Impermeant Anion Research Articles

An Isotonic Model of Neuron Swelling Based on Co-Transport of Salt and Water.

Neurons spend most of their energy building ion gradients across the cell membrane. During energy deprivation the neurons swell, and the concomitant mixing of their ions is commonly assumed to lead toward a Donnan equilibrium, at which the concentration gradients of all permeant ion species have the same Nernst potential. This Donnan equilibrium, however, is not isotonic, as the total concentration of solute will be greater inside than outside the neurons. The present theoretical paper, in contrast, proposes that neurons follow a path along which they swell quasi-isotonically by co-transporting water and ions. The final neuronal volume on the path is taken that at which the concentration of impermeant anions in the shrinking extracellular space equals that inside the swelling neurons. At this final state, which is also a Donnan equilibrium, all permeant ions can mix completely, and their Nernst potentials vanish. This final state is isotonic and electro-neutral, as are all intermediate states along this path. The path is in principle reversible, and maximizes the work of mixing.

The Internal Impermeant Anion, Its Mean Valence and Osmolarity-Charge Asymmetry

Background/Aims: For many years experimental and theoretical studies of the processes controlling the transmembrane potential of living cells and their volume were focused on ions, first of all Na+, K+, and Cl-, that can be moved in and out of the cell by various active and passive mechanisms. But recently more and more attention has been directed toward the internal impermeant anion (Xz-) – a complex entity that is comprised of many very different molecules. The most intriguing feature of the internal impermeant anion is that its amount and, importantly, its mean valence can be changed during the metabolic activity of the cell. The aim of this paper is to computationally investigate how changes in the amount and the mean valence of the internalimpermeant anion influence the concentration of the main ions, the membrane potential, and the cell volume. Methods: The computational analyses were performed using our charge-difference model describe earlier. Results and Conclusion: The results of computational simulations confirm previous results that changes in the amount of Xz- influence nothing but the cell volume if z remains constant, although transient disturbances of concentrations and Em happen and their extent depends on the speed of Xz- changes. Changes of z have more serious consequences. A decrease of |z| leads to a decrease of concentrations of cations ([K+]i and [Na+]i), to an increase of the concentrations of anions ([Cl-]I and [Xz-]i), and to depolarization of the cell membrane; an increase of |z| leads to changes in the opposite directions. Interestingly, even in conditions when Xz- remains unchanged, the normal electrophysiological activity expressed in changes of Em will affect [Cl-]i and consequently [Xz-]i (due to volume changes), inducing feedback effects on the cation concentrations and Em. Accordingly, Xz- is viewed as an important but not the only component of a broader concept of osmolarity-charge asymmetry. The basic physical reasons that determine the interrelations between Xz- on one hand and ionic concentrations, Em, and the cell volume on the other hand are discussed.

The different anion transport capability of prodiginine- and tambjamine-like molecules

Prodiginines and tambjamines are anion-selective ionophores capable of facilitating the transport of anions across the plasma membrane in mammalian cells. One of the potential applications of these anionophores is the possibility of employing them as a substitutive therapy for pathologies involving anion channels, as in cystic fibrosis. We have studied the interaction of a large anion as gluconate with three prodiginine- and two tambjamine-like compounds. Apparent dissociation constants for the chloride, iodide and gluconate complexes were estimated from iodide influx experiments in mammalian cells exposed to different extracellular anion combinations. Our experiments indicate that gluconate is not transported by the prodiginines, leaving the anionophores free to transport chloride and iodide. Conversely, gluconate would be transported to some extent by the tambjamines, competing with halides for the anionophores, and consequently reducing their flux. This might be related to the different structural features of both families of compounds. These data have important implications for the selection of impermeable anions in the analysis of the anionophore mechanism.

Biophysical models reveal the relative importance of transporter proteins and impermeant anions in chloride homeostasis.

Fast synaptic inhibition in the nervous system depends on the transmembrane flux of Cl- ions based on the neuronal Cl- driving force. Established theories regarding the determinants of Cl- driving force have recently been questioned. Here, we present biophysical models of Cl- homeostasis using the pump-leak model. Using numerical and novel analytic solutions, we demonstrate that the Na+/K+-ATPase, ion conductances, impermeant anions, electrodiffusion, water fluxes and cation-chloride cotransporters (CCCs) play roles in setting the Cl- driving force. Our models, together with experimental validation, show that while impermeant anions can contribute to setting [Cl-]i in neurons, they have a negligible effect on the driving force for Cl- locally and cell-wide. In contrast, we demonstrate that CCCs are well-suited for modulating Cl- driving force and hence inhibitory signaling in neurons. Our findings reconcile recent experimental findings and provide a framework for understanding the interplay of different chloride regulatory processes in neurons.

