensation point (9.58). These data indicate that G. lemaneiformis was able to use HCO 3 − as a source of Ci for photosynthesis. Both external and internal carbonic anhydrase (CA) activities, which could be detected potentiometrically, were essential for photosynthesis at alkaline pH values (8.0 and 9.0), because O 2 evolution was inhibited with the addition of CA inhibitors acetazolamide (AZ) and ethoxyzolamide (EZ). By contrast, the anion exchanger inhibitor, 4,4′-diisothiocyano-stilbene-2,2′-disulfonate (DIDS) had no inhibitory effects on photosynthetic O 2 evolution at neither acidic nor alkaline pH values. Therefore, the experiments with CA inhibitors would suggest that the mechanism of extracellular dehydration of HCO 3 − into CO 2 mediated by external CA activity was involved in HCO 3 − use by G. lemaneiformis. The relationship between photosynthetic O 2 evolution rates and Ci concentrations showed that the normal Ci composition in natural seawater could fully saturate photosynthesis of G. lemaneiformis at pH 8.0 or lower. However, the affinity for Ci and the carbon-saturated maximum photosynthesis were dramatically reduced at pH 9.0.

Strategies for carbon acquisition in the red marine macroalga Coccotylus truncatus from the Baltic Sea

Marine macroalgae need carbon-concentrating mechanisms because they have only limited access to CO2 in their natural environment. Previous studies have shown that one important strategy common to many algae is the activity of periplasmic carbonic anhydrases that catalyse the dehydration of HCO3 – into CO2. The latter can then cross the plasma membrane by passive diffusion. We hypothesised that an active (energy-consuming) mechanism might also be involved in the membrane transport of CO2, as is the case in a number of microalgae. Coccotylus truncatus was chosen as a model organism for this study because it belongs to a group of algae that usually lack direct HCO3 – uptake: sublittoral red algae. The method used to study carbon uptake was pH drift of the seawater medium surrounding the algae in a closed vessel, with and without the addition of specific inhibitors or proton buffers. Measured parameters included pH, total inorganic carbon and alkalinity of the seawater medium. Our results suggest that, in C. truncatus, periplasmic carbonic anhydrase as well as H+ extrusion, probably driven by a vanadate-sensitive P-type H+-ATPase (proton pump), are involved in CO2 uptake. No direct uptake of HCO3 – was discovered. This paper also presents data on the buffer capacity of several proton buffers and the carbon-uptake inhibitors acetazolamide, 4,4′-diisothiocyano-stilbene-2,2′-disulfonate (DIDS) and orthovanadate in Baltic Sea water with a salinity of 6.5 psu.

4,4′-Diisothiocyano-2,2′-stilbenedisulfonate protects cultured cerebellar granule neurons from death

We examined the effects of 4,4′-diisothiocyano-2,2′-stilbenedisulfonate (DIDS), an inhibitor of the chloride-bicarbonate exchangers and chloride channels, on death in cultured cerebellar granule neurons. Various stimuli, such as reduction of extracellular K + concentration, removal of growth factors, and staurosporine treatment, induced cell death. This death was blocked by DIDS in a dose dependent manner. In the presence of DIDS, the cells exposed to such stimuli did not show DNA fragmentation, but retained the ability to exclude trypan blue and to metabolize MTT to formazan. On the other hand, pretreatment of the cells with DIDS did not show any protective effects. The neuroprotective effect of DIDS was not influenced by extracellular Na +, Cl −, HCO 3 − or Ca 2+ concentrations, although reduction of extracellular Cl − or Ca 2+ concentrations per se induced neuronal death. Other chloride-bicarbonate exchange blockers like 4-acetamido-4′-isothiocyanatostilmene-2,2′-disulfonic acid (SITS) or 4,4′-dinitrostilbene-2,2′-disulfonic acid (DNDS) showed no significant effects on neuronal survival under these death-inducing stimuli. Dimethylamiloride, an inhibitor of the Na +/H + exchanger, did not influence neuronal death induced by these stimuli. Cells undergoing death showed gradual intracellular acidification, and DIDS did not inhibit this response, although DIDS (2 mM) per se induced transitory acidification followed by recovery within 10 min. DIDS did not influence intracellular Ca 2+ or Cl − levels during the lethal process. DIDS suppressed the cleavage of caspase-3 in the cells exposed to the death-inducing stimuli. These findings suggest that the neuroprotective effect of DIDS is mediated by a novel mechanism other than by nonselective inhibition of transporters or channels, and that DIDS blocks the death program upstream of caspases and downstream of all of the activation processes triggered by various stimuli.

