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1
Yeh, Han-I., Jiunn-Tyng Yeh, and Tzyh-Chang Hwang. "Modulation of CFTR gating by permeant ions." Journal of General Physiology 145, no. 1 (December 15, 2014): 47–60. http://dx.doi.org/10.1085/jgp.201411272.
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Cystic fibrosis transmembrane conductance regulator (CFTR) is unique among ion channels in that after its phosphorylation by protein kinase A (PKA), its ATP-dependent gating violates microscopic reversibility caused by the intimate involvement of ATP hydrolysis in controlling channel closure. Recent studies suggest a gating model featuring an energetic coupling between opening and closing of the gate in CFTR’s transmembrane domains and association and dissociation of its two nucleotide-binding domains (NBDs). We found that permeant ions such as nitrate can increase the open probability (Po) of wild-type (WT) CFTR by increasing the opening rate and decreasing the closing rate. Nearly identical effects were seen with a construct in which activity does not require phosphorylation of the regulatory domain, indicating that nitrate primarily affects ATP-dependent gating steps rather than PKA-dependent phosphorylation. Surprisingly, the effects of nitrate on CFTR gating are remarkably similar to those of VX-770 (N-(2,4-Di-tert-butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide), a potent CFTR potentiator used in clinics. These include effects on single-channel kinetics of WT CFTR, deceleration of the nonhydrolytic closing rate, and potentiation of the Po of the disease-associated mutant G551D. In addition, both VX-770 and nitrate increased the activity of a CFTR construct lacking NBD2 (ΔNBD2), indicating that these gating effects are independent of NBD dimerization. Nonetheless, whereas VX-770 is equally effective when applied from either side of the membrane, nitrate potentiates gating mainly from the cytoplasmic side, implicating a common mechanism for gating modulation mediated through two separate sites of action.
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Piskorowski, Rebecca A., and Richard W. Aldrich. "Relationship between Pore Occupancy and Gating in BK Potassium Channels." Journal of General Physiology 127, no. 5 (April 24, 2006): 557–76. http://dx.doi.org/10.1085/jgp.200509482.
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Permeant ions can have significant effects on ion channel conformational changes. To further understand the relationship between ion occupancy and gating conformational changes, we have studied macroscopic and single-channel gating of BK potassium channels with different permeant monovalent cations. While the slopes of the conductance–voltage curve were reduced with respect to potassium for all permeant ions, BK channels required stronger depolarization to open only when thallium was the permeant ion. Thallium also slowed the activation and deactivation kinetics. Both the change in kinetics and the shift in the GV curve were dependent on the thallium passing through the permeation pathway, as well as on the concentration of thallium. There was a decrease in the mean open time and an increase in the number of short flicker closing events with thallium as the permeating ion. Mean closed durations were unaffected. Application of previously established allosteric gating models indicated that thallium specifically alters the opening and closing transition of the channel and does not alter the calcium activation or voltage activation pathways. Addition of a closed flicker state into the allosteric model can account for the effect of thallium on gating. Consideration of the thallium concentration dependence of the gating effects suggests that the flicker state may correspond to the collapsed selectivity filter seen in crystal structures of the KcsA potassium channel under the condition of low permeant ion concentration.
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Tymianski, Michael, Igor Spigelman, Liang Zhang, Peter L. Carlen, Charles H. Tator, Milton P. Charlton, and M. Christopher Wallace. "Mechanism of Action and Persistence of Neuroprotection by Cell-Permeant Ca2+ Chelators." Journal of Cerebral Blood Flow & Metabolism 14, no. 6 (November 1994): 911–23. http://dx.doi.org/10.1038/jcbfm.1994.122.
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Cell-permeant Ca2+ chelators such as 1,2-bis-(2-aminophenoxy)ethane- N,N,N′,N′-tetraacetic acid acetoxymethyl ester (BAPTA-AM) have been reported to protect neurons in experimental focal cerebral ischemia. However, their in vivo actions are uncertain, and their protective efficacy is proven only in brief cerebral ischemia paradigms. Here we examine their mechanism of action in vitro and duration of efficacy in vivo. Electrophysiological studies were made in CA1 neurons in rat hippocampal slices. When superfused with BAPTA-AM (30–50 μ M), CA1 somatic field potential recordings showed attenuation of the population spike amplitude, and intracellular recordings showed reduced excitatory postsynaptic potentials, indicating inhibition of excitatory synaptic transmission. Also, Ca2+ -dependent accommodation and post-spike-train hyperpolarizations were reduced, indicating Ca2+ chelation hear the internal cell membrane surface. To determine whether Ca2+ chelators reduce the size of cerebral infarction rather than simply delaying its evolution, we studied the effects of BAPTA-AM treatment on infarction size 24 h after permanent middle cerebral artery occlusion. Fischer rats ( n = 8 per group) were pretreated with saline, BAPTA-AM (20 mg/kg), or MK-801 (0.5 mg/kg). Infarction volumes in animals treated with BAPTA-AM were reduced by 50.5% compared with controls ( p = 0.018), whereas animals treated with MK-801 experienced a statistically insignificant infarct volume reduction (26%; p = 0.27). These data show a persistence of neuroprotection by the Ca2+ chelator at 24 h and indicate that it may act by attenuating synaptic transmission and subplasma membrane Ca2+ excess.
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Najib, Omaima N., Stewart B. Kirton, Gary P. Martin, Michelle J. Botha, Al-Sayed Sallam, and Darragh Murnane. "Multivariate Analytical Approaches to Identify Key Molecular Properties of Vehicles, Permeants and Membranes That Affect Permeation through Membranes." Pharmaceutics 12, no. 10 (October 11, 2020): 958. http://dx.doi.org/10.3390/pharmaceutics12100958.
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There has been considerable recent interest in employing computer models to investigate the relationship between the structure of a molecule and its dermal penetration. Molecular permeation across the epidermis has previously been demonstrated to be determined by a number of physicochemical properties, for example, the lipophilicity, molecular weight and hydrogen bonding ability of the permeant. However little attention has been paid to modeling the combined effects of permeant properties in tandem with the properties of vehicles used to deliver those permeants or to whether data obtained using synthetic membranes can be correlated with those obtained using human epidermis. This work uses Principal Components Analysis (PCA) to demonstrate that, for studies of the diffusion of three model permeants (caffeine, methyl paraben and butyl paraben) through synthetic membranes, it is the properties of the oily vehicle in which they are applied that dominated the rates of permeation and flux. Simple robust and predictive descriptor-based quantitative structure–permeability relationship (QSPR) models have been developed to support these findings by utilizing physicochemical descriptors of the oily vehicles to quantify the differences in flux and permeation of the model compounds. Interestingly, PCA showed that, for the flux of co-applied model permeants through human epidermis, the permeation of the model permeants was better described by a balance between the physicochemical properties of the vehicle and the permeant rather than being dominated solely by the vehicle properties as in the case of synthetic model membranes. The important influence of permeant solubility in the vehicle along with the solvent uptake on overall permeant diffusion into the membrane was substantiated. These results confirm that care must be taken in interpreting permeation data when synthetic membranes are employed as surrogates for human epidermis; they also demonstrate the importance of considering not only the permeant properties but also those of both vehicle and membrane when arriving at any conclusions relating to permeation data.
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Swenson, Erik R., Timothy W. Tewson, Per J. Wistrand, Yvonne Ridderstrale, and Chingkuang Tu. "Biochemical, histological, and inhibitor studies of membrane carbonic anhydrase in frog gastric acid secretion." American Journal of Physiology-Gastrointestinal and Liver Physiology 281, no. 1 (July 1, 2001): G61—G68. http://dx.doi.org/10.1152/ajpgi.2001.281.1.g61.
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Gastric acid secretion is dependent on carbonic anhydrase (CA). To define the role of membrane-bound CA, we used biochemical, histochemical, and pharmacological approaches in the frog ( Rana pipiens). CA activity and inhibition by membrane-permeant and -impermeant agents were studied in stomach homogenates and microsomal fractions. H+secretion in the histamine-stimulated isolated mucosa was measured before and after mucosal addition of a permeant CA inhibitor (methazolamide) and before and after mucosal or serosal addition of two impermeant CA inhibitors of differing molecular mass: a 3,500-kDa polymer linked to aminobenzolamide and p-fluorobenzyl-aminobenzolamide (molecular mass, 454 kDa). Total CA activity of frog gastric mucosa is 2,280 U/g, of which 10% is due to membrane-bound CA. Membrane-bound CA retains detectable activity below pH 4. Histochemically, there is membrane-associated CA in surface epithelial, oxynticopeptic, and capillary endothelial cells. Methazolamide reduced H+secretion by 100%, whereas the two impermeant inhibitors equally blocked secretion by 40% when applied to the mucosal side and by 55% when applied to the serosal side. The presence of membrane-bound CA in frog oxynticopeptic cells and its relative resistance to acid inactivation and inhibition by impermeant inhibitors demonstrate that it subserves acid secretion at both the apical and basolateral sides.
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Eddins, Donnie, Lisa K. Lyford, Jung Weon Lee, Sanjay A. Desai, and Robert L. Rosenberg. "Permeant but not impermeant divalent cations enhance activation of nondesensitizing α7 nicotinic receptors." American Journal of Physiology-Cell Physiology 282, no. 4 (April 1, 2002): C796—C804. http://dx.doi.org/10.1152/ajpcell.00453.2001.
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Neuronal α7 nicotinic acetylcholine receptors (nAChRs) are permeable to Ca2+ and other divalent cations. We characterized the modulation of the pharmacological properties of nondesensitizing mutant (L247T and S240T/L247T) α7 nAChRs by permeant (Ca2+, Ba2+, and Sr2+) and impermeant (Cd2+ and Zn2+) divalent cations. α7 receptors were expressed in Xenopus oocytes and studied with two-electrode voltage clamp. Extracellular permeant divalent cations increased the potency and maximal efficacy of ACh, whereas impermeant divalent cations decreased potency and maximal efficacy. The antagonist dihydro-β-erythroidine (DHβE) was a strong partial agonist of L247T and S240T/L247T α7 receptors in the presence of divalent cations but was a weak partial agonist in the presence of impermeant divalent cations. Mutation of the “intermediate ring” glutamates (E237A) in L247T α7 nAChRs eliminated Ca2+conductance but did not alter the Ca2+-dependent increase in ACh potency, suggesting that site(s) required for modulation are on the extracellular side of the intermediate ring. The difference between permeant and impermeant divalent cations suggests that sites within the pore are important for modulation by divalent cations.
