Journal articles on the topic 'Permeant anion'
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1
Lai, Xiao-Gang, Jun Yang, Shi-Sheng Zhou, Jun Zhu, Gui-Rong Li, and Tak-Ming Wong. "Involvement of anion channel(s) in the modulation of the transient outward K+ channel in rat ventricular myocytes." American Journal of Physiology-Cell Physiology 287, no. 1 (July 2004): C163—C170. http://dx.doi.org/10.1152/ajpcell.00297.2003.
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The cardiac Ca2+-independent transient outward K+ current ( Ito), a major repolarizing ionic current, is markedly affected by Cl− substitution and anion channel blockers. We reexplored the mechanism of the action of anions on Ito by using whole cell patch-clamp in single isolated rat cardiac ventricular myocytes. The transient outward current was sensitive to blockade by 4-aminopyridine (4-AP) and was abolished by Cs+ substitution for intracellular K+. Replacement of most of the extracellular Cl− with less permeant anions, aspartate (Asp−) and glutamate (Glu−), markedly suppressed the current. Removal of external Na+ or stabilization of F-actin with phalloidin did not significantly affect the inhibitory action of less permeant anions on Ito. In contrast, the permeant Cl− substitute Br− did not markedly affect the current, whereas F− substitution for Cl− induced a slight inhibition. The Ito elicited during Br− substitution for Cl− was also sensitive to blockade by 4-AP. The ability of Cl− substitutes to induce rightward shifts of the steady-state inactivation curve of Ito was in the following sequence: NO3− > Cl− ≈ Br− > gluconate− > Glu− > Asp−. Depolymerization of actin filaments with cytochalasin D (CytD) induced an effect on the steady-state inactivation of Ito similar to that of less permeant anions. Fluorescent phalloidin staining experiments revealed that CytD-pretreatment significantly decreased the intensity of FITC-phalloidin staining of F-actin, whereas Asp− substitution for Cl− was without significant effect on the intensity. These results suggest that the Ito channel is modulated by anion channel(s), in which the actin cytoskeleton may be implicated.
2
De Jesús-Pérez, José J., Alejandra Castro-Chong, Ru-Chi Shieh, Carmen Y. Hernández-Carballo, José A. De Santiago-Castillo, and Jorge Arreola. "Gating the glutamate gate of CLC-2 chloride channel by pore occupancy." Journal of General Physiology 147, no. 1 (December 14, 2015): 25–37. http://dx.doi.org/10.1085/jgp.201511424.
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CLC-2 channels are dimeric double-barreled chloride channels that open in response to hyperpolarization. Hyperpolarization activates protopore gates that independently regulate the permeability of the pore in each subunit and the common gate that affects the permeability through both pores. CLC-2 channels lack classic transmembrane voltage–sensing domains; instead, their protopore gates (residing within the pore and each formed by the side chain of a glutamate residue) open under repulsion by permeant intracellular anions or protonation by extracellular H+. Here, we show that voltage-dependent gating of CLC-2: (a) is facilitated when permeant anions (Cl−, Br−, SCN−, and I−) are present in the cytosolic side; (b) happens with poorly permeant anions fluoride, glutamate, gluconate, and methanesulfonate present in the cytosolic side; (c) depends on pore occupancy by permeant and poorly permeant anions; (d) is strongly facilitated by multi-ion occupancy; (e) is absent under likely protonation conditions (pHe = 5.5 or 6.5) in cells dialyzed with acetate (an impermeant anion); and (f) was the same at intracellular pH 7.3 and 4.2; and (g) is observed in both whole-cell and inside-out patches exposed to increasing [Cl−]i under unlikely protonation conditions (pHe = 10). Thus, based on our results we propose that hyperpolarization activates CLC-2 mainly by driving intracellular anions into the channel pores, and that protonation by extracellular H+ plays a minor role in dislodging the glutamate gate.
3
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.
4
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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Linsdell, Paul, and John W. Hanrahan. "Adenosine Triphosphate–dependent Asymmetry of Anion Permeation in the Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel." Journal of General Physiology 111, no. 4 (April 1, 1998): 601–14. http://dx.doi.org/10.1085/jgp.111.4.601.
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The cystic fibrosis transmembrane conductance regulator (CFTR) forms a tightly regulated channel that mediates the passive diffusion of Cl− ions. Here we show, using macroscopic current recording from excised membrane patches, that CFTR also shows significant, but highly asymmetrical, permeability to a broad range of large organic anions. Thus, all large organic anions tested were permeant when present in the intracellular solution under biionic conditions (PX/PCl = 0.048–0.25), whereas most were not measurably permeant when present in the extracellular solution. This asymmetry was not observed for smaller anions. ATPase inhibitors that “lock” CFTR channels in the open state (pyrophosphate, 5′-adenylylimidodiphosphate) disrupted the asymmetry of large anion permeation by allowing their influx from the extracellular solution, which suggests that ATP hydrolysis is required to maintain asymmetric permeability. The ability of CFTR to allow efflux of large organic anions represents a novel function of CFTR. Loss of this function may contribute to the pleiotropic symptoms seen in cystic fibrosis.
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DAWSON, DAVID C., STEPHEN S. SMITH, and MONIQUE K. MANSOURA. "CFTR: Mechanism of Anion Conduction." Physiological Reviews 79, no. 1 (January 1, 1999): S47—S75. http://dx.doi.org/10.1152/physrev.1999.79.1.s47.
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Dawson, David C., Stephen S. Smith, and Monique K. Mansoura. CFTR: Mechanism of Anion Conduction. Physiol. Rev. 79, Suppl.: S47–S75, 1999. — The purpose of this review is to collect together the results of recent investigations of anion conductance by the cystic fibrosis transmembrane conductance regulator along with some of the basic background that is a prerequisite for developing some physical picture of the conduction process. The review begins with an introduction to the concepts of permeability and conductance and the Nernst-Planck and rate theory models that are used to interpret these parameters. Some of the physical forces that impinge on anion conductance are considered in the context of permeability selectivity and anion binding to proteins. Probes of the conduction process are considered, particularly permeant anions that bind tightly within the pore and block anion flow. Finally, structure-function studies are reviewed in the context of some predictions for the origin of pore properties.
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Linsdell, P., and J. W. Hanrahan. "Flickery block of single CFTR chloride channels by intracellular anions and osmolytes." American Journal of Physiology-Cell Physiology 271, no. 2 (August 1, 1996): C628—C634. http://dx.doi.org/10.1152/ajpcell.1996.271.2.c628.
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Cystic fibrosis transmembrane conductance regulator (CFTR) is a phosphorylation- and nucleotide-dependent chloride channel. Single CFTR currents recorded on cell show slight outward rectification, which has previously been suggested to be due to an asymmetrical chloride ion gradient or to a specific interaction between permeant intracellular anions and the channel. Using a single-channel recording from Chinese hamster ovary cells stably expressing CFTR, we have found that both the sparingly permeant anion glutamate and the impermeant anion gluconate cause a rapid, voltage-dependent block of CFTR channels when applied to the intracellular, but not the extracellular, face of excised patches. Both the affinity and the voltage dependence of block were affected by the extracellular chloride concentration in a manner consistent with chloride ions being able to repel these blocking ions from the pore. These results are discussed in terms of previous models of CFTR current outward rectification, and it is suggested that this rectification may result from a combination of asymmetrical chloride concentrations and voltage-dependent block of the channel by large cytoplasmic anions. In addition, we find that CFTR conductance is decreased by high concentrations of intracellular sucrose, sorbitol, and urea in a manner consistent with a rapid block of the channel by these molecules.
8
Qu, Zhiqiang, and H. Criss Hartzell. "Anion Permeation in Ca2+-Activated Cl− Channels." Journal of General Physiology 116, no. 6 (December 1, 2000): 825–44. http://dx.doi.org/10.1085/jgp.116.6.825.
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Ca2+-activated Cl channels (ClCaCs) are an important class of anion channels that are opened by increases in cytosolic [Ca2+]. Here, we examine the mechanisms of anion permeation through ClCaCs from Xenopus oocytes in excised inside-out and outside-out patches. ClCaCs exhibited moderate selectivity for Cl over Na: PNa/PCl = 0.1. The apparent affinity of ClCaCs for Cl was low: Kd = 73 mM. The channel had an estimated pore diameter >0.6 nm. The relative permeabilities measured under bi-ionic conditions by changes in Erev were as follows: C(CN)3 > SCN > N(CN)2 > ClO4 > I > N3 > Br > Cl > formate > HCO3 > acetate = F > gluconate. The conductance sequence was as follows: N3 > Br > Cl > N(CN)2 > I > SCN > COOH > ClO4 > acetate > HCO3 = C(CN)3 > gluconate. Permeant anions block in a voltage-dependent manner with the following affinities: C(CN)3 > SCN = ClO4 > N(CN)2 > I > N3 > Br > HCO3 > Cl > gluconate > formate > acetate. Although these data suggest that anionic selectivity is determined by ionic hydration energy, other factors contribute, because the energy barrier for permeation is exponentially related to anion hydration energy. ClCaCs exhibit weak anomalous mole fraction behavior, implying that the channel may be a multi-ion pore, but that ions interact weakly in the pore. The affinity of the channel for Ca2+ depended on the permeant anion at low [Ca2+] (100–500 nM). Apparently, occupancy of the pore by a permeant anion increased the affinity of the channel for Ca2+. The current was strongly dependent on pH. Increasing pH on the cytoplasmic side decreased the inward current, whereas increasing pH on the external side decreased the outward current. In both cases, the apparent pKa was voltage-dependent with apparent pKa at 0 mV = ∼9.2. The channel may be blocked by OH− ions, or protons may titrate a site in the pore necessary for ion permeation. These data demonstrate that the permeation properties of ClCaCs are different from those of CFTR or ClC-1, and provide insights into the nature of the ClCaC pore.