Ammonia-induced extracellular accumulation of taurine in the rat striatum in vivo: role of ionotropic glutamate receptors.

Accumulation of taurine (Tau), glutamate (Glu) and glutamine (Gln) was measured in vivo in microdialysates of the rat striatum following a direct application to the microdialysis tube of 60 mM ammonium chloride which renders the final ammonia concentration in the extracellular space to approximately 5 mM. The following compounds were coadministered with ammonia to distinguish between the different mechanisms that may underlie the accumulation of amino acids: ion transport inhibitors, diisothiocyanostilbene-2,2'-disulfonate (DIDS) and furosemide, a Glu transport inhibitor L-trans-pyrrolidine-2,4-dicarboxylate (PDC), an NMDA receptor antagonist dizocilpine (MK-801) and an 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/kainate (KA) receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX). Ammonia stimulated Tau accumulation in the microdialysates to approximately 250% of the basal value. Furosemide did not significantly affect the stimulation by ammonia and DIDS only moderately depressed the effect. The ammonia-dependent Tau accumulation was increased by approximately 50% in the presence of PDC and reduced by approximately 35% in the presence dizocilpine and DNQX. In the microdialysates ammonia stimulated Glu and Gln accumulation somewhat less than Tau accumulation. Except for stimulation of Gln accumulation by DNQX, the effects were not modified by any of the cotreatments. The results are consistent with the assumption that ammonia stimulates Tau efflux mainly via activation of ionotropic Glu receptors.

Analysis of Integral Membrane Protein Contributions to the Deformability and Stability of the Human Erythrocyte Membrane

Three major hypotheses have been proposed to explain the role of membrane-spanning proteins in establishing/maintaining membrane stability. These hypotheses ascribe the essential contribution of integral membrane proteins to (i) their ability to anchor the membrane skeleton to the lipid bilayer, (ii) their capacity to bind and stabilize membrane lipids, and (iii) their ability to influence and regulate local membrane curvature. In an effort to test these hypotheses in greater detail, we have modified both the membrane skeletal and lipid binding interactions of band 3 (the major membrane-spanning and skeletal binding protein of the human erythrocyte membrane) and have examined the impact of these modifications on erythrocyte membrane morphology, deformability, and stability. The desired changes in membrane skeletal and protein-lipid interactions were induced by 1) reaction of the cells with 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS), an inhibitor of band 3-mediated anion transport that dissociates band 3 into dimers (increasing its surface area in contact with lipid) and severs band 3 linkages to the membrane skeleton; 2) a fragment of ankyrin that ruptures the same ankyrin-band 3 bridge to the membrane skeleton, but drives the band 3 subunit equilibrium toward the tetramer (i.e. decreasing the band 3 surface area in contact with lipid); and 3) an antibody to the ankyrin-binding site on band 3 that promotes the same changes in band 3 skeletal and lipid interactions as the ankyrin fragment. We observed that although DIDS induced echinocytic morphological changes in the treated erythrocytes, it had little impact on either membrane deformability or stability. In contrast, resealing of either the ankyrin fragment or anti-band 3 IgG into erythrocytes caused spontaneous membrane fragmentation and loss of deformability/stability. Because these and other new observations cannot all be reconciled with any single hypothesis on membrane stability, we suggest that more than one hypothesis may be operative and provide an explanation of how each might individually contribute to net membrane stability.