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Thompson, Jill, and Ted Begenisich. "Selectivity filter gating in large-conductance Ca2+-activated K+ channels." Journal of General Physiology 139, no. 3 (February 27, 2012): 235–44. http://dx.doi.org/10.1085/jgp.201110748.
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Membrane voltage controls the passage of ions through voltage-gated K (Kv) channels, and many studies have demonstrated that this is accomplished by a physical gate located at the cytoplasmic end of the pore. Critical to this determination were the findings that quaternary ammonium ions and certain peptides have access to their internal pore-blocking sites only when the channel gates are open, and that large blocking ions interfere with channel closing. Although an intracellular location for the physical gate of Kv channels is well established, it is not clear if such a cytoplasmic gate exists in all K+ channels. Some studies on large-conductance, voltage- and Ca2+-activated K+ (BK) channels suggest a cytoplasmic location for the gate, but other findings question this conclusion and, instead, support the concept that BK channels are gated by the pore selectivity filter. If the BK channel is gated by the selectivity filter, the interactions between the blocking ions and channel gating should be influenced by the permeant ion. Thus, we tested tetrabutyl ammonium (TBA) and the Shaker “ball” peptide (BP) on BK channels with either K+ or Rb+ as the permeant ion. When tested in K+ solutions, both TBA and the BP acted as open-channel blockers of BK channels, and the BP interfered with channel closing. In contrast, when Rb+ replaced K+ as the permeant ion, TBA and the BP blocked both closed and open BK channels, and the BP no longer interfered with channel closing. We also tested the cytoplasmically gated Shaker K channels and found the opposite behavior: the interactions of TBA and the BP with these Kv channels were independent of the permeant ion. Our results add significantly to the evidence against a cytoplasmic gate in BK channels and represent a positive test for selectivity filter gating.
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Yoshikawa, Masakazu, Takeshi Matsuura, and David Cooney. "Studies on the state of permeant in the membrane and its effect on pervaporation phenomena." Journal of Applied Polymer Science 42, no. 5 (March 5, 1991): 1417–21. http://dx.doi.org/10.1002/app.1991.070420526.
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Otto, Daniel, Johann Combrinck, Anja Otto, Louwrens Tiedt, and Melgardt de Villiers. "Dissipative Particle Dynamics Investigation of the Transport of Salicylic Acid through a Simulated In Vitro Skin Permeation Model." Pharmaceuticals 11, no. 4 (December 5, 2018): 134. http://dx.doi.org/10.3390/ph11040134.
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Permeation models are often used to determine diffusion properties of a drug through a membrane as it is released from a delivery system. In order to circumvent problematic in vivo studies, diffusion studies can be performed in vitro, using (semi-)synthetic membranes. In this study salicylic acid permeation was studied, employing a nitrocellulose membrane. Both saturated and unsaturated salicylic acid solutions were studied. Additionally, the transport of salicylic acid through the nitrocellulose membrane was simulated by computational modelling. Experimental observations could be explained by the transport mechanism that was revealed by dissipative particle dynamics (DPD) simulations. The DPD model was developed with the aid of atomistic scale molecular dynamics (AA-MD). The choice of a suitable model membrane can therefore, be predicted by AA-MD and DPD simulations. Additionally, the difference in the magnitude of release from saturated and unsaturated salicylic acid and solutions could also be observed with DPD. Moreover, computational studies can reveal hidden variables such as membrane-permeant interaction that cannot be measured experimentally. A recommendation is made for the development of future model permeation membranes is to incorporate computational modelling to aid the choice of model.
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Shapiro, M. S., and T. E. DeCoursey. "Permeant ion effects on the gating kinetics of the type L potassium channel in mouse lymphocytes." Journal of General Physiology 97, no. 6 (June 1, 1991): 1251–78. http://dx.doi.org/10.1085/jgp.97.6.1251.
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Permeant ion species was found to profoundly affect the gating kinetics of type l K+ currents in mouse T lymphocytes studied with the whole-cell or on-cell patch gigaohm-seal techniques. Replacing external K+ with Rb+ (as the sole monovalent cation, at 160 mM) shifted the peak conductance voltage (g-V) relation by approximately 20 mV to more negative potentials, while NH4+ shifted the g-V curve by 15 mV to more positive potentials. Deactivation (the tail current time constant, tau tail) was slowed by an average of 14-fold at -70 mV in external Rb+, by approximately 8-fold in Cs+, and by a factor of two to three in NH4+. Changing the external K+ concentration, [K+]o, from 4.5 to 160 mM or [Rb+]o from 10 to 160 mM had no effect on tau tail. With all the internal K+ replaced by Rb+ or Cs+ and either isotonic Rb+ or K+ in the bath, tau tail was indistinguishable from that with K+ in the cell. With the exception of NH4+, activation time constants were insensitive to permeant ion species. These results indicate that external permeant ions have stronger effects than internal permeant ions, suggesting an external modulatory site that influences K+ channel gating. However, in bi-ionic experiments with reduced external permeant ion concentrations, tau tail was sensitive to the direction of current flow, indicating that the modulatory site is either within the permeation pathway or in the outer vestibule of the channel. The latter interpretation implies that outward current through an open type l K+ channel significantly alters local ion concentrations at the modulatory site in the outer vestibule, and consequently at the mouth of the channel. Experiments with mixtures of K+ and Rb+ in the external solution reveal that deactivation kinetics are minimally affected by addition of Rb+ until the Rb+ mole fraction approaches unity. This relationship between mole fraction and tau tail, together with the concentration independence of tau tail, was hard to reconcile with simple models in which occupancy of a site within the permeation pathway prevents channel closing, but is consistent with a model in which a permeant ion binding site in the outer vestibule modulates gating depending on the species of ion occupying the site. A description of the ionic selectivity of the type l K+ channel is presented in the companion paper (Shapiro and DeCoursey, 1991b).
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O'Brodovich, H., X. Wang, C. Li, B. Rafii, J. Correa, and C. Bear. "Novobiocin forms cation-permeable ion channels in rat fetal distal lung epithelium." American Journal of Physiology-Cell Physiology 264, no. 6 (June 1, 1993): C1532—C1537. http://dx.doi.org/10.1152/ajpcell.1993.264.6.c1532.
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The antibiotic novobiocin has been previously reported to increase Na+ transport in frog skin, presumably by attenuation of Na+ self-inhibition of Na+ channels. To determine whether novobiocin had similar effects and utilized a similar mechanism in mammalian Na(+)-transporting tissues, we studied its effect on ion transport by primary cultures of fetal distal lung epithelium (FDLE) cultured from 20-day gestationally aged rats (term = 22 days). Novobiocin (10 mM) increased short-circuit current and markedly decreased the resistance in FDLE monolayers mounted in Ussing chambers. Fura-2 single-cell studies showed that novobiocin increased intracellular Ca2+ concentration and that this resulted from extracellular sources. Nystatin-perforated patch-clamp techniques demonstrated that novobiocin increased nonrectifying cation whole cell currents without inducing detectable anion currents. Novobiocin created nonrectifying monovalent cation-selective channels in lipid bilayers. These studies demonstrated that novobiocin affects the bioelectric properties of Na+ transporting lung epithelium and that this likely occurs by the formation of ion-permeant channels in their lipid membranes.
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Wells, Lisa A., P. E. Cassidy, T. M. Aminabhavi, and R. B. Perry. "A Study of Permeation and Diffusion of Aqueous Salt Solutions through Polyurethane and Polysiloxane and Their Laminates." Rubber Chemistry and Technology 63, no. 1 (March 1, 1990): 66–76. http://dx.doi.org/10.5254/1.3538243.
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Abstract Effect of ion size on water permeation through polyurethane, polysiloxane, and their laminates has been studied. The results of these studies for the polyurethane membranes correlated well with the reported values of water permeation through a variety of other polyurethane elastomers. Permeation rates and the Arrhenius relationship of polyurethane and polysiloxane membranes were found to be similar. Solute size showed no observable effect on water permeation through polyurethane, polysiloxane, or their bilayer laminates, within experimental error. A small effect of solute size was observed for diffusion of water into polyurethane. Permeation rates of the bilayer laminates demonstrated a small directional dependence or “valving” effect. In the light of this evidence, it may be suggested that highly permeable systems, such as polyurethane and polysiloxane, are relatively insensitive to permeant composition.
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Lytton, SD, B. Mester, J. Libman, A. Shanzer, and ZI Cabantchik. "Mode of action of iron (III) chelators as antimalarials: II. Evidence for differential effects on parasite iron-dependent nucleic acid synthesis." Blood 84, no. 3 (August 1, 1994): 910–15. http://dx.doi.org/10.1182/blood.v84.3.910.910.
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Abstract Iron chelation treatment of red blood cells infected with Plasmodium falciparum selectively intervenes with iron-dependent metabolism of malaria parasites and inhibits their development. Highly permeant hydroxamate iron chelator RSFileum2 affects all parasite stages when cultures are continuously exposed to drug, but affects primarily ring stages when assessed for irreversible effects, ie, sustained inhibition remaining after drug removal. On the other hand, the hydrophilic and poorly permeant desferrioxamine (DFO) affects primarily trophozoite/schizont stages when tested either in the continuous mode or irreversible mode. Unlike parasites, mammalian cells subjected to similar drug treatment show complete growth recovery once drugs are removed. Our studies indicate that parasites display a limited capacity to recover from intracellular iron depletion evoked by iron chelators. Based on these findings we provide a working model in which the irreversible effects of RSFs on rings are explained by the absence of pathways for iron acquisition/utilization by early forms of parasites. Trophozoite/schizonts can partially recover from RSFileum2 treatments, but show no DNA synthesis following DFO treatment even after drug removal and iron replenishment by permeant iron carriers. At trophozoite stage, the parasite uses a limited pathway for refurnishing its iron-containing enzymes, thus overcoming iron deprivation caused by permeant RSFileum2, but not by DFO because this latter drug is not easily removable from parasites. Their DNA synthesis is blocked by the hydroxamate iron chelators probably by affecting synthesis of ribonucleotide reductase (RNRase). Presumably in parasites, prolonged repression of the enzyme leads also to irreversible loss of activity. The action profiles of RSFileum2 and DFO presented in this study have implications for improved chemotherapeutic performance by combined drug treatment and future drug design based on specific intervention at parasite DNA synthesis.