9
Franciolini, F., and W. Nonner. "A multi-ion permeation mechanism in neuronal background chloride channels." Journal of General Physiology 104, no. 4 (October 1, 1994): 725–46. http://dx.doi.org/10.1085/jgp.104.4.725.
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Unitary current/voltage relationships of background Cl channels of rat hippocampal neurons were determined for varied gradients and absolute concentrations of NaCl. The channels revealed permeabilities for both Cl and Na ions. A hyperlinear increase of unitary conductance, observed for a symmetrical increase of salt concentration from 300 and 600 mM, indicated a multi-ion permeation mechanism. A variety of kinetic models of permeation were tested against the experimental current/voltage relationships. Models involving a pore occupied by mixed complexes of up to five ions were necessary to reproduce all measurements. A minimal model included four equilibrium states and four rate-limiting transitions, such that the empty pore accepts first an anion and then can acquire one or two cation/anion pairs. Three transport cycles are formed: a slow anion cycle (between the empty and single-anion states), a slow cation cycle (between the one- and three-ion states), and a fast anion cycle (between the three- and five-ion states). Thus, permeant anions are required for cation permeation, and several bound anions and cations promote a high rate of anion permeation. The optimized free-energy and electrical charge parameters yielded a self-consistent molecular interpretation, which can account for the particular order in which the pore accepts ions from the solutions. Although the model describes the mixed anion/cation permeability of the channel observed at elevated concentrations, it predicts a high selectivity for Cl anion at physiological ionic conditions.
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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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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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Voets, Thomas, Guy Droogmans, and Bernd Nilius. "Modulation of Voltage-dependent Properties of a Swelling-activated Cl− Current." Journal of General Physiology 110, no. 3 (September 1, 1997): 313–25. http://dx.doi.org/10.1085/jgp.110.3.313.
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We used the patch-clamp technique to study the voltage-dependent properties of the swelling-activated Cl− current (ICl,swell) in BC3H1 myoblasts. This Cl− current is outwardly rectifying and exhibits time-dependent inactivation at positive potentials (potential for half-maximal inactivation of +75 mV). Single-channel Cl− currents with similar voltage-dependent characteristics could be measured in outside-out patches pulled from swollen cells. The estimated single-channel slope conductance in the region between +60 and +140 mV was 47 pS. The time course of inactivation was well described by a double exponential function, with a voltage-independent fast time constant (∼60 ms) and a voltage-dependent slow time constant (>200 ms). Recovery from inactivation, which occurred over the physiological voltage range, was also well described by a double exponential function, with a voltage-dependent fast time constant (10–80 ms) and a voltage-dependent slow time constant (>100 ms). The inactivation process was significantly accelerated by reducing the pH, increasing the Mg2+ concentration or reducing the Cl− concentration of the extracellular solution. Replacing extracellular Cl− by other permeant anions shifted the inactivation curve in parallel with their relative permeabilities (SCN− > I− > NO3− > Cl− >> gluconate). A leftward shift of the inactivation curve could also be induced by channel blockers. Additionally, the permeant anion and the channel blockers, but not external pH or Mg2+, modulated the recovery from inactivation. In conclusion, our results show that the voltage-dependent properties of ICl,swell are strongly influenced by external pH , external divalent cations, and by the nature of the permeant anion.
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Smith, Stephen S., Erich D. Steinle, Mark E. Meyerhoff, and David C. Dawson. "Cystic Fibrosis Transmembrane Conductance Regulator." Journal of General Physiology 114, no. 6 (November 29, 1999): 799–818. http://dx.doi.org/10.1085/jgp.114.6.799.
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The cystic fibrosis transmembrane conductance regulator (CFTR) Cl channel exhibits lyotropic anion selectivity. Anions that are more readily dehydrated than Cl exhibit permeability ratios (PS/PCl) greater than unity and also bind more tightly in the channel. We compared the selectivity of CFTR to that of a synthetic anion-selective membrane [poly(vinyl chloride)–tridodecylmethylammonium chloride; PVC-TDMAC] for which the nature of the physical process that governs the anion-selective response is more readily apparent. The permeability and binding selectivity patterns of CFTR differed only by a multiplicative constant from that of the PVC-TDMAC membrane; and a continuum electrostatic model suggested that both patterns could be understood in terms of the differences in the relative stabilization of anions by water and the polarizable interior of the channel or synthetic membrane. The calculated energies of anion–channel interaction, derived from measurements of either permeability or binding, varied as a linear function of inverse ionic radius (1/r), as expected from a Born-type model of ion charging in a medium characterized by an effective dielectric constant of 19. The model predicts that large anions, like SCN, although they experience weaker interactions (relative to Cl) with water and also with the channel, are more permeant than Cl because anion–water energy is a steeper function of 1/r than is the anion–channel energy. These large anions also bind more tightly for the same reason: the reduced energy of hydration allows the net transfer energy (the well depth) to be more negative. This simple selectivity mechanism that governs permeability and binding acts to optimize the function of CFTR as a Cl filter. Anions that are smaller (more difficult to dehydrate) than Cl are energetically retarded from entering the channel, while the larger (more readily dehydrated) anions are retarded in their passage by “sticking” within the channel.
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Halm, D. R., and R. A. Frizzell. "Anion permeation in an apical membrane chloride channel of a secretory epithelial cell." Journal of General Physiology 99, no. 3 (March 1, 1992): 339–66. http://dx.doi.org/10.1085/jgp.99.3.339.
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Single channel currents though apical membrane Cl channels of the secretory epithelial cell line T84 were measured to determine the anionic selectivity and concentration dependence of permeation. The current-voltage relation was rectified with single channel conductance increasing at positive potentials. At 0 mV the single channel conductance was 41 +/- 2 pS. Permeability, determined from reversal potentials, was optimal for anions with diameters between 0.4 and 0.5 nm. Anions of larger diameter had low permeability, consistent with a minimum pore diameter of 0.55 nm. Permeability for anions of similar size was largest for those ions with a more symmetrical charge distribution. Both HCO3 and H2PO4 had lower permeability than the similar-sized symmetrical anions, NO3 and ClO4. The permeability sequence was SCN greater than I approximately NO3 approximately ClO4 greater than Br greater than Cl greater than PF6 greater than HCO3 approximately F much greater than H2PO4. Highly permeant anions had lower relative single channel conductance, consistent with longer times of residence in the channel for these ions. The conductance sequence for anion efflux was NO3 greater than SCN approximately ClO4 greater than Cl approximately I approximately Br greater than PF6 greater than F approximately HCO3 much greater than H2PO4. At high internal concentrations, anions with low permeability and conductance reduced Cl influx consistent with block of the pore. The dependence of current on Cl concentration indicated that Cl can also occupy the channel long enough to limit current flow. Interaction of Cl and SCN within the conduction pathway is supported by the presence of a minimum in the conductance vs. mole fraction relation. These results indicate that this 40-pS Cl channel behaves as a multi-ion pathway in which other permeant anions could alter Cl flow across the apical membrane.
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Steinberg, Benjamin E., Kassidy K. Huynh, Alexandre Brodovitch, Sabrina Jabs, Tobias Stauber, Thomas J. Jentsch, and Sergio Grinstein. "A cation counterflux supports lysosomal acidification." Journal of Cell Biology 189, no. 7 (June 21, 2010): 1171–86. http://dx.doi.org/10.1083/jcb.200911083.
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The profound luminal acidification essential for the degradative function of lysosomes requires a counter-ion flux to dissipate an opposing voltage that would prohibit proton accumulation. It has generally been assumed that a parallel anion influx is the main or only counter-ion transport that enables acidification. Indeed, defective anion conductance has been suggested as the mechanism underlying attenuated lysosome acidification in cells deficient in CFTR or ClC-7. To assess the individual contribution of counter-ions to acidification, we devised means of reversibly and separately permeabilizing the plasma and lysosomal membranes to dialyze the cytosol and lysosome lumen in intact cells, while ratiometrically monitoring lysosomal pH. Replacement of cytosolic Cl− with impermeant anions did not significantly alter proton pumping, while the presence of permeant cations in the lysosomal lumen supported acidification. Accordingly, the lysosomes were found to acidify to the same pH in both CFTR- and ClC-7–deficient cells. We conclude that cations, in addition to chloride, can support lysosomal acidification and defects in lysosomal anion conductance cannot explain the impaired microbicidal capacity of CF phagocytes.
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Fykse, E. M., and F. Fonnum. "Transport of γ-aminobutyrate and l-glutamate into synaptic vesicles. Effect of different inhibitors on the vesicular uptake of neurotransmitters and on the Mg2+-ATPase." Biochemical Journal 276, no. 2 (June 1, 1991): 363–67. http://dx.doi.org/10.1042/bj2760363.