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Lytton, SD, B. Mester, J. Libman, A. Shanzer, and ZI Cabantchik. "Mode of action of iron (III) chelators as antimalarials: II. Evidence for differential effects on parasite iron-dependent nucleic acid synthesis." Blood 84, no. 3 (August 1, 1994): 910–15. http://dx.doi.org/10.1182/blood.v84.3.910.bloodjournal843910.
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Iron chelation treatment of red blood cells infected with Plasmodium falciparum selectively intervenes with iron-dependent metabolism of malaria parasites and inhibits their development. Highly permeant hydroxamate iron chelator RSFileum2 affects all parasite stages when cultures are continuously exposed to drug, but affects primarily ring stages when assessed for irreversible effects, ie, sustained inhibition remaining after drug removal. On the other hand, the hydrophilic and poorly permeant desferrioxamine (DFO) affects primarily trophozoite/schizont stages when tested either in the continuous mode or irreversible mode. Unlike parasites, mammalian cells subjected to similar drug treatment show complete growth recovery once drugs are removed. Our studies indicate that parasites display a limited capacity to recover from intracellular iron depletion evoked by iron chelators. Based on these findings we provide a working model in which the irreversible effects of RSFs on rings are explained by the absence of pathways for iron acquisition/utilization by early forms of parasites. Trophozoite/schizonts can partially recover from RSFileum2 treatments, but show no DNA synthesis following DFO treatment even after drug removal and iron replenishment by permeant iron carriers. At trophozoite stage, the parasite uses a limited pathway for refurnishing its iron-containing enzymes, thus overcoming iron deprivation caused by permeant RSFileum2, but not by DFO because this latter drug is not easily removable from parasites. Their DNA synthesis is blocked by the hydroxamate iron chelators probably by affecting synthesis of ribonucleotide reductase (RNRase). Presumably in parasites, prolonged repression of the enzyme leads also to irreversible loss of activity. The action profiles of RSFileum2 and DFO presented in this study have implications for improved chemotherapeutic performance by combined drug treatment and future drug design based on specific intervention at parasite DNA synthesis.
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Yue, Lixia, Betsy Navarro, Dejian Ren, Arnolt Ramos, and David E. Clapham. "The Cation Selectivity Filter of the Bacterial Sodium Channel, NaChBac." Journal of General Physiology 120, no. 6 (November 11, 2002): 845–53. http://dx.doi.org/10.1085/jgp.20028699.
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The Bacillus halodurans voltage-gated sodium-selective channel (NaChBac) (Ren, D., B. Navarro, H. Xu, L. Yue, Q. Shi, and D.E. Clapham. 2001b. Science. 294:2372–2375), is an ideal candidate for high resolution structural studies because it can be expressed in mammalian cells and its functional properties studied in detail. It has the added advantage of being a single six transmembrane (6TM) orthologue of a single repeat of mammalian voltage-gated Ca2+ (CaV) and Na+ (NaV) channels. Here we report that six amino acids in the pore domain (LESWAS) participate in the selectivity filter. Replacing the amino acid residues adjacent to glutamatic acid (E) by a negatively charged aspartate (D; LEDWAS) converted the Na+-selective NaChBac to a Ca2+- and Na+-permeant channel. When additional aspartates were incorporated (LDDWAD), the mutant channel resulted in a highly expressing voltage-gated Ca2+-selective conductance.
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Yang, J. "Ion permeation through 5-hydroxytryptamine-gated channels in neuroblastoma N18 cells." Journal of General Physiology 96, no. 6 (December 1, 1990): 1177–98. http://dx.doi.org/10.1085/jgp.96.6.1177.
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Ionic currents induced by 5-hydroxytryptamine (5-HT) in cultured neuroblastoma N18 cells were studied using whole-cell voltage clamp. The response was blocked by 1-10 nM 5-HT3 receptor-specific antagonists MDL 7222 or ICS 205-930, but not by 1 microM 5-HT1/5-HT2 receptor antagonist spiperone or 5-HT2 receptor-specific antagonist ketanserin. These 5-HT3 receptors seem to be ligand-gated channels because the response (a) did not require internal ATP or GTP, (b) persisted with long internal dialysis of CsF (90 mM), A1F4- (100 microM), or GTP gamma S (100 microM), and (c) with ionophoretic delivery of 5-HT developed with a delay of less than 10 ms and rose to a peak in 34-130 ms. Fluctuation analysis yielded an apparent single-channel conductance of 593 fS. The relative permeabilities of the channel for a variety of ions were determined from reversal potentials. The channel was only weakly selective among small cations, with permeability ratios PX/PNa of 1.22, 1.10, 1.01, 1.00, and 0.99 for Cs+, K+, Li+, Na+, and Rb+, and 1.12, 0.79, and 0.73 for Ca2+, Ba2+, and Mg2+ (when studied in mixtures of 20 mM divalent ions and 120 mM N-methyl-D-glucamine). Apparent permeability ratios for the divalent ions decreased as the concentration of divalent ions was increased. Small monovalent organic cations were highly permeant. Large organic cations such as Tris and glucosamine were measurably permeant with permeability ratios of 0.20 and 0.08, and N-methyl-D-glucamine was almost impermeant. Small anions, NO3-, Cl-, and F-, were slightly permeant with permeability ratios of 0.08, 0.04, and 0.03. The results indicate that the open 5-HT3 receptor channel has an effective minimum circular pore size of 7.6 A and that ionic interactions in the channel may involve negative charges near the pore mouth.
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LeMasurier, Meredith, Lise Heginbotham, and Christopher Miller. "Kcsa." Journal of General Physiology 118, no. 3 (August 27, 2001): 303–14. http://dx.doi.org/10.1085/jgp.118.3.303.
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Ion conduction and selectivity properties of KcsA, a bacterial ion channel of known structure, were studied in a planar lipid bilayer system at the single-channel level. Selectivity sequences for permeant ions were determined by symmetrical solution conductance (K+ > Rb+, NH4+, Tl+ ≫ Cs+, Na+, Li+) and by reversal potentials under bi-ionic or mixed-ion conditions (Tl+ > K+ > Rb+ > NH4+ ≫ Na+, Li+). Determination of reversal potentials with submillivolt accuracy shows that K+ is over 150-fold more permeant than Na+. Variation of conductance with concentration under symmetrical salt conditions is complex, with at least two ion-binding processes revealing themselves: a high affinity process below 20 mM and a low affinity process over the range 100–1,000 mM. These properties are analogous to those seen in many eukaryotic K+ channels, and they establish KcsA as a faithful structural model for ion permeation in eukaryotic K+ channels.
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Sanchez, J. A., J. A. Dani, D. Siemen, and B. Hille. "Slow permeation of organic cations in acetylcholine receptor channels." Journal of General Physiology 87, no. 6 (June 1, 1986): 985–1001. http://dx.doi.org/10.1085/jgp.87.6.985.
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Block, permeation, and agonist action of small organic amine compounds were studied in acetylcholine receptor (AChR) channels. Single channel conductances were calculated from fluctuation analysis at the frog neuromuscular junction and measured by patch clamp of cultured rat myotubes. The conductance was depressed by a few millimolar external dimethylammonium, arginine, dimethyldiethanolammonium, and Tris. Except with dimethylammonium, the block was intensified with hyperpolarization. A two-barrier Eyring model describes the slowed permeation and voltage dependence well for the three less permeant test cations. The cations were assumed to pause at a site halfway across the electric field of the channel while passing through it. For the voltage-independent action of highly permeant dimethylammonium, a more appropriate model might be a superficial binding site that did not prevent the flow of other ions, but depressed it. Solutions of several amine compounds were found to have agonist activity at millimolar concentrations, inducing brief openings of AChR channels on rat myotubes in the absence of ACh.
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Dias, M., J. Hadgraft, S. L. Raghavan, and J. Tetteh. "The effect of solvent on permeant diffusion through membranes studied using ATR‐FTIR and chemometric data analysis." Journal of Pharmaceutical Sciences 93, no. 1 (January 2004): 186–96. http://dx.doi.org/10.1002/jps.10530.
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Franciolini, F., and W. Nonner. "Anion and cation permeability of a chloride channel in rat hippocampal neurons." Journal of General Physiology 90, no. 4 (October 1, 1987): 453–78. http://dx.doi.org/10.1085/jgp.90.4.453.
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The ionic permeability of a voltage-dependent Cl channel of rat hippocampal neurons was studied with the patch-clamp method. The unitary conductance of this channel was approximately 30 pS in symmetrical 150 mM NaCl saline. Reversal potentials interpreted in terms of the Goldman-Hodgkin-Katz voltage equation indicate a Cl:Na permeability ratio of approximately 5:1 for conditions where there is a salt gradient. Many anions are permeant; permeability generally follows a lyotropic sequence. Permeant cations include Li, Na, K, and Cs. The unitary conductance does not saturate for NaCl concentrations up to 1 M. No Na current is observed when the anion Cl is replaced by the impermeant anion SO4. Unitary conductance depends on the cation species present. The channel is reversibly blocked by extracellular Zn or 9-anthracene carboxylic acid. Physiological concentrations of Ca or Mg do not affect the Na:Cl permeability ratio. The permeability properties of the channel are consistent with a permeation mechanism that involves an activated complex of an anionic site, an extrinsic cation, and an extrinsic anion.
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Stutzin, Andrés, Rubén Torres, Macarena Oporto, Patricio Pacheco, Ana Luisa Eguiguren, L. Pablo Cid, and Francisco V. Sepúlveda. "Separate taurine and chloride efflux pathways activated during regulatory volume decrease." American Journal of Physiology-Cell Physiology 277, no. 3 (September 1, 1999): C392—C402. http://dx.doi.org/10.1152/ajpcell.1999.277.3.c392.