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The uptakes of gamma-aminobutyrate (GABA) and L-glutamate into synaptic vesicles isolated from rat brain were compared with respect to the effects of 4-acetamido-4′-isothiocyanostilbene-2,2′- disulphonic acid (SITS), 4,4′-di-isothiocyanostilbene-2,2′-disulphonic acid (DIDS) and 5-nitro-2-(3-phenylpropylamino)benzoic acid (N144), agents known to block anion channels. The uptake of glutamate was inhibited by low micromolar concentrations of SITS, DIDS and N144. GABA uptake was much less sensitive to these agents than was glutamate uptake. SITS and N144 inhibited the vacuolar H(+)-ATPase of synaptic vesicles to a smaller extent than the glutamate uptake. The uptake of GABA was not affected by the permeant anions Cl- and Br-, whereas the uptake of glutamate was highly stimulated by low concentrations of these ions. The uptakes of both glutamate and GABA were inhibited by similar, but not identical, concentrations of the lipophilic anion SCN-.
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Franciolini, F., and W. Nonner. "Anion-cation interactions in the pore of neuronal background chloride channels." Journal of General Physiology 104, no. 4 (October 1, 1994): 711–23. http://dx.doi.org/10.1085/jgp.104.4.711.
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Background Cl channels in neurons and skeletal muscle are significantly permeable for alkali cations when tested with asymmetrical concentrations of the same salt. Both anion and cation permeation were proposed to require binding of an alkali cation with the pore (Franciolini, F., and W. Nonner. 1987. Journal of General Physiology. 90:453-478). We tested this hypothesis by bilaterally substituting large alkali cations for Na and found no significant changes of unitary conductance at 300 mM symmetrical concentrations. In addition, all organic cations examined were permeant in a salt gradient test (1,000 mM internal@300 mM external), including triethanolamine, benzyltrimethylamine, and bis-tris-propane (BTP, which is divalent at the tested pH of 6.2). Inward currents were detected following substitution of internal NaCl by the Na salts of the divalent anions of phosphoric, fumaric, and malic acid. Zero-current potentials in gradients of the Na and BTP salts of varied anions (propionate, F, Br, nitrate) that have different permeabilities under bi-ionic conditions, were approximately constant, as if the permeation of either cation were coupled to the permeation of the anion. These results rule out our earlier hypothesis of anion permeation dependent on a bound alkali cation, but they are consistent with the idea that the tested anions and cations form mixed complexes while traversing the Cl channel.
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Zhou, Shi-Sheng, Zhan Gao, Ling Dong, Yan-Feng Ding, Xiao-Dong Zhang, Yue-Min Wang, Jian-Ming Pei, Feng Gao, and Xin-Liang Ma. "Anion channels influence ECC by modulating L-type Ca2+ channel in ventricular myocytes." Journal of Applied Physiology 93, no. 5 (November 1, 2002): 1660–68. http://dx.doi.org/10.1152/japplphysiol.00220.2002.
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Anion channels are extensively expressed in the heart, but their roles in cardiac excitation-contraction coupling (ECC) are poorly understood. We, therefore, investigated the effects of anion channels on cardiac ventricular ECC. Edge detection, fura 2 fluorescence measurements, and whole cell patch-clamp techniques were used to measure cell shortening, the intracellular Ca2+ transient, and the L-type Ca2+ current ( I Ca,L) in single rat ventricular myocytes. The anion channel blockers 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and niflumic acid reversibly inhibited the Ca2+ transients and cell shortening in a dose-dependent manner. Comparable results were observed when the majority of the extracellular Cl− was replaced with the relatively impermeant anions glutamate (Glt−) and aspartate (Asp−). NPPB and niflumic acid or the Cl− substitutes did not affect the resting intracellular Ca2+ concentration but significantly inhibited I Ca,L. In contrast, replacement of extracellular Cl− with the permeant anions NO[Formula: see text], SCN−, and Br− supported the ECC and I Ca,L, which were still sensitive to blockade by NPPB. Exposure of cardiac ventricular myocytes to a hypotonic bath solution enhanced the amplitude of cell shortening and supported I Ca,L, whereas hypertonic stress depressed the contraction and I Ca,L. Moreover, cardiac contraction was completely abolished by NPPB (50 μM) under hypotonic conditions. It is concluded that a swelling-activated anion channel may be involved in the regulation of cardiac ECC through modulating L-type Ca2+ channel activity.
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Skott, O., and B. L. Jensen. "Involvement of chloride in renin secretion from isolated rat glomeruli." American Journal of Physiology-Renal Physiology 262, no. 3 (March 1, 1992): F403—F410. http://dx.doi.org/10.1152/ajprenal.1992.262.3.f403.
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The sensitivity of renin release to changes in anion and calcium concentrations was assessed in superfused rat glomeruli with attached juxtaglomerular cells. Isosmotic substitution of Cl-with gluconate (1/12, 1/6, 1/3, 2/3, or total exchange), isethionate (15 or 101 mM), or sulfate (10 mM) inhibited renin release reversibly. Substitution of Cl- with nitrate (101 mM) stimulated renin secretion. Substitution with iodide (15 or 101 mM) had no consistent effect. The stimulation induced by calcium-free solutions was high in May and low in September. In the absence of chloride, the response to calcium-free solution was inhibited similarly all year. In May reintroduction of calcium and chloride stimulated renin release, suggesting that releasable renin had been stockpiled during the exposure to calcium-free solution. In September reintroduction of calcium and chloride inhibited renin release. It is concluded that the renin secretory process has a demand for permeant anions. The stimulation caused by low external calcium involves at least two mechanisms: one is anion sensitive, powerful, varies with the season, and includes a recruitment phenomenon; another is anion insensitive and weak.
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Qu, Zhiqiang, Rodolphe Fischmeister, and Criss Hartzell. "Mouse Bestrophin-2 Is a Bona fide Cl− Channel." Journal of General Physiology 123, no. 4 (March 29, 2004): 327–40. http://dx.doi.org/10.1085/jgp.200409031.
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Bestrophins have recently been proposed to comprise a new family of Cl− channels. Our goal was to test whether mouse bestrophin-2 (mBest2) is a bona fide Cl− channel. We expressed mBest2 in three different mammalian cell lines. mBest2 was trafficked to the plasma membrane as shown by biotinylation and immunoprecipitation, and induced a Ca2+-activated Cl− current in all three cell lines (EC50 for Ca2+ = 230 nM). The permeability sequence was SCN−: I−: Br−: Cl−: F− (8.2: 1.9: 1.4: 1: 0.5). Although SCN− was highly permeant, its conductance was ∼10% that of Cl− and SCN− blocked Cl− conductance (IC50 = 12 mM). Therefore, SCN− entered the pore more easily than Cl−, but bound more tightly than Cl−. Mutations in S79 altered the relative permeability and conductance for SCN− as expected if S79 contributed to an anion binding site in the channel. PSCN/PCl = 8.2 ± 1.3 for wild-type and 3.9 ± 0.4 for S79C. GSCN/GCl = 0.14 ± 0.03 for wild-type and 0.94 ± 0.04 for S79C. In the S79 mutants, SCN− did not block Cl− conductance. This suggested that the S79C mutation altered the affinity of an anion binding site for SCN−. Additional evidence that S79 was located in the conduction pathway was provided by the finding that modification of the sulfhydryl group in S79C with MTSET+ or MTSES− increased conductance significantly. Because the effect of positively and negatively charged MTS reagents was similar, electrostatic interactions between the permeant anion and the channel at this residue were probably not critical in anion selectivity. These data provide strong evidence that mBest2 forms part of the novel Cl− conduction pathway in mBest2-transfected cells and that S79 plays an important role in anion binding in the pore of the channel.
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Qu, Zhiqiang, and Criss Hartzell. "Determinants of Anion Permeation in the Second Transmembrane Domain of the Mouse Bestrophin-2 Chloride Channel." Journal of General Physiology 124, no. 4 (September 27, 2004): 371–82. http://dx.doi.org/10.1085/jgp.200409108.
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Bestrophins have been proposed to constitute a new family of Cl channels that are activated by cytosolic Ca. We showed previously that mutation of serine-79 to cysteine in mouse bestrophin-2 (mBest2) altered the relative permeability and conductance to SCN. In this paper, we have overexpressed various mutant constructs of mBest2 in HEK-293 cells to explore the contributions to anion selectivity of serine-79 and other amino acids (V78, F80, G83, F84, V86, and T87) located in the putative second transmembrane domain (TMD2). Residues selected for mutagenesis were distributed throughout TMD2, but mutations at all positions changed the selectivity. The effects on selectivity were rather modest. Replacement of residues 78, 79, 80, 83, 84, 86, or 87 with cysteine had similar effects: the permeability of the channel to SCN relative to Cl (PSCN/PCl) was decreased three- to fourfold and the relative SCN conductance (GSCN/GCl) was increased five- to tenfold. Side chains at positions 78 and 80 appeared to be situated close to the permeant anion, because the electrostatic charge at these positions affected permeation in specific ways. The effects of charged sulfhydryl-reactive MTS reagents were the opposite in the V78C and F80C mutants and the effects were partially mimicked by substitution of F80 with charged amino acids. In S79T, switching from Cl to SCN caused slow changes in GSCN/GCl (τ = 16.6 s), suggesting that SCN binding to the channel altered channel gating as well as conductance. The data in this paper and other data support a model in which TMD2 plays an important role in forming the bestrophin pore. We suggest that the major determinant in anion permeation involves partitioning of the permeant anion into an aqueous pore whose structural features are rather flexible. Furthermore, anion permeation and gating may be linked.