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Organic osmolyte and halide permeability pathways activated in epithelial HeLa cells by cell swelling were studied by radiotracer efflux techniques and single-cell volume measurements. The replacement of extracellular Cl− by anions that are more permeant through the volume-activated Cl− channel, as indicated by electrophysiological measurements, significantly decreased taurine efflux. In the presence of less-permeant anions, an increase in taurine efflux was observed. Simultaneous measurement of the125I, used as a tracer for Cl−, and [3H]taurine efflux showed that the time courses for the two effluxes differed. In Cl−-rich medium the increase in I− efflux was transient, whereas that for taurine was sustained. Osmosensitive Cl− conductance, assessed by measuring changes in cell volume, increased rapidly after hypotonic shock. The influx of taurine was able to counteract Cl− conductance-dependent cell shrinkage but only ∼4 min after triggering cell swelling. This taurine-induced effect was blocked by DIDS. Differences in anion sensitivity, the time course of activation, and sensitivity to DIDS suggest that the main cell swelling-activated permeability pathways for taurine and Cl− are separate.
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Almansour, Khaled, Alistair Taverner, Ian M. Eggleston, and Randall J. Mrsny. "Mechanistic studies of a cell-permeant peptide designed to enhance myosin light chain phosphorylation in polarized intestinal epithelia." Journal of Controlled Release 279 (June 2018): 208–19. http://dx.doi.org/10.1016/j.jconrel.2018.03.033.
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Lu, Tao, Li Wu, Jun Xiao, and Jian Yang. "Permeant Ion-Dependent Changes in Gating of Kir2.1 Inward Rectifier Potassium Channels." Journal of General Physiology 118, no. 5 (November 1, 2001): 509–22. http://dx.doi.org/10.1085/jgp.118.5.509.
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We studied the effect of monovalent thallium ion (Tl+) on the gating of single Kir2.1 channels, which open and close spontaneously at a constant membrane potential. In cell-attached recordings of single-channel inward current, changing the external permeant ion from K+ to Tl+ decreases the mean open-time by ∼20-fold. Furthermore, the channel resides predominantly at a subconductance level, which results from a slow decay (τ = 2.7 ms at −100 mV) from the fully open level immediately following channel opening. Mutation of a pore-lining cysteine (C169) to valine abolishes the slow decay and subconductance level, and single-channel recordings from channels formed by tandem tetramers containing one to three C169V mutant subunits indicate that Tl+ must interact with at least three C169 residues to induce these effects. However, the C169V mutation does not alter the single-channel closing kinetics of Tl+ current. These results suggest that Tl+ ions change the conformation of the ion conduction pathway during permeation and alter gating by two distinct mechanisms. First, they interact with the thiolate groups of C169 lining the cavity to induce conformational changes of the ion passageway, and thereby produce a slow decay of single-channel current and a dominant subconductance state. Second, they interact more strongly than K+ with the main chain carbonyl oxygens lining the selectivity filter to destabilize the open state of the channel and, thus, alter the open/close kinetics of gating. In addition to altering gating, Tl+ greatly diminishes Ba2+ block. The unblocking rate of Ba2+ is increased by >22-fold when the external permeant ion is switched from K+ to Tl+ regardless of the direction of Ba2+ exit. This effect cannot be explained solely by ion–ion interactions, but is consistent with the notion that Tl+ induces conformational changes in the selectivity filter.
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van Acker, Gijs J. D., Ashok K. Saluja, Lakshmi Bhagat, Vijay P. Singh, Albert M. Song, and Michael L. Steer. "Cathepsin B inhibition prevents trypsinogen activation and reduces pancreatitis severity." American Journal of Physiology-Gastrointestinal and Liver Physiology 283, no. 3 (September 1, 2002): G794—G800. http://dx.doi.org/10.1152/ajpgi.00363.2001.
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Intrapancreatic activation of trypsinogen is believed to play a critical role in the initiation of acute pancreatitis, but mechanisms responsible for intrapancreatic trypsinogen activation during pancreatitis have not been clearly defined. In previous in vitro studies, we have shown that intra-acinar cell activation of trypsinogen and acinar cell injury in response to supramaximal secretagogue stimulation could be prevented by the cell permeant cathepsin B inhibitor E64d (Saluja A, Donovan EA, Yamanaka K, Yamaguchi Y, Hofbauer B, and Steer ML. Gastroenterology 113: 304–310, 1997). The present studies evaluated the role of intrapancreatic trypsinogen activation, this time under in vivo conditions, in two models of pancreatitis by using another highly soluble cell permeant cathepsin B inhibitor,l-3-trans-(propylcarbamoyl)oxirane-2-carbonyl-l-isoleucyl-l-proline methyl ester (CA-074me). Intravenous administration of CA-074me (10 mg/kg) before induction of either secretagogue-elicited pancreatitis in mice or duct infusion-elicited pancreatitis in rats markedly reduced the extent of intrapancreatic trypsinogen activation and substantially reduced the severity of both pancreatitis models. These observations support the hypothesis that, during the early stages of pancreatitis, trypsinogen activation in the pancreas is mediated by the lysosomal enzyme cathepsin B. Our findings also suggest that pharmacological interventions that inhibit cathepsin B may prove useful in preventing acute pancreatitis or reducing its severity.
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Landrito, Queenie Charisse, Gianina Krisha Rodymae Menor, Fortunato Sevilla III, Leah Tolosa, and Cristina Tiangco. "Development of noninvasive method of measuring copper and zinc by passive diffusion: an in vitro model." MATEC Web of Conferences 268 (2019): 01008. http://dx.doi.org/10.1051/matecconf/201926801008.
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There has been increasing interest in the determination of essential or toxic elements in human body fluids in relation to health and nutrition. The human body is composed of almost every natural element found in nature. This includes copper and zinc that are both essential if obtained at a right amount and toxic when exceeds. This study aims to develop a noninvasive method for measuring trace amounts of copper and zinc by passive diffusion. A cellulose acetate membrane was used as a model representing human skin. Different amount of permeant concentration of copper and zinc solutions such as 0.1, 0.5, and 1mM were placed inside the membrane and the samples were collected and analyzed using Atomic Absorption Spectroscopy. The amount of copper and zinc diffusing shows proportionality with time. The permeability and steady state flux were computed. Permeability of -2.39×10-7 cm/s and 6.67×10-7 cm/s were computed for copper at 0.1 mM and 0.5 mM concentrations respectively and 3.11×10-7, 4.61×10-7 and 1.75×10-6 cm/s were computed for zinc at 1, 0.5 and 0.1 mM permeant concentrations respectively. For steady state flux, the values computed are 1.22×10-3, 9.03×10-4 and 6.84×10-4 µM/cm2min for zinc at 1, 0.5 and 0.1 mM respectively and -7.84×10-4 and 1.27×10-3 µM/cm2min for copper at 0.5 and 0.1 mM permeant concentrations. The values gathered are all acceptable taking into the account previous studies regarding skin diffusions. This method presents a noninvasive alternative method to track copper and zinc trends.
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Betto, Giulia, O. Lijo Cherian, Simone Pifferi, Valentina Cenedese, Anna Boccaccio, and Anna Menini. "Interactions between permeation and gating in the TMEM16B/anoctamin2 calcium-activated chloride channel." Journal of General Physiology 143, no. 6 (May 26, 2014): 703–18. http://dx.doi.org/10.1085/jgp.201411182.
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At least two members of the TMEM16/anoctamin family, TMEM16A (also known as anoctamin1) and TMEM16B (also known as anoctamin2), encode Ca2+-activated Cl− channels (CaCCs), which are found in various cell types and mediate numerous physiological functions. Here, we used whole-cell and excised inside-out patch-clamp to investigate the relationship between anion permeation and gating, two processes typically viewed as independent, in TMEM16B expressed in HEK 293T cells. The permeability ratio sequence determined by substituting Cl− with other anions (PX/PCl) was SCN− > I− > NO3− > Br− > Cl− > F− > gluconate. When external Cl− was substituted with other anions, TMEM16B activation and deactivation kinetics at 0.5 µM Ca2+ were modified according to the sequence of permeability ratios, with anions more permeant than Cl− slowing both activation and deactivation and anions less permeant than Cl− accelerating them. Moreover, replacement of external Cl− with gluconate, or sucrose, shifted the voltage dependence of steady-state activation (G-V relation) to more positive potentials, whereas substitution of extracellular or intracellular Cl− with SCN− shifted G-V to more negative potentials. Dose–response relationships for Ca2+ in the presence of different extracellular anions indicated that the apparent affinity for Ca2+ at +100 mV increased with increasing permeability ratio. The apparent affinity for Ca2+ in the presence of intracellular SCN− also increased compared with that in Cl−. Our results provide the first evidence that TMEM16B gating is modulated by permeant anions and provide the basis for future studies aimed at identifying the molecular determinants of TMEM16B ion selectivity and gating.
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Vickers, Mark F., Rakesh Kumar, Frank Visser, Jing Zhang, Jahangir Charania, R. Taylor Raborn, Stephen A. Baldwin, James D. Young, and Carol E. Cass. "Comparison of the interaction of uridine, cytidine, and other pyrimidine nucleoside analogues with recombinant human equilibrative nucleoside transporter 2 (hENT2) produced in Saccharomyces cerevisiae." Biochemistry and Cell Biology 80, no. 5 (October 1, 2002): 639–44. http://dx.doi.org/10.1139/o02-148.
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The human equilibrative nucleoside transporters 1 and 2 (hENT1, hENT2) share 50% amino acid identity and exhibit broad selectivities, accepting purine and pyrimidine nucleosides as permeants. The permeant selectivity of hENT2 is less well understood because of the low abundance of the native transporter in cells amenable to functional analysis. Recent studies of hENT2 produced in recombinant form in functional expression systems have shown that it differs from hENT1 in that it transports nucleobases. To further understand the structural requirements for permeant interaction with hENT2, we compared the relative abilities of uridine, cytidine, and their analogues to inhibit transport of [3H]uridine by recombinant hENT1 and hENT2 produced in yeast. hENT1 and hENT2 tolerated halogen modification at the 5 position of the base and the 2' and 5' positions of the ribose moieties of uridine whereas removal of the hydroxyl group at the 3' position of the ribose moiety of uridine eliminated interaction with both transporters. hENT2 displayed a lower ability, compared with hENT1, to interact with cytidine and cytidine analogues, suggesting a low tolerance for the presence of the amino group at the 4 position of the base.Key words: hENT2, hENT1, araC, uridine, NBMPR.