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Thomas, S. A., and R. I. Hume. "Permeation of both cations and anions through a single class of ATP-activated ion channels in developing chick skeletal muscle." Journal of General Physiology 95, no. 4 (April 1, 1990): 569–90. http://dx.doi.org/10.1085/jgp.95.4.569.
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Micromolar concentrations of extracellular adenosine 5'-triphosphate (ATP) elicit a rapid excitatory response in developing chick skeletal muscle. Excitation is the result of a simultaneous increase in membrane permeability to sodium, potassium, and chloride ions. In the present study we quantify the selectivity of the ATP response, and provide evidence that a single class of ATP-activated ion channels conducts both cations and anions. Experiments were performed on myoballs using the whole-cell patch-clamp technique. We estimated permeability ratios by measuring the shift in reversal potential when one ion was substituted for another. We found that monovalent cations, divalent cations, and monovalent anions all permeate the membrane during the ATP response, and that there was only moderate selectivity between many of these ions. Calcium was the most permeant ion tested. To determine if ATP activates a single class of channels that conducts both cations and anions, or if ATP activates separate classes of cation and anion channels, we analyzed the fluctuations about the mean current induced by ATP. Ionic conditions were arranged so that the reversal potential for cations was +50 mV and the reversal potential for anions was -50 mV. Under these conditions, if ATP activates a single class of channels, ATP should not evoke an increase in noise at the reversal potential of the ATP current. However, if ATP activates separate classes of cation and anion channels, ATP should evoke a significant increase in noise at the reversal potential of the ATP current. At both +40 and -50 mV ATP elicited a clear increase in noise, but at the reversal potential of the ATP current (-5 mV), no increase in noise above background was seen. These results indicate that there is only a single class of excitatory ATP-activated channels, which do not select by charge. Based on analysis of the noise spectrum, the conductance of individual channels is estimated to be 0.2-0.4 pS.
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Beblo, Dolores A., and Richard D. Veenstra. "Monovalent Cation Permeation through the Connexin40 Gap Junction Channel." Journal of General Physiology 109, no. 4 (April 1, 1997): 509–22. http://dx.doi.org/10.1085/jgp.109.4.509.
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The unitary conductances and permeability sequences of the rat connexin40 (rCx40) gap junction channels to seven monovalent cations and anions were studied in rCx40-transfected neuroblastoma 2A (N2A) cell pairs using the dual whole cell recording technique. Chloride salt cation substitutions (115 mM principal salt) resulted in the following junctional maximal single channel current-voltage relationship slope conductances (γj in pS): CsCl (153), RbCl (148), KCl (142), NaCl (115), LiCl (86), TMACl (71), TEACl (63). Reversible block of the rCx40 channel was observed with TBA. Potassium anion salt γj are: Kglutamate (160), Kacetate (160), Kaspartate (158), KNO3 (157), KF (148), KCl (142), and KBr (132). Ion selectivity was verified by measuring reversal potentials for current in rCx40 gap junction channels with asymmetric salt solutions in the two electrodes and using the Goldman-Hodgkin-Katz equation to calculate relative permeabilities. The permeabilities relative to Li+ are: Cs+ (1.38), Rb+ (1.32), K+ (1.31), Na+ (1.16), TMA+ (0.53), TEA+ (0.45), TBA+ (0.03), Cl− (0.19), glutamate− (0.04), and NO3− (0.14), assuming that the monovalent anions permeate the channel by forming ion pairs with permeant monovalent cations within the pore thereby causing proportionate decreases in the channel conductance. This hypothesis can account for why the predicted increasing conductances with increasing ion mobilities in an essentially aqueous channel were not observed for anions in the rCx40 channel. The rCx40 effective channel radius is estimated to be 6.6 Å from a theoretical fit of the relationship of relative permeability and cation radius.
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Pusch, Michael, Uwe Ludewig, Annett Rehfeldt, and Thomas J. Jentsch. "Gating of the voltage-dependent chloride channel CIC-0 by the permeant anion." Nature 373, no. 6514 (February 1995): 527–31. http://dx.doi.org/10.1038/373527a0.
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Kolen, Bettina, Daniel Kortzak, Arne Franzen, and Christoph Fahlke. "An amino-terminal point mutation increases EAAT2 anion currents without affecting glutamate transport rates." Journal of Biological Chemistry 295, no. 44 (August 20, 2020): 14936–47. http://dx.doi.org/10.1074/jbc.ra120.013704.
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Excitatory amino acid transporters (EAATs) are prototypical dual function proteins that function as coupled glutamate/Na+/H+/K+ transporters and as anion-selective channels. Both transport functions are intimately intertwined at the structural level: Secondary active glutamate transport is based on elevator-like movements of the mobile transport domain across the membrane, and the lateral movement of this domain results in anion channel opening. This particular anion channel gating mechanism predicts the existence of mutant transporters with changed anion channel properties, but without alteration in glutamate transport. We here report that the L46P mutation in the human EAAT2 transporter fulfills this prediction. L46 is a pore-forming residue of the EAAT2 anion channels at the cytoplasmic entrance into the ion conduction pathway. In whole-cell patch clamp recordings, we observed larger macroscopic anion current amplitudes for L46P than for WT EAAT2. Rapid l-glutamate application under forward transport conditions demonstrated that L46P does not reduce the transport rate of individual transporters. In contrast, changes in selectivity made gluconate permeant in L46P EAAT2, and nonstationary noise analysis revealed slightly increased unitary current amplitudes in mutant EAAT2 anion channels. We used unitary current amplitudes and individual transport rates to quantify absolute open probabilities of EAAT2 anion channels from ratios of anion currents by glutamate uptake currents. This analysis revealed up to 7-fold increased absolute open probability of L46P EAAT2 anion channels. Our results reveal an important determinant of the diameter of EAAT2 anion pore and demonstrate the existence of anion channel gating processes outside the EAAT uptake cycle.
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Payne, J. A., C. Lytle, and T. J. McManus. "Foreign anion substitution for chloride in human red blood cells: effect on ionic and osmotic equilibria." American Journal of Physiology-Cell Physiology 259, no. 5 (November 1, 1990): C819—C827. http://dx.doi.org/10.1152/ajpcell.1990.259.5.c819.
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In human red blood cells, when chloride was replaced isosmotically with a permeant chaotropic anion of the lyotropic series (NO3, I, or SCN), an immediate and significant loss of cell water was observed. In contrast, replacement of chloride by a substituted monovalent sulfonate, such as methanesulfonate or sulfamate, had no significant effect on cell water. Cell water loss in the presence of lyotropic anions was not the result of hemolysis or cation loss but was associated with a significant fall in the distribution ratios of protons (out/in) and chloride (in/out), suggesting an increase in nondiffusible intracellular negative charges. This hypothesis was examined using the equilibrium dialysis technique of Freedman and Hoffman (J. Gen. Physiol. 74: 157-185, 1979) in which fixed charges are titrated in cells permeabilized by nystatin. The equilibrium concentration ratios (in/out) of potassium, sodium, and chloride were determined at various external pH (pHo) values. The point at which anion and cation ratios are equal is the effective isoelectric point for the intracellular charges. In normal chloride-containing medium at 24 degrees C, this point was found at a pHo of 6.93. When chloride was replaced by a chaotropic anion, the isoelectric point at 24 degrees C shifted to a lower pHo: NO3 (6.38), I (5.98), and SCN (5.70). The substituted monovalent sulfonates had little effect on isoelectric point: methyl sulfate (6.81), sulfamate (7.00), and methanesulfonate (7.07). Calculation of the intracellular charges from titration data, as well as equilibrium distribution studies with [14C]SCN, suggests that lyotropic anion binding to intracellular sites (mainly hemoglobin) is responsible for the observed changes in cell water, cell pH, and chloride distribution.
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Donaldson, P. J., L. K. Chen, and S. A. Lewis. "Effects of serosal anion composition on the permeability properties of rabbit urinary bladder." American Journal of Physiology-Renal Physiology 256, no. 6 (June 1, 1989): F1125—F1134. http://dx.doi.org/10.1152/ajprenal.1989.256.6.f1125.
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This study describes the effects of serosal Cl- and HCO3- substitution on transepithelial Na+ transport and basolateral membrane properties of the rabbit urinary bladder. Replacement of Cl- with NO3-, SCN-, and Br- had no effect on transepithelial Na+ transport or the basolateral membrane potential (Vbl). However, gluconate, isethionate, and cyclamate (anions that were shown previously to be not as permeable as Cl- through the basolateral membrane anion channel), decreased transepithelial Na+ transport and depolarized Vbl. Replacement of HCO3- also produced a decrease in transepithelial Na+ transport and a depolarization of Vbl. Utilizing conventional and K+-specific microelectrodes, we found the depolarization to be due to a reduction in basolateral K+ conductance. This depolarization was reversible only when Cl- was returned to the serosal solution, the normally permeant anion NO3- being unable to affect repolarization, suggesting that both the K+ and Cl- conductance are reduced during depolarization. A lag period of some 4 min preceded the repolarization of Vbl. The Na+-H+ exchange blocker amiloride prolonged the lag phase associated with repolarization, whereas niflumic acid, a Cl-(-)HCO3- exchange blocker (in red blood cells) reduced the magnitude of Vbl repolarization. Because of the possible involvement of the exchangers it is believed that the lag phase represents a volume-dependent and/or pH-dependent reactivation of the basolateral membrane conductances.
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Steinberg, Benjamin E., Alexandre Brodovitch, Kassidy K. Huynh, and Sergio Grinstein. "ROLE OF THE CYSTIC FIBROSIS TRANSMEMBRANE REGULATOR (CFTR) CHLORIDE CHANNEL IN MACROPHAGE LYSOSOME ACIDIFICATION." Clinical & Investigative Medicine 31, no. 4 (August 1, 2008): 23. http://dx.doi.org/10.25011/cim.v31i4.4828.
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Background: Lysosome acidification is the result of proton pumping by thevacuolar-type ATPase (V-ATPase). Because the V-ATPase is electrogenic, a substantial lysosomal membrane potential can develop if left uncompensated by counterions. An increasing membrane potential will oppose further proton pumping, limiting the acidification. It has generally been assumed that a parallel anion influx accompanies proton pumping to enable acidification. Indeed, defective anion channel function in cystic fibrosis (CF) has been suggested as the mechanism underlying attenuated lysosomal acidification and impaired microbial killing in the lung (Di A, et al., 2006, Nature Cell Biol. 8, 933-944). Chronic lung inflammation and infection represent the major source of morbidity and mortality in CF, and understanding the mechanism underlying the disease will therefore have far-reaching therapeutic implications. As such, it is important to accurately evaluate the reported role of CFTR in lysosome acidification. Methods: To assess the individual contribution of counterions to acidification, we carried out cytosolic and lysosomal ion substitution experiments in intact cells while monitoring lysosomal pH by ratiometric imaging. Results: Replacement of cytosolic Cl^- with impermeant anions did not alter the rate or extent of proton pumping. In contrast, permeant luminalcations were required for normal acidification. Because cations are the main counterion for lysosomal proton uptake, defects in the lysosomal pH are not anticipated in CF cells. Accordingly, the lysosomes of CFTR-deficient alveolar macrophages were found to acidify normally. Conclusion: We conclude that cations are the primary counterions responsible for lysosomal acidification and that defects in lysosomal anion conductance cannot explain the impaired microbicidal capacity of CF phagocytes.
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Berkowitz, L. R. "Loop diuretic and anion modification of NEM-induced K transport in human red blood cells." American Journal of Physiology-Cell Physiology 258, no. 4 (April 1, 1990): C622—C629. http://dx.doi.org/10.1152/ajpcell.1990.258.4.c622.
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The thioalkylating agent N-ethylmaleimide (NEM) causes ouabain-insensitive K loss from human red blood cells. This K loss is inhibited when intracellular Cl is replaced by another permeant anion or when loop diuretics are placed in the incubation medium after NEM exposure. In this report, we have tested the possibility that Cl replacement or loop diuretics not only influence the transport of K induced by NEM but also the interaction of NEM with its target sulfhydryl group. This possibility was examined by replacing intracellular Cl or exposing the cells to loop diuretics before NEM exposure, then measuring K loss in a Cl medium free of loop diuretics. We found that such pretreatment with either Cl substitution or loop diuretics stimulated, rather than inhibited, NEM-induced K loss. This enhancement was not additive in that the increase in K loss induced by anion substitution was not increased further when loop diuretics were also present. These data suggest that anion substitution and loop diuretics enhance the interaction of NEM with its cellular target but inhibit the K loss induced by NEM.
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Wilson, Martin, and Evanna Gleason. "An unusual voltage-gated anion channel found in the cone cells of the chicken retina." Visual Neuroscience 6, no. 1 (January 1991): 19–23. http://dx.doi.org/10.1017/s0952523800000870.
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AbstractUsing the whole-cell patch clamp technique, we have examined the voltage-gated currents present in adult chicken cone cells. When calcium and calcium-gated currents are blocked, hyperpolarizing voltage steps elicit slowly increasing inward currents as has been shown for photoreceptors in other species. Unlike the case for other species, chicken cones appear to lack the inward-rectifying cationic current Ih that contributes to the shaping of the photovoltage. Instead of Ih, these cones possess an anionic inward-rectifying current that in kinetics, activation range and probably function is remarkably similar to Ih. This anion channel is unusual in that both nitrate and acetate are more permeant than chloride ions.
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Musch, M. W., T. R. Leffingwell, and L. Goldstein. "Band 3 modulation and hypotonic-stimulated taurine efflux in skate erythrocytes." American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 266, no. 1 (January 1, 1994): R65—R74. http://dx.doi.org/10.1152/ajpregu.1994.266.1.r65.
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Previous studies have shown that exposure of skate erythrocytes to hypotonic medium or isosmotic medium with permeant solutes such as ethylene glycol or ammonium chloride increases cell volume. Initial swelling is followed by a regulatory volume decrease accomplished by efflux of cell solutes, including the beta-amino acid taurine. Taurine efflux, as well as the cell volume recovery, is inhibited by stilbene disulfonates and other anion exchange inhibitors, suggesting involvement of the erythrocyte anion exchanger band 3. In the present study we show that binding of the stilbene dihydro 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (H2DIDS) to intact cells doubles after exposure to one-half hypotonic medium. Binding also increases after exposure of erythrocytes to medium of one-third hypotonic medium and ethylene glycol medium, but DIDS binding is not significantly elevated in ammonium chloride medium. To investigate possible mechanisms for modulation of band 3, cells were labeled with 32PO4 and levels of protein phosphorylation determined. Phosphorylation of three proteins of molecular masses of 99, 65, and 32 kDa increases with hypotonic stress. Because the anion exchanger migrates with molecular mass near 100 kDa, the anion exchanger was immunoprecipitated. Hypotonic stress induces a rapid and persistent increase in the 32PO4 phosphorylation of the exchanger. This modification may be responsible for the increased DIDS binding and could be related to stimulated taurine efflux.
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Laver, Derek R., and Katherine M. Bradley. "Disulfonic stilbene permeation and block of the anion channel from the sarcoplasmic reticulum of rabbit skeletal muscle." American Journal of Physiology-Cell Physiology 290, no. 6 (June 2006): C1666—C1677. http://dx.doi.org/10.1152/ajpcell.00299.2005.
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Block of a sarcoplasmic reticulum anion channel (SCl channel) by disulfonic stilbene derivatives [DIDS, dibenzamidostilbene-2,2′-disulfonic acid (DBDS), and 4,4′-dinitrostilbene-2,2′-disulfonic acid (DNDS)] was investigated in planar bilayers using SO[Formula: see text] as the conducting ion. All molecules caused reversible voltage-dependent channel block when applied to either side of the membrane. DIDS also produced nonreversible channel block from both sides within 1–3 min. Reversible inhibition was associated with a decrease in channel open probability and mean open duration but not with any change in channel conductance. The half inhibitory concentration for cis- and trans-inhibition had voltage dependencies with minima of 190 nM and 33 μM for DBDS and 3.4 and 55 μM for DNDS. Our data supports a permeant blocker mechanism, in which stilbenes block SCl channels by lodging in the permeation pathway, where they may dissociate to either side of the membrane and thus permeate the channel. The stilbenes acted as open channel blockers where the binding of a single molecule occludes the channel. DBDS and DNDS, from opposite sides of the membrane, competed for common sites on the channel. Dissociation rates exhibited biphasic voltage dependence, indicative of two dissociation processes associated with ion movement in opposite directions within the trans-membrane electric field. The kinetics of DNDS and DBDS inhibition predict that there are two stilbene sites in the channel that are separated by 14–24 Å and that the pore constriction is ∼10 Å in diameter.
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Rubaiy, Hussein N., and Paul Linsdell. "Location of a permeant anion binding site in the cystic fibrosis transmembrane conductance regulator chloride channel pore." Journal of Physiological Sciences 65, no. 3 (February 12, 2015): 233–41. http://dx.doi.org/10.1007/s12576-015-0359-6.
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Nguyen, Dung, Hwoi Kwon, and Tsung-Yu Chen. "Divalent Cation Modulation of Ion Permeation in TMEM16 Proteins." International Journal of Molecular Sciences 22, no. 4 (February 23, 2021): 2209. http://dx.doi.org/10.3390/ijms22042209.
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Intracellular divalent cations control the molecular function of transmembrane protein 16 (TMEM16) family members. Both anion channels (such as TMEM16A) and phospholipid scramblases (such as TMEM16F) in this family are activated by intracellular Ca2+ in the low µM range. In addition, intracellular Ca2+ or Co2+ at mM concentrations have been shown to further potentiate the saturated Ca2+-activated current of TMEM16A. In this study, we found that all alkaline earth divalent cations in mM concentrations can generate similar potentiation effects in TMEM16A when applied intracellularly, and that manipulations thought to deplete membrane phospholipids weaken the effect. In comparison, mM concentrations of divalent cations minimally potentiate the current of TMEM16F but significantly change its cation/anion selectivity. We suggest that divalent cations may increase local concentrations of permeant ions via a change in pore electrostatic potential, possibly acting through phospholipid head groups in or near the pore. Monovalent cations appear to exert a similar effect, although with a much lower affinity. Our findings resolve controversies regarding the ion selectivity of TMEM16 proteins. The physiological role of this mechanism, however, remains elusive because of the nearly constant high cation concentrations in cytosols.