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Dwyer, T. M. "Guanidine block of single channel currents activated by acetylcholine." Journal of General Physiology 88, no. 5 (November 1, 1986): 635–50. http://dx.doi.org/10.1085/jgp.88.5.635.
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The acetylcholine-activated channel of chick myotube was studied using the patch-clamp method. Single channel current amplitudes were measured between -300 and +250 mV in solutions containing the permeant ions Cs+ and guanidine (G+). G+ has a relative permeability, PG/PCs, of 1.6, but carries no more than half the current that Cs+ does, with an equivalent electrochemical driving force. Experiments using G+ revealed an asymmetry of the acetylcholine-activated channel, with G+ being more effective at reducing Cs+ currents when added to the outside than when added to the inside. The block caused by outside, but not inside, G+ was evident for both inward and outward currents. The block caused by outside G+ was voltage dependent, first increasing and then being partially relieved when the driving force was made more negative. Experiments with mixtures of Cs+ and G+ revealed anomalously low magnitudes for reversal potentials, relative to predictions based on the Goldman-Hodgkin-Katz equation. These findings are consistent with a two-well, three-barrier Eyring rate model for ion flow, and demonstrate that a highly permeant ion, guanidine, can block asymmetrically by acting from within the voltage field of the acetylcholine-activated channel.
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Eksi, S., B. Czesny, G. J. van Gemert, R. W. Sauerwein, W. Eling, and K. C. Williamson. "Inhibition of Plasmodium falciparum Oocyst Production by Membrane-Permeant Cysteine Protease Inhibitor E64d." Antimicrobial Agents and Chemotherapy 51, no. 3 (December 18, 2006): 1064–70. http://dx.doi.org/10.1128/aac.01012-06.
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ABSTRACT During asexual intraerythrocytic growth, Plasmodium falciparum utilizes hemoglobin obtained from the host red blood cell (RBC) as a nutrient source. Papain-like cysteine proteases, falcipains 2 and 3, have been reported to be involved in hemoglobin digestion and are targets of current antimalarial drug development efforts. However, their expression during gametocytogenesis, which is required for malaria parasite transmission, has not been studied. Many of the available antimalarials do not inhibit development of sexual stage parasites, and therefore, the persistence of gametocytes after drug treatment allows continued transmission of the disease. In the work reported here, incubation of stage V gametocytes with membrane-permeant cysteine protease inhibitor E64d significantly inhibited oocyst production (80 to 100%). The same conditions inhibited processing of gametocyte-surface antigen Pfs230 during gametogenesis but did not alter the morphology of the food vacuole in gametocytes, inhibit emergence, or block male exflagellation. E64d reduced the level of oocyst production more effectively than that reported previously for falcipain 1-knockout parasites, suggesting that falcipains 2 and 3 may also be involved in malaria parasite transmission. However, in this study only falcipain 3 and not falcipain 2 was found to be expressed in stage V gametocytes. Interestingly, during gametocytogenesis falcipain 3 was transported into the red blood cell and by stage V was localized in vesicles along the RBC surface, consistent with a role during gamete emergence. The ability of a membrane-permeant cysteine protease inhibitor to significantly reduce malaria parasite transmission suggests that future drug design should include evaluation of gametogenesis and sporogonic development.
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Nimigean, Crina M., and Christopher Miller. "Na+ Block and Permeation in a K+ Channel of Known Structure." Journal of General Physiology 120, no. 3 (August 12, 2002): 323–35. http://dx.doi.org/10.1085/jgp.20028614.
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The effects of intracellular Na+ were studied on K+ and Rb+ currents through single KcsA channels. At low voltage, Na+ produces voltage-dependent block, which becomes relieved at high voltage by a “punchthrough” mechanism representing Na+ escaping from its blocking site through the selectivity filter. The Na+ blocking site is located in the wide, hydrated vestibule, and it displays unexpected selectivity for K+ and Rb+ against Na+. The voltage dependence of Na+ block reflects coordinated movements of the blocker with permeant ions in the selectivity filter.
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Garty, H., T. J. Furlong, D. E. Ellis, and K. R. Spring. "Sorbitol permease: an apical membrane transporter in cultured renal papillary epithelial cells." American Journal of Physiology-Renal Physiology 260, no. 5 (May 1, 1991): F650—F656. http://dx.doi.org/10.1152/ajprenal.1991.260.5.f650.
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The efflux of sorbitol from the rabbit papillary epithelial cell line PAP-HT25 occurs through a specific transport pathway, which we denote the "sorbitol permease." The permease was studied by measuring cell volume changes that accompanied osmotic swelling and by determination of the sorbitol efflux from plasma membrane vesicles. The cell volume studies showed that sorbitol efflux in response to hypotonicity occurred only across the apical membrane of the cells and that loss of sorbitol was the primary mechanism for regulatory volume decrease (RVD) by these cells. Quinidine, a permeant inhibitor of the sorbitol permease, was shown to prevent RVD when added to either apical or basolateral bathing solution. Cell volume experiments also showed that the permease was present only on the apical membrane of cells that had been grown in isotonic medium and did not accumulate sorbitol. The permease could be demonstrated in membrane vesicles obtained from cells exposed to a hypotonic environment before being homogenized. Quinidine blocked the sorbitol efflux from vesicles indicating that it either directly inhibited the permease or a membrane-associated activation step.
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Ma, Gary S., Nicolas Aznar, Nicholas Kalogriopoulos, Krishna K. Midde, Inmaculada Lopez-Sanchez, Emi Sato, Ying Dunkel, Richard L. Gallo, and Pradipta Ghosh. "Therapeutic effects of cell-permeant peptides that activate G proteins downstream of growth factors." Proceedings of the National Academy of Sciences 112, no. 20 (April 29, 2015): E2602—E2610. http://dx.doi.org/10.1073/pnas.1505543112.
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In eukaryotes, receptor tyrosine kinases (RTKs) and trimeric G proteins are two major signaling hubs. Signal transduction via trimeric G proteins has long been believed to be triggered exclusively by G protein-coupled receptors (GPCRs). This paradigm has recently been challenged by several studies on a multimodular signal transducer, Gα-Interacting Vesicle associated protein (GIV/Girdin). We recently demonstrated that GIV’s C terminus (CT) serves as a platform for dynamic association of ligand-activated RTKs with Gαi, and for noncanonical transactivation of G proteins. However, exogenous manipulation of this platform has remained beyond reach. Here we developed cell-permeable GIV-CT peptides by fusing a TAT-peptide transduction domain (TAT-PTD) to the minimal modular elements of GIV that are necessary and sufficient for activation of Gi downstream of RTKs, and used them to engineer signaling networks and alter cell behavior. In the presence of an intact GEF motif, TAT-GIV-CT peptides enhanced diverse processes in which GIV’s GEF function has previously been implicated, e.g., 2D cell migration after scratch-wounding, invasion of cancer cells, and finally, myofibroblast activation and collagen production. Furthermore, topical application of TAT-GIV-CT peptides enhanced the complex, multireceptor-driven process of wound repair in mice in a GEF-dependent manner. Thus, TAT-GIV peptides provide a novel and versatile tool to manipulate Gαi activation downstream of growth factors in a diverse array of pathophysiologic conditions.
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Grover, A. K., A. P. Singh, P. K. Rangachari, and P. Nicholls. "Ion movements in membrane vesicles: a new fluorescence method and application to smooth muscle." American Journal of Physiology-Cell Physiology 248, no. 3 (March 1, 1985): C372—C378. http://dx.doi.org/10.1152/ajpcell.1985.248.3.c372.
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A method is described for studying ion permeabilities of membrane vesicles based on the principle that when membrane permeability to H+ is very high, the H+ movement is determined by the membrane potential generated by the H+ movement. The rate of H+ movement under these conditions thus gives a measure of the rate of dissipation of this membrane potential by comovement of anions or countermovement of cations present. Thus, by studying the H+ efflux using an impermeant cation and different anions, the membrane permeability to the anions can be assessed. Similarly, the use of an impermeant anion allows the study of the permeation of various cations. H+ movement was followed across the membranes by monitoring a change in the fluorescence intensity of the pH-sensitive dye pyranine trapped inside the membranes. This method when tested using phosphatidylcholine liposomes yielded the expected results, i.e., permeability of the liposomal membrane was: Cl- greater than SO2-4 and K+ greater than Na+. A plasma membrane-enriched fraction loaded with pyranine was isolated from estrogen-dominant rat myometrium. The anion permeability characteristics of this membrane were studied using tetramethylammonium (TMA+) as the poorly permeant cation, and the cation permeability was studied using L-glutamate- as the poorly permeant anion. The anion permeabilities were D-glutamate- less than L-glutamate- less than glutarate2- less than Cl- less than or equal to SO2-4, and the cation permeabilities were TMA+ less than K+ less than Na+. It is hypothesized that the observed anomalously higher Na+ and SO2-4 movements may involve special mechanisms.
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Deng, Jiaojiao, Sophia Chernikova, Wolf-Nicolas Fischer, Kerry Koller, Bernd Jandeleit, Gordon Ringold, and Melanie Gephart. "LPTO-06. A NOVEL BRAIN-PERMEANT CHEMOTHERAPEUTIC AGENT FOR THE TREATMENT OF BREAST CANCER LEPTOMENINGEAL METASTASIS." Neuro-Oncology Advances 1, Supplement_1 (August 2019): i7. http://dx.doi.org/10.1093/noajnl/vdz014.029.
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Abstract Leptomeningeal metastasis (LM), a spread of cancer to the cerebrospinal fluid and meninges, is universally and rapidly fatal due to poor detection and no effective treatment. Breast cancers account for a majority of LMs from solid tumors, with triple-negative breast cancers (TNBCs) having the highest propensity to metastasize to LM. The treatment of LM is challenged by poor drug penetration into CNS and high neurotoxicity. Therefore, there is an urgent need for new modalities and targeted therapies able to overcome the limitations of current treatment options. Quadriga has discovered a novel, brain-permeant chemotherapeutic agent that is currently in development as a potential treatment for glioblastoma (GBM). The compound is active in suppressing the growth of GBM tumor cell lines implanted into the brain. Radiolabel distribution studies have shown significant tumor accumulation in intracranial brain tumors while sparing the adjacent normal brain tissue. Recently, we have demonstrated dose-dependent in vitro and in vivo anti-tumor activity with various breast cancer cell lines including the human TNBC cell line MDA-MB-231. To evaluate the in vivo antitumor activity of the compound on LM, we used the mouse model of LM based on the internal carotid injection of luciferase-expressing MDA-MB-231-BR3 cells. Once the bioluminescence signal intensity from the metastatic spread reached (0.2 - 0.5) x 106 photons/sec, mice were dosed i.p. twice a week with either 4 or 8 mg/kg for nine weeks. Tumor growth was monitored by bioluminescence. The compound was well tolerated and caused a significant delay in metastatic growth resulting in significant extension of survival. Tumors regressed completely in ~ 28 % of treated animals. Given that current treatments for LM are palliative with only few studies reporting a survival benefit, Quadriga’s new agent could be effective as a therapeutic for both primary and metastatic brain tumors such as LM. REF: https://onlinelibrary.wiley.com/doi/full/10.1002/pro6.43
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Ogura, Tatsuya, and Sue C. Kinnamon. "IP3-Independent Release of Ca2+ From Intracellular Stores: A Novel Mechanism for Transduction of Bitter Stimuli." Journal of Neurophysiology 82, no. 5 (November 1, 1999): 2657–66. http://dx.doi.org/10.1152/jn.1999.82.5.2657.
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A variety of substances with different chemical structures elicits a bitter taste. Several different transduction mechanisms underlie detection of bitter tastants; however, these have been described in detail for only a few compounds. In addition, most studies have focused on mammalian taste cells, of which only a small subset is responsive to any particular bitter compound. In contrast, ∼80% of the taste cells in the mudpuppy, Necturus maculosus, are bitter-responsive. In this study, we used Ca2+ imaging and giga-seal whole cell recording to compare the transduction of dextromethorphan (DEX), a bitter antitussive, with transduction of the well-studied bitter compound denatonium. Bath perfusion of DEX (2.5 mM) increased the intracellular Ca2+level in most taste cells. The DEX-induced Ca2+ increase was inhibited by thapsigargin, an inhibitor of Ca2+transport into intracellular stores, but not by U73122, an inhibitor of phospholipase C, or by ryanodine, an inhibitor of ryanodine-sensitive Ca2+ stores. Increasing intracellular cAMP levels with a cell-permeant cAMP analogue and a phosphodiesterase inhibitor enhanced the DEX-induced Ca2+ increase, which was inhibited partially by H89, a protein kinase A inhibitor. Electrophysiological measurements showed that DEX depolarized the membrane potential and inhibited voltage-gated Na+ and K+ currents in the presence of GDP-β-S, a blocker of G-protein activation. DEX also inhibited voltage-gated Ca2+ channels. We suggest that DEX, like quinine, depolarizes taste cells by block of voltage-gated K channels, which are localized to the apical membrane in mudpuppy. In addition, DEX causes release of Ca2+ from intracellular stores by a phospholipase C-independent mechanism. We speculate that the membrane-permeant DEX may enter taste cells and interact directly with Ca2+ stores. Comparing transduction of DEX with that of denatonium, both compounds release Ca2+ from intracellular stores. However, denatonium requires activation of phospholipase C, and the mechanism results in a hyperpolarization rather than a depolarization of the membrane potential. These data support the hypothesis that single taste receptor cells can use multiple mechanisms for transducing the same bitter compound.
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Brunet, Sylvain, Todd Scheuer, and William A. Catterall. "Cooperative regulation of Cav1.2 channels by intracellular Mg2+, the proximal C-terminal EF-hand, and the distal C-terminal domain." Journal of General Physiology 134, no. 2 (July 13, 2009): 81–94. http://dx.doi.org/10.1085/jgp.200910209.
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L-type Ca2+ currents conducted by Cav1.2 channels initiate excitation–contraction coupling in cardiac myocytes. Intracellular Mg2+ (Mgi) inhibits the ionic current of Cav1.2 channels. Because Mgi is altered in ischemia and heart failure, its regulation of Cav1.2 channels is important in understanding cardiac pathophysiology. Here, we studied the effects of Mgi on voltage-dependent inactivation (VDI) of Cav1.2 channels using Na+ as permeant ion to eliminate the effects of permeant divalent cations that engage the Ca2+-dependent inactivation process. We confirmed that increased Mgi reduces peak ionic currents and increases VDI of Cav1.2 channels in ventricular myocytes and in transfected cells when measured with Na+ as permeant ion. The increased rate and extent of VDI caused by increased Mgi were substantially reduced by mutations of a cation-binding residue in the proximal C-terminal EF-hand, consistent with the conclusion that both reduction of peak currents and enhancement of VDI result from the binding of Mgi to the EF-hand (KD ≈ 0.9 mM) near the resting level of Mgi in ventricular myocytes. VDI was more rapid for L-type Ca2+ currents in ventricular myocytes than for Cav1.2 channels in transfected cells. Coexpression of Cavβ2b subunits and formation of an autoinhibitory complex of truncated Cav1.2 channels with noncovalently bound distal C-terminal domain (DCT) both increased VDI in transfected cells, indicating that the subunit structure of the Cav1.2 channel greatly influences its VDI. The effects of noncovalently bound DCT on peak current amplitude and VDI required Mgi binding to the proximal C-terminal EF-hand and were prevented by mutations of a key divalent cation-binding amino acid residue. Our results demonstrate cooperative regulation of peak current amplitude and VDI of Cav1.2 channels by Mgi, the proximal C-terminal EF-hand, and the DCT, and suggest that conformational changes that regulate VDI are propagated from the DCT through the proximal C-terminal EF-hand to the channel-gating mechanism.
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Bartsch, M., M. Zorn-Kruppa, N. Kühl, H. G. Genieser, F. Schwede, and B. Jastorff. "Bioactivatable, Membrane-Permeant Analogs of Cyclic Nucleotides as Biological Tools for Growth Control of C6 Glioma Cells." Biological Chemistry 384, no. 9 (September 28, 2003): 1321–26. http://dx.doi.org/10.1515/bc.2003.148.
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Abstract In the present study, the cAMP analogs 8-bromocAMP (8-Brc-AMP), N6-2'O-dibutyryl-cAMP (DBcAMP) and 8-parachlorophenylthio-cAMP (8-CPT-cAMP), as well as the corresponding cAMP-acetoxymethyl (AM)-ester-prodrugs were tested in a HPLC study for their membrane permeability, intracellular accumulation and biotransformation. Antiproliferative activities of these compounds were studied in the rat C6 glioma cell line. Chromatographic analysis revealed that the AM-ester analogs of the cyclic nucleotides penetrate quantitatively into rat C6 glioma cells and generate high amounts of their parent cyclic nucleotides intracellularly within 60 min; however, longterm growth inhibition tested in C6 cells is only slightly enhanced with the AM-ester prodrugs of 8-Br-cAMP or DBcAMP.
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Siflinger-Birnboim, A., H. Lum, P. J. Del Vecchio, and A. B. Malik. "Involvement of Ca2+ in the H2O2-induced increase in endothelial permeability." American Journal of Physiology-Lung Cellular and Molecular Physiology 270, no. 6 (June 1, 1996): L973—L978. http://dx.doi.org/10.1152/ajplung.1996.270.6.l973.
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We studied the role of Ca2+ in mediating the hydrogen peroxide (H2O2)-induced increase in endothelial permeability to 125I-labeled albumin using bovine pulmonary microvessel endothelial cells (BMVEC). Changes in cytosolic-free Ca2+ ([Ca2+]i) were monitored in BMVEC monolayers loaded with the Ca(2+)-sensitive membrane permeant fluorescent dye fura 2-AM. H2O2 (100 microM) produced a rise in [Ca2+]i within 10 s that was reduced by the addition of EGTA to the medium. Uptake of 45Ca2+ from the extracellular medium increased in the presence of H2O2 (100 microM) compared with control monolayers, suggesting that the H2O2-induced rise in [Ca2+]i is partly the result of extracellular Ca2+ influx. The effects of [Ca2+]i on endothelial permeability were addressed by pretreatment of BMVEC monolayers with BAPTA-AM (3-5 microM), a membrane permeant Ca2+ chelator, before the H2O2 exposure. BAPTA-AM produced an approximately 50% decrease in the H2O2-induced increase in endothelial permeability compared with endothelial cell monolayers exposed to H2O2 alone. The increase in endothelial permeability was independent of Ca2+ influx, since LaCl3 (0-100 microM), which displaces Ca2+ from binding sites on the cell surface, did not modify the permeability response. These results indicate that the rise in [Ca2+]i produced by H2O2 is a critical determinant of the increase in endothelial permeability.
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Phillippe, M., E. M. Chien, M. Freij, and T. Saunders. "Ionomycin-stimulated phasic myometrial contractions." American Journal of Physiology-Endocrinology and Metabolism 269, no. 4 (October 1, 1995): E779—E785. http://dx.doi.org/10.1152/ajpendo.1995.269.4.e779.
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Ionomycin, a calcium ionophore, facilitates the sustained entry of extracellular calcium; however, in myometrial tissue it stimulates phasic contractions. This study sought to define further this unanticipated effect of ionomycin and to begin to explore the possible mechanism(s) involved. Utilizing rat uterine strips, in vitro isometric contraction studies were performed to determine the effects of ionomycin with and without membrane-permeant inhibitors of cytosolic calcium oscillations. To determine the effects of ionomycin on phospholipase C, qualitative inositol phosphate production studies were performed. The in vitro contraction studies confirmed that ionomycin-stimulated phasic myometrial contractions were potentially dependent on stimulation of phospholipase C, calcium-induced calcium release, and additional calcium influx through dihydropyridine-sensitive membrane calcium channels. The inositol phosphate production studies confirmed that ionomycin stimulated phospholipase C in a dose-related fashion to levels comparable to oxytocin. In summary, these observations have confirmed the ability of ionomycin to generate dose-related phasic myometrial contractions through mechanisms potentially involving the phosphatidylinositol-signaling pathway.