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Serrano, José R., Xuehong Liu, Erik R. Borg, Christopher S. Alexander, C. Frank Shaw, and David C. Dawson. "CFTR: Ligand Exchange between a Permeant Anion ([Au(CN)2]−) and an Engineered Cysteine (T338C) Blocks the Pore." Biophysical Journal 91, no. 5 (September 2006): 1737–48. http://dx.doi.org/10.1529/biophysj.105.078899.
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Amorim, J. B. O., M. A. Bailey, R. Musa-Aziz, G. Giebisch, and G. Malnic. "Role of luminal anion and pH in distal tubule potassium secretion." American Journal of Physiology-Renal Physiology 284, no. 2 (February 1, 2003): F381—F388. http://dx.doi.org/10.1152/ajprenal.00236.2002.
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Potassium secretory flux ( J K) by the distal nephron is regulated by systemic and luminal factors. In the present investigation, J K was measured with a double-barreled K+ electrode during paired microperfusion of superficial segments of the rat distal nephron. We used control solutions (100 mM NaCl, pH 7.0) and experimental solutions in which Cl− had been replaced with a less permeant anion and/or pH had been increased to 8.0. J K increased when Cl− was replaced by either acetate (∼37%), sulfate (∼32%), or bicarbonate (∼62%), and also when the pH of the control perfusate was increased (∼26%). The majority (80%) of acetate-stimulated J K was Ba2+sensitive, but furosemide (1 mM) further reduced secretion (∼10% of total), suggesting that K+-Cl− cotransport was operative. Progressive reduction in luminal Cl−concentration from 100 to 20 to 2 mM caused increments in J K that were abolished by inhibitors of K+-Cl− cortransport, i.e., furosemide and [(dihydroindenyl)oxy]alkanoic acid. Increasing the pH of the luminal perfusion fluid also increased J K even in the presence of Ba2+, suggesting that this effect cannot be accounted for only by K+ channel modulation of K+ secretion in the distal nephron of the rat. Collectively, these data suggest a role for K+-Cl− cotransport in distal nephron K+ secretion.
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Stewart, A. K., M. N. Chernova, Y. Z. Kunes, and S. L. Alper. "Regulation of AE2 anion exchanger by intracellular pH: critical regions of the NH2-terminal cytoplasmic domain." American Journal of Physiology-Cell Physiology 281, no. 4 (October 1, 2001): C1344—C1354. http://dx.doi.org/10.1152/ajpcell.2001.281.4.c1344.
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The role of intracellular pH (pHi) in regulation of AE2 function in Xenopus oocytes remains unclear. We therefore compared AE2-mediated 36Cl− efflux from Xenopus oocytes during imposed variation of extracellular pH (pHo) or variation of pHi at constant pHo. Wild-type AE2-mediated 36Cl−efflux displayed a steep pHo vs. activity curve, with pHo(50) = 6.91 ± 0.04. Sequential NH2-terminal deletion of amino acid residues in two regions, between amino acids 328 and 347 or between amino acids 391 and 510, shifted pHo(50) to more acidic values by nearly 0.6 units. Permeant weak acids were then used to alter oocyte pHi at constant pHo and were shown to be neither substrates nor inhibitors of AE2-mediated Cl−transport. At constant pHo, AE2 was inhibited by intracellular acidification and activated by intracellular alkalinization. Our data define structure-function relationships within the AE2 NH2-terminal cytoplasmic domain, which demonstrates distinct structural requirements for AE2 regulation by intracellular and extracellular protons.
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Bekar, Lane K., and Wolfgang Walz. "Evidence for Chloride Ions as Intracellular Messenger Substances in Astrocytes." Journal of Neurophysiology 82, no. 1 (July 1, 1999): 248–54. http://dx.doi.org/10.1152/jn.1999.82.1.248.
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Cultured rat hippocampal astrocytes were used to investigate the mechanism underlying the suppression of Ba2+-sensitive K+ currents by GABAAreceptor activation. Muscimol application had two effects on whole cell currents: opening of the well-known Cl− channel of the GABAA receptor and a secondary longer-lasting blockade of outward K+ currents displaying both peak and plateau phases. This blockade was independent of both Na+ (inside and outside) and ATP in the pipette. It also seemed to be independent of muscimol binding to the receptor because picrotoxin application showed no effect on the K+ conductance. The effect is blocked when anion efflux is prevented by replacing Cl−with gluconate (both inside and out) and is enhanced with more permeant anions such as Br− and I−. Moreover, the effect is reproduced in the absence of muscimol by promoting Cl− efflux via lowering of extracellular Cl−levels. These results, along with the requirement for Cl−efflux in muscimol experiments, show a strong dependency of the secondary blockade on Cl− efflux through the Cl− channel of the GABAA receptor. We therefore conclude that changes in the intracellular Cl−concentration alter the outward K+ conductances of astrocytes. Such a Cl−-mediated modulation of an astrocytic K+ conductance will have important consequences for the progression of spreading depression through brain tissue and for astrocytic swelling in pathological situations.
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Yi, Sheng, Fernando Pierucci-Alves, and Bruce D. Schultz. "Transforming growth factor-β1 impairs CFTR-mediated anion secretion across cultured porcine vas deferens epithelial monolayer via the p38 MAPK pathway." American Journal of Physiology-Cell Physiology 305, no. 8 (October 15, 2013): C867—C876. http://dx.doi.org/10.1152/ajpcell.00121.2013.
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The goal of this study was to determine whether transforming growth factor-β1 (TGF-β1) affects epithelial cells lining the vas deferens, an organ that is universally affected in cystic fibrosis male patients. In PVD9902 cells, which are derived from porcine vas deferens epithelium, TGF-β1 exposure significantly reduced short-circuit current ( Isc) stimulated by forskolin or a cell membrane-permeant cAMP analog, 8-pCPT-cAMP, suggesting that TGF-β1 affects targets of the cAMP signaling pathway. Electrophysiological results indicated that TGF-β1 reduces the magnitude of current inhibited by cystic fibrosis transmembrane conductance regulator (CFTR) channel blockers. Real-time RT-PCR revealed that TGF-β1 downregulates the abundance of mRNA coding for CFTR, while biotinylation and Western blot showed that TGF-β1 reduces both total CFTR and apical cell surface CFTR abundance. These results suggest that TGF-β1 causes a reduction in CFTR expression, which limits CFTR-mediated anion secretion. TGF-β1-associated attenuation of anion secretion was abrogated by SB431542, a TGF-β1 receptor I inhibitor. Signaling pathway studies showed that the effect of TGF-β1 on Isc was reduced by SB203580, an inhibitor of p38 mitogen-activated protein kinase (MAPK). TGF-β1 exposure also increased the amount of phospho-p38 MAPK substantially. In addition, anisomycin, a p38 MAPK activator, mimicked the effect of TGF-β1, which further suggests that TGF-β1 affects PVD9902 cells through a p38 MAPK pathway. These observations suggest that TGF-β1, via TGF-β1 receptor I and p38 MAPK signaling, reduces CFTR expression to impair CFTR-mediated anion secretion, which would likely compound the effects associated with mild CFTR mutations and ultimately would compromise male fertility.
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Halperin, J. A., C. Brugnara, M. T. Tosteson, T. Van Ha, and D. C. Tosteson. "Voltage-activated cation transport in human erythrocytes." American Journal of Physiology-Cell Physiology 257, no. 5 (November 1, 1989): C986—C996. http://dx.doi.org/10.1152/ajpcell.1989.257.5.c986.
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We report here the effects of membrane potential on the permeability of the human erythrocyte to Na, K, and Ca. Membrane potential was changed either by varying the K concentration gradient in the presence of valinomycin or by varying the concentration gradient of the permeant anion nitrate in the presence of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. When the membrane potential was changed from inside negative (-10 mV) to inside positive (greater than 40 mV), influx, efflux, and net flux of Na and K increased. Marked net cation loss and cell shrinkage occurred in the absence of a chemical gradient for Na and K. This voltage-dependent increase in Na and K conductance is partially inhibited by 10 microM ruthenium red and persists when the membrane potential is returned to -10 mV after transient exposure to inside-positive potentials. A similar voltage-dependent behavior was found for Ca influx. The voltage-activated Ca influx is almost completely inhibited by 10 microM ruthenium red.
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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.
Full textAbstract:
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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Mangoo-Karim, R., M. Ye, D. P. Wallace, J. J. Grantham, and L. P. Sullivan. "Anion secretion drives fluid secretion by monolayers of cultured human polycystic cells." American Journal of Physiology-Renal Physiology 269, no. 3 (September 1, 1995): F381—F388. http://dx.doi.org/10.1152/ajprenal.1995.269.3.f381.