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Díaz-Guerra, M. J. M., and L. Boscá. "Lack of translocation of protein kinase C from the cytosol to the membranes in vasopressin-stimulated hepatocytes." Biochemical Journal 269, no. 1 (July 1, 1990): 163–68. http://dx.doi.org/10.1042/bj2690163.
Full textAbstract:
The ability of Ca2(+)-mobilizing hormones to promote changes in the subcellular distribution of protein kinase C (PKC) was studied in isolated hepatocytes. In recently isolated cells the distribution of PKC between the soluble and particulate fractions was 47 and 53% respectively. Exposure of the hepatocytes to 100 nM-vasopressin produced an increased phosphoinositide turnover, as reflected by the changes in the concentrations of inositol trisphosphate and Ca2+, and in glycogen phosphorylase a activity. However, the distribution of both PKC activity and [3H]phorbol dibutyrate binding between the cytosol and the membranes remained unchanged under these conditions. To determine the threshold values of the concentrations of Ca2+ and diacylglycerol required to produce a redistribution of PKC, the hepatocytes were treated with the Ca2+ ionophore ionomycin, and with permeant diacylglycerol derivatives. Hepatocytes incubated in the presence of 100 nM-vasopressin required concentrations of Ca2+ 2.5 times those produced physiologically by the hormone to produce translocation of PKC from the cytosol to the membranes. These studies suggest that, at least in hepatocytes, activation of PKC in response to Ca2(+)-mobilizing hormones involves only the pre-existent membrane-bound enzyme without affecting the soluble enzyme.
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Haddad, Elie, José L. Zugaza, Fawzia Louache, Najet Debili, Catherine Crouin, Klaus Schwarz, Alain Fischer, William Vainchenker, and Jacques Bertoglio. "The interaction between Cdc42 and WASP is required for SDF-1–induced T-lymphocyte chemotaxis." Blood 97, no. 1 (January 1, 2001): 33–38. http://dx.doi.org/10.1182/blood.v97.1.33.
Full textAbstract:
Abstract In studies aimed at further characterizing the cellular immunodeficiency of the Wiskott-Aldrich syndrome (WAS), we found that T lymphocytes from WAS patients display abnormal chemotaxis in response to the T-cell chemoattractant stromal cell–derived factor (SDF)-1. The Wiskott- Aldrich syndrome protein (WASP), together with the Rho family GTPase Cdc42, control stimulus-induced actin cytoskeleton rearrangements that are involved in cell motility. Because WASP is an effector of Cdc42, we further studied how Cdc42 and WASP are involved in SDF-1–induced chemotaxis of T lymphocytes. We provide here direct evidence that SDF-1 activates Cdc42. We then specifically investigated the role of the interaction between Cdc42 and WASP in SDF-1–responsive cells. This was achieved by abrogating this interaction with a recombinant polypeptide (TAT-CRIB), comprising the Cdc42/Rac interactive binding (CRIB) domain of WASP and a human immunodeficiency virus–TAT peptide that renders the fusion protein cell-permeant. This TAT-CRIB protein was shown to bind specifically to Cdc42-GTP and to inhibit the chemotactic response of a T-cell line to SDF-1. Altogether, these data demonstrate that Cdc42-WASP interaction is critical for SDF-1–induced chemotaxis of T cells.
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Sáez, Juan C., Aníbal A. Vargas, Diego E. Hernández, Fernando C. Ortiz, Christian Giaume, and Juan A. Orellana. "Permeation of Molecules through Astroglial Connexin 43 Hemichannels Is Modulated by Cytokines with Parameters Depending on the Permeant Species." International Journal of Molecular Sciences 21, no. 11 (June 1, 2020): 3970. http://dx.doi.org/10.3390/ijms21113970.
Full textAbstract:
Recent studies indicate that connexin hemichannels do not act as freely permeable non-selective pores, but they select permeants in an isoform-specific manner with cooperative, competitive and saturable kinetics. The aim of this study was to investigate whether the treatment with a mixture of IL-1β plus TNF-α, a well-known pro-inflammatory condition that activates astroglial connexin 43 (Cx43) hemichannels, could alter their permeability to molecules. We found that IL-1β plus TNF-α left-shifted the dye uptake rate vs. dye concentration relationship for Etd and 2-NBDG, but the opposite took place for DAPI or YO-PRO-1, whereas no alterations were observed for Prd. The latter modifications were accompanied of changes in Kd (Etd, DAPI, YO-PRO-1 or 2-NBDG) and Hill coefficients (Etd and YO-PRO-1), but not in alterations of Vmax. We speculate that IL-1β plus TNF-α may distinctively affect the binding sites to permeants in astroglial Cx43 hemichannels rather than their number in the cell surface. Alternatively, IL-1β plus TNF-α could induce the production of endogenous permeants that may favor or compete for in the pore-lining residues of Cx43 hemichannels. Future studies shall elucidate whether the differential ionic/molecule permeation of Cx43 hemichannels in astrocytes could impact their communication with neurons in the normal and inflamed nervous system.
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Wang, X., V. A. Briner, and R. W. Schrier. "Parathyroid hormone inhibition of vasopressin-induced vascular smooth muscle contraction." American Journal of Physiology-Renal Physiology 264, no. 3 (March 1, 1993): F453—F457. http://dx.doi.org/10.1152/ajprenal.1993.264.3.f453.
Full textAbstract:
In various cells, parathyroid hormone (PTH) has been shown to initiate both polyphosphoinositide (PI) breakdown and activation of adenylate cyclase (AC). In vascular smooth muscle cells (VSMC), PI hydrolysis is known to induce contraction, whereas a rise in adenosine 3',5'-cyclic monophosphate (cAMP) causes relaxation. In the present study, the effect of PTH on arginine vasopressin (AVP)-induced VSMC contraction and signal transduction was studied. PTH (10(-7) M) attenuated the percentage of VSMC contracting in response to AVP (10(-7) M; 40 to 26.5%, P < 0.05). This loss of VSMC contractility was not the result of PTH-induced changes in AVP receptor binding. PTH did, however, stimulate VSMC cAMP production in a dose-dependent manner. The effect of PTH on AVP-induced contraction could be mimicked by treating VSMC with the cell-permeant cAMP analogue, 8-(4-chlorophenylthio)-cAMP (ClPheScAMP). The effect of PTH on AVP-induced VSMC contraction was blocked by H-8, an inhibitor of protein kinase A and thus cAMP production. In parallel to the inhibitory effects of ClPheScAMP on VSMC contraction, AVP-stimulated inositol trisphosphate production was also reduced by this permeant cAMP (4,415 to 2,592 cpm/mg protein, P < 0.01). PTH-induced production of cAMP was not blocked by an inhibition of prostaglandin synthesis [PTH 203 vs. PTH + ibuprofen 161 fmol/micrograms protein, not significant (NS)]. In contrast, AVP also stimulated cAMP, but this increase was blocked by ibuprofen.(ABSTRACT TRUNCATED AT 250 WORDS)
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Neyton, J., and C. Miller. "Potassium blocks barium permeation through a calcium-activated potassium channel." Journal of General Physiology 92, no. 5 (November 1, 1988): 549–67. http://dx.doi.org/10.1085/jgp.92.5.549.
Full textAbstract:
Single high-conductance Ca2+-activated K+ channels from rat skeletal muscle were inserted into planar lipid bilayers, and discrete blocking by the Ba2+ ion was studied. Specifically, the ability of external K+ to reduce the Ba2+ dissociation rate was investigated. In the presence of 150 mM internal K+, 1-5 microM internal Ba2+, and 150 mM external Na+, Ba2+ dissociation is rapid (5 s-1) in external solutions that are kept rigorously K+ free. The addition of external K+ in the low millimolar range reduces the Ba2+ off-rate 20-fold. Other permeant ions, such as Tl+, Rb+, and NH4+ show a similar effect. The half-inhibition constants rise in the order: Tl+ (0.08 mM) less than Rb+ (0.1 mM) less than K+ (0.3 mM) less than Cs+ (0.5 mM) less than NH4+ (3 mM). When external Na+ is replaced by 150 mM N-methyl glucamine, the Ba2+ off-rate is even higher, 20 s-1. External K+ and other permeant ions reduce this rate by approximately 100-fold in the micromolar range of concentrations. Na+ also reduces the Ba2+ off-rate, but at much higher concentrations. The half-inhibition concentrations rise in the order: Rb+ (4 microM) less than K+ (19 microM) much less than Na+ (27 mM) less than Li+ (greater than 50 mM). The results require that the conduction pore of this channel contains at least three sites that may all be occupied simultaneously by conducting ions.
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Pietrobon, D., B. Prod'hom, and P. Hess. "Interactions of protons with single open L-type calcium channels. pH dependence of proton-induced current fluctuations with Cs+, K+, and Na+ as permeant ions." Journal of General Physiology 94, no. 1 (July 1, 1989): 1–21. http://dx.doi.org/10.1085/jgp.94.1.1.
Full textAbstract:
We studied the pH dependence of the proton-induced current fluctuations that appear in single open L-type Ca channels when monovalent ions are the charge carriers. We used different methods of analysis to obtain kinetic measurements even under conditions where the individual transitions were too fast to be resolved directly as discrete current steps between two conductance levels. The reciprocal of the dwell times at the high conductance level increased linearly with the pipette proton activity, with a slope that was similar for Cs, K, and Na as permeant ions. Contrary to the expectation for a simple model in which the high and low conductances represent the unprotonated and protonated states of the channel, respectively, the dwell times at the low conductance level were also pH dependent and lengthened with increasing proton activity. At all pH values the dwell times at the low conductance level were longest with Cs as permeant ion and shortened in the order Cs greater than K greater than Na. We introduce a more general model of the protonation cycle in which the channel is represented by four states and can be protonated and deprotonated both at the high and low conductance levels. The conductance change is represented by a conformational change of the channel protein. We discuss the validity of this model and its implications for the mechanism by which protons interact with ion permeation through L-type Ca channels.