Full textAbstract:
We have investigated the hypothesis that active anion transport drives fluid secretion by the cystic epithelium in autosomal dominant polycystic kidney disease (ADPKD). We prepared monolayers of a primary culture derived from cystic tissue removed from ADPKD patients. The monolayers were grown on permeant supports, and fluid secretion was initiated by forskolin. The results were compared with those obtained with monolayers of Madin-Darby canine kidney (MDCK) cells, known to secrete Cl-. In the absence of the agonist, ADPKD monolayers absorbed fluid (0.20 +/- 0.02 microliter.cm surface area-2.h-1). Forskolin reversed this to secretion (0.60 +/- 0.03 microliter.cm-2.h-1). Control MDCK monolayers did not transport fluid in either direction, but forskolin induced secretion (0.48 +/- 0.03 microliter.cm-2.h-1). The electrical properties of the monolayers were monitored in Ussing chambers. Forskolin increased the transepithelial potential difference (Vte) of ADPKD monolayers (-0.9 +/- 0.1 to -1.1 +/- 0.1 mV) and the short-circuit current (Isc) (6.6 +/- 0.7 to 9.2 +/- 0.8 microA/cm2). The transepithelial resistance (Rte) fell (156 +/- 9 to 138 +/- 10 omega.cm2). Similar results were obtained with MDCK monolayers. The polarity of Vte and the direction of the Isc are compatible with the hypothesis that active secretion of anion drives fluid secretion. Basolateral application of the Na-K-2Cl cotransporter, bumetanide, reduced forskolin-stimulated fluid secretion by ADPKD monolayers (0.56 +/- 0.05 to 0.28 +/- 0.03), depolarized Vte, and inhibited Isc without affecting Rte. Apical application of the Cl- channel blocker, diphenylamine-2-carboxylate, also inhibited fluid secretion by ADPKD monolayers (0.65 +/- 0.03 to 0.27 +/- 0.02 microliter.cm-2.h-1).(ABSTRACT TRUNCATED AT 250 WORDS)
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Takuma, T., T. Ichida, K. Okumura, Y. Sasaki, and M. Kanazawa. "Effects of valinomycin on osmotic lysis of zymogen granules and amylase exocytosis from parotid acini." American Journal of Physiology-Gastrointestinal and Liver Physiology 264, no. 5 (May 1, 1993): G895—G901. http://dx.doi.org/10.1152/ajpgi.1993.264.5.g895.
Full textAbstract:
The role of osmotic swelling of the secretory granules in adenosine 3',5'-cyclic monophosphate (cAMP)-mediated amylase exocytosis was evaluated by use of isolated zymogen granules and saponin-permeabilized acini of the rat parotid gland. The osmotic lysis of the isolated granules was markedly enhanced by the addition of valinomycin (> 10(-9) M) in the presence of isosmotic KSCN or KI medium. However, valinomycin (up to 10(-5) M) did not increase the granule lysis in KCl medium, although the granules were slightly less stable in KCl medium than in K2SO4 or potassium gluconate medium. Guanosine 5'-O-(3-thiotriphosphate) did not affect the granule lysis. Valinomycin alone had no effect on amylase release from saponin-permeabilized parotid acini incubated in KCl medium, but completely abolished cAMP-mediated amylase release in all K+ media used. The inhibition was clearly detected at 0.1 microM valinomycin in KCl medium, not blocked by the addition of 1 mM MgATP to the medium, and was greatly reduced in NaCl medium. cAMP-evoked amylase release was completely inhibited by SCN- and I- (permeant anions), the mean inhibitory dosages of which were approximately 25 and 50 mM, respectively. These results suggest that 1) the membrane of parotid zymogen granules has no detectable Cl- channels responsible for osmotic swelling of the granules, and 2) increase in K+ or anion conductance of the granule does not enhance but inhibits cAMP-mediated amylase exocytosis from parotid acini.
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O'Donnell, M. J., J. A. Dow, G. R. Huesmann, N. J. Tublitz, and S. H. Maddrell. "Separate control of anion and cation transport in malpighian tubules of Drosophila Melanogaster." Journal of Experimental Biology 199, no. 5 (May 1, 1996): 1163–75. http://dx.doi.org/10.1242/jeb.199.5.1163.
Full textAbstract:
Microelectrode measurements of basal, apical and transepithelial potentials in the Malpighian tubules of Drosophila melanogaster were obtained under a range of conditions in order to investigate whether each of the three main second messenger systems known to act in the tubules (cyclic AMP, cyclic GMP and Ca2+) acted specifically on either cation or anion transport, or whether they activated both systems. Ion-selective microelectrode determinations of K+ concentration and pH of secreted fluid allowed the role of each signalling system to be analysed further. Stimulation with cyclic nucleotides markedly alters the potential profile across principal cells through the selective activation of an apical electrogenic V-ATPase. By contrast, manipulation of extracellular chloride levels, combined with stimulation with leucokinin, does not affect the potential profile across the principal cells, showing that chloride must pass through another route. The cell-permeant Ca2+ chelator BAPTA-AM was shown to suppress the action of leucokinins (insect peptides that induce rapid fluid secretion), but not those of cyclic AMP, the neuronally derived insect peptide cardioacceleratory peptide 2b (CAP2b) or its intracellular messenger cyclic GMP. This shows that leucokinins act through Ca2+ and not through cyclic nucleotides and that the cyclic nucleotide pathways do not co-activate the intracellular Ca2+ pathway to exert their effects. Taken together, these results show that leucokinin acts through intracellular Ca2+, independently of cyclic AMP or cyclic GMP, to raise the chloride permeability of the epithelium. By contrast, either cyclic AMP or cyclic GMP (upon CAP2b stimulation) acts on the electrogenic cation-transporting apical V-ATPase, with only a negligible effect on anion conductance and without perturbing intracellular [Ca2+]. There is thus a clear functional separation between the control pathways acting on cation and anion transport in the tubules. Given the evidence from D. melanogaster and other species that chloride does not pass through the principal cells, we speculate that these two pathways may also be physically separated within cell subtypes of the tubules.
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Cymes, Gisela D., and Claudio Grosman. "Identifying the elusive link between amino acid sequence and charge selectivity in pentameric ligand-gated ion channels." Proceedings of the National Academy of Sciences 113, no. 45 (October 10, 2016): E7106—E7115. http://dx.doi.org/10.1073/pnas.1608519113.
Full textAbstract:
Among neurotransmitter-gated ion channels, the superfamily of pentameric ligand-gated ion channels (pLGICs) is unique in that its members display opposite permeant-ion charge selectivities despite sharing the same structural fold. Although much effort has been devoted to the identification of the mechanism underlying the cation-versus-anion selectivity of these channels, a careful analysis of past work reveals that discrepancies exist, that different explanations for the same phenomenon have often been put forth, and that no consensus view has yet been reached. To elucidate the molecular basis of charge selectivity for the superfamily as a whole, we performed extensive mutagenesis and electrophysiological recordings on six different cation-selective and anion-selective homologs from vertebrate, invertebrate, and bacterial origin. We present compelling evidence for the critical involvement of ionized side chains—whether pore-facing or buried—rather than backbone atoms and propose a mechanism whereby not only their charge sign but also their conformation determines charge selectivity. Insertions, deletions, and residue-to-residue mutations involving nonionizable residues in the intracellular end of the pore seem to affect charge selectivity by changing the rotamer preferences of the ionized side chains in the first turn of the M2 α-helices. We also found that, upon neutralization of the charged residues in the first turn of M2, the control of charge selectivity is handed over to the many other ionized side chains that decorate the pore. This explains the long-standing puzzle as to why the neutralization of the intracellular-mouth glutamates affects charge selectivity to markedly different extents in different cation-selective pLGICs.
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Matsumoto, Takayuki, Keisuke Takayanagi, Mihoka Kojima, Kumiko Taguchi, and Tsuneo Kobayashi. "Acute Exposure to Indoxyl Sulfate Impairs Endothelium-Dependent Vasorelaxation in Rat Aorta." International Journal of Molecular Sciences 20, no. 2 (January 15, 2019): 338. http://dx.doi.org/10.3390/ijms20020338.
Full textAbstract:
Gut microbiota are emerging as potential contributors to the regulation of host homeostasis. Dysbiosis of the gut microbiota associated with increased intestinal permeability facilitates the passage of endotoxins and other microbial products, including indoxyl sulfate in the circulation. Although an emerging body of evidence has suggested that indoxyl sulfate is a key substance for the development of chronic kidney disease, few studies have investigated the direct association of indoxyl sulfate with vascular function. We hypothesized that indoxyl sulfate adversely affects vascular function. Aortas isolated from male Wistar rat were examined in the presence or absence of indoxyl sulfate to assess the vascular function, including vasorelaxation and vasocontraction. Indoxyl sulfate (vs. vehicle) (1) decreased vasorelaxation induced by acetylcholine (ACh) but not by sodium nitroprusside; (2) had no significant alterations of noradrenaline-induced vasocontraction in the absence and presence of endothelium; (3) decreased adenylyl cyclase activator (forskolin)-induced vasorelaxation, while such a difference was eliminated by endothelial denudation; and (4) decreased vasorelaxations induced by calcium ionophore (A23187) and transient receptor potential vanilloid 4 agonist (GSK1016790A). The indoxyl sulfate-induced decrease in the vasorelaxations induced by ACh and A23187 increased by cell-permeant superoxide dismutase or by organic anion transporter inhibitor. However, apocynin, an inhibitor of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, had no effects on vasorelaxations induced by ACh, A23187, forskolin, and GSK1016790A in the presence of indoxyl sulfate. These results suggest that indoxyl sulfate directly affects the vascular function, particularly, endothelium-dependent vasorelaxation, and this effect may be attributable to increased oxidative stress after cell transportion via organic anion transporter, and such increased oxidative stress may not be attributable to activation of NADPH oxidase activation.