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WANG, Suwei, Srigiridhar KOTAMRAJU, Eugene KONOREV, Shasi KALIVENDI, Joy JOSEPH, and Balaraman KALYANARAMAN. "Activation of nuclear factor-κB during doxorubicin-induced apoptosis in endothelial cells and myocytes is pro-apoptotic: the role of hydrogen peroxide." Biochemical Journal 367, no. 3 (November 1, 2002): 729–40. http://dx.doi.org/10.1042/bj20020752.
Full textAbstract:
Doxorubicin (DOX) is a widely used anti-tumour drug. Cardiotoxicity is a major toxic side effect of DOX therapy. Although recent studies implicated an apoptotic pathway in DOX-induced cardiotoxicity, the mechanism of DOX-induced apoptosis remains unclear. In the present study, we investigated the role of reactive oxygen species and the nuclear transcription factor nuclear factor κB (NF-κB) during apoptosis induced by DOX in bovine aortic endothelial cells (BAECs) and adult rat cardiomyocytes. DOX-induced NF-κB activation is both dose- and time-dependent, as demonstrated using electrophoretic mobility-shift assay and luciferase and p65 (Rel A) nuclear-translocation assays. Addition of a cell-permeant iron metalloporphyrin significantly suppressed NF-κB activation and apoptosis induced by DOX. Overexpression of glutathione peroxidase, which detoxifies cellular H2O2, significantly decreased DOX-induced NF-κB activation and apoptosis. Inhibition of DOX-induced NF-κB activation by a cell-permeant peptide SN50 that blocks translocation of the NF-κB complex into the nucleus greatly diminished DOX-induced apoptosis. Apoptosis was inhibited when IκB mutant vector, another NF-κB inhibitor, was added to DOX-treated BAECs. These results suggest that NF-κB activation in DOX-treated endothelial cells and myocytes is pro-apoptotic, in contrast with DOX-treated cancer cells, where NF-κB activation is anti-apoptotic. Removal of intracellular H2O2 protects endothelial cells and myocytes from DOX-induced apoptosis, possibly by inhibiting NF-κB activation. These findings suggest a novel mechanism for enhancing the therapeutic efficacy of DOX.
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Takami, M., S. L. Preston, V. A. Toyloy, and Harold R. Behrman. "Antioxidants reversibly inhibit the spontaneous resumption of meiosis." American Journal of Physiology-Endocrinology and Metabolism 276, no. 4 (April 1, 1999): E684—E688. http://dx.doi.org/10.1152/ajpendo.1999.276.4.e684.
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We previously showed that the cell-permeant antioxidant 2(3)- tert-butyl-4-hydroxyanisole (BHA) inhibited germinal vesicle breakdown (GVBD) in oocyte-cumulus complexes (OCC) of the rat. The objective of the present studies was to assess other antioxidants and whether such inhibition was reversible. Spontaneous GVBD in OCC incubated for 2 h was significantly inhibited ( P < 0.005) by nordihydroguaiaretic acid (NDGA; GVBD = 19.4%), BHA (GVBD = 25.7%), octyl gallate (OG; GVBD = 52.2%), ethoxyquin (EQ; GVBD = 58.8%), 2,6-di- tert-butyl-hydroxymethyl phenol (TBHMP; GVBD = 59%), butylated hydroxytoluene (BHT; GVBD = 59.5%), and tert-butyl hydroperoxide (TBHP; GVBD = 60.0%). Other antioxidants that produced lower but significant ( P < 0.05) inhibition of oocyte maturation included propyl gallate (PG; GVBD = 70.3%), 2,4,5-trihydroxybutrophenone (THBP; GVBD = 71.4%), and lauryl gallate (LG; GVBD = 71.4%). Antioxidants that had no effect on oocyte maturation at the same concentration (100 μM) included ascorbic acid, vitamin E, and Trolox. Inhibition of GVBD was evident for up to 8 h of incubation of OCC and denuded oocytes (DO) with BHA or NDGA and was reversed by washing. NDGA was less potent than BHA for inhibition of GVBD in DO, unlike that seen with OCC. Oocyte maturation was induced by incubation of follicles for 3 h with human chorionic gonadotropin (hCG), and this response was inhibited by BHA or NDGA. These findings support the conclusion that cell-permeant antioxidants inhibit spontaneous resumption of meiosis, which may implicate a role of oxygen radicals in oocyte maturation.
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Goisnard, Antoine, Pierre Daumar, Clémence Dubois, Corinne Aubel, Manon Roux, Marie Depresle, Jean Gauthier, et al. "LightSpot®-FL-1 Fluorescent Probe: An Innovative Tool for Cancer Drug Resistance Analysis by Direct Detection and Quantification of the P-glycoprotein (P-gp) on Monolayer Culture and Spheroid Triple Negative Breast Cancer Models." Cancers 13, no. 16 (August 11, 2021): 4050. http://dx.doi.org/10.3390/cancers13164050.
Full textAbstract:
P-gp is the most widely studied MDR protein conferring cellular resistance to many standard or targeted therapeutic agents. For this reason, P-gp chemoresistance evaluation, established before or during chemotherapy, can be very relevant in order to optimize the efficacy of treatments, particularly for aggressive tumoral subtypes such as triple-negative breast cancer (TNBC). In this context, our team developed an innovative cell-permeant fluorescent probe called the LightSpot®-FL-1, which is able to specifically localize and quantify the P-gp in cells or cell masses, as evidenced on different TNBC cell models. First, flow cytometry analysis showed LightSpot®-FL-1 cell penetration and persistence in time, in TNBC cells. Then, LightSpot®-FL-1 staining was compared to anti-P-gp immunostaining by fluorescence microscopy on five TNBC cell lines. Results showed a clear similarity of P-gp localization and expression level, confirmed by Pearson’s and Mander’s colocalization coefficients with 92.1% and 100.0%, and a strong correlation coefficient of R2 = 0.99. In addition, the LightSpot®-FL-1 staining allowed the quantification of a P-gp induction (33% expression increase) following a 6-hour spheroid model exposure to the anti-PARP Olaparib. Thus, the new LightSpot®-FL-1 cell-permeant probe, targeting P-gp, appears to be an effective tool for drug resistance evaluation in preclinical models and shows promising possibilities for future use in clinical diagnosis.
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Tinker, A., and A. J. Williams. "Probing the structure of the conduction pathway of the sheep cardiac sarcoplasmic reticulum calcium-release channel with permeant and impermeant organic cations." Journal of General Physiology 102, no. 6 (December 1, 1993): 1107–29. http://dx.doi.org/10.1085/jgp.102.6.1107.
Full textAbstract:
The sarcoplasmic reticulum Ca(2+)-release channel plays a central role in cardiac muscle function by providing a ligand-regulated pathway for the release of sequestered Ca2+ to initiate contraction following cell excitation. The efficiency of the channel as a Ca(2+)-release pathway will be influenced by both gating and conductance properties of the system. In the past we have investigated conduction and discrimination of inorganic mono- and divalent cations with the aim of describing the mechanisms governing ion handling in the channel (Tinker, A., A.R. G. Lindsay, and A.J. Williams. 1992. Journal of General Physiology. 100:495-517.). In the present study, we have used permeant and impermeant organic cations to provide additional information on structural features of the conduction pathway. The use of permeant organic cations in biological channels to explore structural motifs underlying selectivity has been an important tool for the electrophysiologist. We have examined the conduction properties of a series of monovalent organic cations of varying size in the purified sheep cardiac sarcoplasmic reticulum Ca(2+)-release channel. Relative permeability, determined from the reversal potential measured under bi-ionic conditions with 210-mM test cation at the cytoplasmic face of the channel and 210 mM K+ at the luminal, was related inversely to the minimum circular cation radius. The reversal potential was concentration-independent. The excluded area hypothesis, with and without a term for solute-wall friction, described the data well and gave a lower estimate for minimum pore radius of 3.3-3.5 A. Blocking studies with the impermeant charged derivative of triethylamine reveal that this narrowing occurs over the first 10-20% of the voltage drop when crossing from the lumen of the SR to the cytoplasm. Single-channel conductances were measured in symmetrical 210 mM salt. Factors other than relative permeability determine conductance as ions with similar relative permeability can have widely varying single-channel conductance. Permeant ions, such as the charged derivatives of trimethylamine and diethylmethylamine, can also inhibit K+ current. The reduction in relative conductance with increasing concentrations of these two ions at a holding potential of 60 mV was described by a rectangular hyperbola and revealed higher affinity binding for diethylmethylamine as compared to trimethylamine. It was possible to describe the complex permeation properties of these two ions using a single-ion four barrier, three binding site Eyring rate theory model. In conclusion, these studies reveal that the cardiac Ca(2+)-release channel has a selectivity filter of approximately 3.5-A radius located at the luminal face of the protein.(ABSTRACT TRUNCATED AT 400 WORDS)
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Garvin, J. L. "Inhibition of Jv by ANF in rat proximal straight tubules requires angiotensin." American Journal of Physiology-Renal Physiology 257, no. 5 (November 1, 1989): F907—F911. http://dx.doi.org/10.1152/ajprenal.1989.257.5.f907.
Full textAbstract:
The effects of atrial natriuretic factor (ANF) on fluid absorption (Jv) by isolated perfused proximal straight tubules of rats were investigated. ANF alone (10(-8) M) added to the bath had no significant effect on absorption. In contrast, when tubules were first treated with 1.6 X 10(-10) M angiotensin II, this same concentration of ANF lowered fluid absorption from 0.99 +/- 0.03 to 0.69 +/- 0.02 nl.mm-1.min-1. A lower dose of ANF, 2 X 10(-10) M, reduced fluid absorption in the presence of angiotensin II from 1.13 +/- 0.06 to 0.65 +/- 0.05 nl.mm-1.min-1, an inhibition of 40%. Since guanosine 3',5'-cyclic monophosphate (cGMP) is reportedly part of the second messenger system of ANF, the effects of dibutyryl-cGMP (DBcGMP) on fluid absorption were studied. This membrane-permeant form of cGMP mimicked the effects of ANF, reducing fluid absorption from 1.15 +/- 0.18 to 0.54 +/- 0.08 nl.mm-1.min-1. These studies suggested the following: 1) ANF can regulate fluid absorption in the proximal nephron; 2) this inhibition occurs only in the presence of angiotensin; and 3) cGMP is part of the second messenger system of ANF in the rat proximal straight tubule, as it is in other tissues.