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Reenstra, W. W., and J. G. Forte. "Characterization of K+ and Cl- conductances in apical membrane vesicles from stimulated rabbit oxyntic cells." American Journal of Physiology-Gastrointestinal and Liver Physiology 259, no. 5 (November 1, 1990): G850—G858. http://dx.doi.org/10.1152/ajpgi.1990.259.5.g850.
Full textAbstract:
K+ and Cl- conductance pathways in apical membrane vesicles (SA vesicles) of stimulated oxyntic cells have been characterized. SA vesicles were prepared from rabbit fundic mucosa after stimulation of acid secretion with histamine. Conductive K+ and Cl- fluxes were assayed by several methods: by their effects on pH gradient formation by endogenous H(+)-K(+)-ATPase, by the protonophore-induced dissipation of preformed pH gradients, and by the effects of channel blockers. pH gradient formation by H(+)-K(+)-ATPase required K+ and was greatly reduced when the permeant anion chloride was replaced by gluconate or sulfate. In the presence of 75 mM K+, 1 mM Cl- was sufficient for generation of near maximal pH gradients, as was 5 mM K+ in the presence of 75 mM Cl-. At all K+ and Cl- concentrations tested, the ATP-generated formation of pH gradients was inhibited and the dissipation of these pH gradients stimulated by the protonophore tetrachlorosalicylanilide (TCS). Similar effects of TCS were also seen when Cl- was replaced by impermeant anions. Both processes were blocked by the K+ channel inhibitor Ba2+. The Ki for Ba2+ inhibition of pH gradient formation was 1.5 microM at 5 mM K+ and was proportional to the 3rd power of the K+ concentration. At 75 mM K+ the Cl- channel blocker diphenylamine-2-carboxylate inhibited ATP-dependent pH gradient formation when the Cl- concentration was 1 mM; however, when the Cl- concentration was greater than 5 mM no inhibition was observed. The membrane potential-sensitive dye DISC (5) was used to measure membrane potential generated by K+ gradients.(ABSTRACT TRUNCATED AT 250 WORDS)
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Clarke, L. L., and M. C. Harline. "CFTR is required for cAMP inhibition of intestinal Na+ absorption in a cystic fibrosis mouse model." American Journal of Physiology-Gastrointestinal and Liver Physiology 270, no. 2 (February 1, 1996): G259—G267. http://dx.doi.org/10.1152/ajpgi.1996.270.2.g259.
Full textAbstract:
Acute adenosine 3',5'-cyclic monophosphate (cAMP) stimulation of intestinal epithelium induces net transepithelial Cl- secretion and inhibits neutral coupled NaCl absorption. To investigate the role that the cystic fibrosis transmembrane conductance regulator (CFTR) plays in these events, we measured bioelectric changes and radioisotopic NaCl flux across jejunal tissues from gene-targeted cftr "knockout" mice [cftr(-/-) homozygotes] and their normal littermates [cftr(+/+) homozygotes and cftr(+/-) heterozygotes]. Before stimulation, the short-circuit current (Isc, an index of Cl- secretion) of the cftr(-/-) jejunum was essentially zero and significantly less than in the cftr(+/+) or cftr(+/-) intestine. Acute cAMP stimulation had little effect on the bioelectric parameters of the cftr(-/-) intestine but induced a marked increase of Isc and decrease of total tissue conductance in both the cftr(+/+) and cftr(+/-) intestine. Differences in the magnitude of the cAMP-induced Isc between the cftr(+/+) and cftr(+/-) intestine were only observed when the cell-to-lumen anion concentration gradient was maximized by removal of permeant anions from the luminal bath. Radioisotope flux measurements revealed that Na+ and Cl- were absorbed equally across the cftr(-/-) jejunum under basal conditions. In cftr(+/+) and cftr(+/-) intestine, Na+ was absorbed at a similar rate, but net Cl- absorption was reduced from that in cftr(-/-) intestine by an amount approximating the Isc. Acute cAMP stimulation of the cftr(+/+) and cftr(+/-) intestine abolished net NaCl absorption and induced electrogenic Cl- secretion. In contrast, net NaCl absorption was unchanged from the preceding flux period in the cftr(-/-) jejunum. The data suggest that CFTR not only mediates cAMP-induced transepithelial Cl- secretion but is also required for cAMP inhibition of neutral NaCl absorption in the intestine.
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Lamsa, Karri, and Kai Kaila. "Ionic Mechanisms of Spontaneous GABAergic Events in Rat Hippocampal Slices Exposed to 4-Aminopyridine." Journal of Neurophysiology 78, no. 5 (November 1, 1997): 2582–91. http://dx.doi.org/10.1152/jn.1997.78.5.2582.
Full textAbstract:
Lamsa, Karri and Kai Kaila. Ionic mechanisms of spontaneous GABAergic events in rat hippocampal slices exposed to 4-aminopyridine. J. Neurophysiol. 78: 2582–2591, 1997. Ion-selective (H+ and K+) microelectrode techniques as well as conventional extra- and intracellular recordings were used to study the ionic mechanisms of propagating spontaneous GABAergic events (SGEs) in rat hippocampal slices exposed to 4-aminopyridine (4-AP, 50–100 μM). All experiments were made in the presence of antagonists of ionotropic glutamate receptors [10 μM 6-nitro-7-sulphamoylbenzoquinoxaline-2,3-dione (NBQX) and 40 μM dl-2-amino-5-phosphonopentanoic acid (AP5)]. The SGEs were composed of a negative-going change in field potential with a temporally coincident increase (0.7 ± 0.3 mM; mean ± SE) in extracellular K+ ([K+]o) and an alkaline transient (0.01–0.08 units) in extracellular pH (pHo) in stratum radiatum of the area CA1. Simultaneous intracellular recordings showed a triphasic hyperpolarization-depolarization–late hyperpolarization response in pyramidal cells. Application of pentobarbital sodium (PB, 100 μM) decreased the interval between SGEs from a mean value of 35 to ∼20 s and shortened the period of refractoriness of stimulus-evoked propagating events. This was accompanied by an increase in the amplitude of the field potential response of the [K+]o and the pHo shifts and of the depolarizing phase of the pyramidal-cell response. The SGEs were completely blocked by the γ-aminobutyric acid-A (GABAA) receptor antagonist, picrotoxin (PiTX; 100 μM). The amplitudes of the negative-going field potential and of the depolarizing phase of the pyramidal-cell response as well as the ionic shifts associated with SGEs were strongly suppressed in the nominal absence of CO2/HCO− 3. There was a five-fold increase in the interevent interval, and propagating SGEs could not be evoked by stimuli given at intervals shorter than ∼2–3 min. Exposure to inhibitors of carbonic anhydrase, benzolamide (BA; 10 μM) or ethoxyzolamide (EZA; 50 μM) fully blocked the alkaline pHo transients and turned them into acid shifts. The poorly membrane-permeant BA had no discernible effect on the other components of the SGEs, but application of EZA had effects reminiscent to those of CO2/HCO− 3-free medium. Addition of the GABAA receptor–permeant weak-acid anion, formate (20 mM) reestablished the SGEs that were first suppressed by exposure to the CO2/HCO− 3-free medium. No SGEs were seen in the presence of a similar concentration of the GABAA receptor–impermeant anion propionate. Unlike the alkaline transients associated with HCO− 3-driven SGEs, those supported by formate were not blocked by BA. The present data suggest that an inward current carried by bicarbonate is necessary for the generation of SGEs and that the GABAA receptor–mediated excitatory coupling among GABAergic interneurons is essentially dependent on the availability of intracellular bicarbonate.
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Herness, M. S. "Neurophysiological and biophysical evidence on the mechanism of electric taste." Journal of General Physiology 86, no. 1 (July 1, 1985): 59–87. http://dx.doi.org/10.1085/jgp.86.1.59.
Full textAbstract:
The phenomenon of electric taste was investigated by recording from the chorda tympani nerve of the rat in response to both electrical and chemical stimulations of the tongue with electrolytes in order to gain some insight into its mechanism on both a neurophysiological and biophysical basis. The maximum neural response levels were identical for an individual salt (LiCl, NaCl, KCl, or CaCl2), whether it was presented as a chemical solution or as an anodal stimulus through a subthreshold solution. These observations support the idea that stimulation occurs by iontophoresis of ions to the receptors at these current densities (less than 100 microA/cm2). Electric responses through dilute HCl were smaller than the chemically applied stimulations, but the integrated anodal responses appeared similar to chemical acid responses, as evidenced by an OFF response to both forms of stimuli. Hydrogen may be more permeant to the lingual epithelium and would thus be shunted away from the taste receptors during anodal stimulation. When the anion of electric taste was varied via subthreshold salt solutions, the response magnitude increased as the mobility of the anion decreased. The transport numbers of the salts involved adequately explains these differences. The physical aspects of ion migration occurring within the adapting fluid on the tongue are also discussed. Direct neural stimulation by the current appears to occur only at higher current densities (greater than 300 microA/cm2). If the taste cells of the tongue were inactivated with either iodoacetic acid (IAA) or N-ethyl maleimide (NEM), or removed with collagenase, then responses from the chorda tympani could be obtained only at these higher current densities. Latency measurements before and after IAA or NEM treatment corroborated these findings. The results are discussed in terms of several proposed mechanisms of electric taste and it is concluded that an ion accumulation mechanism can adequately explain the data.