Spontaneous and α-adrenoceptor-induced contractility in human collecting lymphatic vessels require chloride.

Human lymphatic vessels are myogenically active and respond to sympathetic stimulation. The role of various cations in this behavior has recently been investigated, but whether the anion Cl- is essential is unclear. With ethical approval and informed consent, human thoracic duct and mesenteric lymphatic vessels were obtained from surgical patients. Spontaneous or norepinephrine-induced isometric force production from isolated vessels was measured by wire myography; the transmembrane Cl- gradient and Cl- channels were investigated by substitution of extracellular Cl- with the impermeant anion aspartate and inhibition of Cl- transport and channels with the clinical diuretics furosemide and bendroflumethiazide as well as DIDS and 5-nitro-2-(3-phenylpropylamino)benzoic acid. The molecular expression of Ca2+-activated Cl- channels was investigated by RT-PCR, and proteins were localized using immunoreactivity. Spontaneous and norepinephrine-induced contractility in human lymphatic vessels was highly abrogated after Cl- substitution with aspartate. About 100-300 µM DIDS or 5-nitro-2-(3-phenylpropylamino)benzoic acid inhibited spontaneous contractile behavior. Norepinephrine-stimulated tone was furthermore markedly abrogated by 200 µM DIDS. Furosemide lowered only spontaneous constrictions, whereas bendroflumethiazide had nonspecific inhibitory effects. Consistent expression of transmembrane member 16A [TMEM16A (anoctamin-1)] was found in both the thoracic duct and mesenteric lymphatic vessels, and immunoreactivity with different antibodies localized TMEM16A to lymphatic smooth muscle cells and interstitial cells. The significant change in contractile function observed with inhibitors and anion substitution suggests that Cl- movement over the plasma membrane of lymphatic myocytes is integral for spontaneous and α-adrenoceptor-evoked contractility in human collecting lymphatic vessels. Consistent detection and localization of TMEM16A to myocytes suggests that this channel could play a major functional role. NEW & NOTEWORTHY In this study, we report the first observations of Cl- being a critical ionic component of spontaneous and agonist-evoked contractility in human lymphatics. The most consistently expressed Ca2+-activated Cl- channel gene in the human thoracic duct and mesenteric lymphatic vessels appears to be transmembrane member 16A, suggesting that this channel plays a major role.

Tenuous Inhibitory GABAergic Signaling in the Reticular Thalamus.

Maintenance of a low intracellular Cl- concentration ([Cl-]i) is critical for enabling inhibitory neuronal responses to GABAA receptor-mediated signaling. Cl- transporters, including KCC2, and extracellular impermeant anions ([A]o) of the extracellular matrix are both proposed to be important regulators of [Cl-]i Neurons of the reticular thalamic (RT) nucleus express reduced levels of KCC2, indicating that GABAergic signaling may produce excitation in RT neurons. However, by performing perforated patch recordings and calcium imaging experiments in rats (male and female), we find that [Cl-]i remains relatively low in RT neurons. Although we identify a small contribution of [A]o to a low [Cl-]i in RT neurons, our results also demonstrate that reduced levels of KCC2 remain sufficient to maintain low levels of Cl- Reduced KCC2 levels, however, restrict the capacity of RT neurons to rapidly extrude Cl- following periods of elevated GABAergic signaling. In a computational model of a local RT network featuring slow Cl- extrusion kinetics, similar to those we found experimentally, model RT neurons are predisposed to an activity-dependent switch from GABA-mediated inhibition to excitation. By decreasing the activity threshold required to produce excitatory GABAergic signaling, weaker stimuli are able to propagate activity within the model RT nucleus. Our results indicate the importance of even diminished levels of KCC2 in maintaining inhibitory signaling within the RT nucleus and suggest how this important activity choke point may be easily overcome in disorders such as epilepsy.SIGNIFICANCE STATEMENT Precise regulation of intracellular Cl- levels ([Cl-]i) preserves appropriate, often inhibitory, GABAergic signaling within the brain. However, there is disagreement over the relative contribution of various mechanisms that maintain low [Cl-]i We found that the Cl- transporter KCC2 is an important Cl- extruder in the reticular thalamic (RT) nucleus, despite this nucleus having remarkably low KCC2 immunoreactivity relative to other regions of the adult brain. We also identified a smaller contribution of fixed, impermeant anions ([A]o) to lowering [Cl-]i in RT neurons. Inhibitory signaling among RT neurons is important for preventing excessive activation of RT neurons, which can be responsible for generating seizures. Our work suggests that KCC2 critically restricts the spread of activity within the RT nucleus.

Extracellular Cl- regulates electrical slow waves and setting of smooth muscle membrane potential by interstitial cells of Cajal in mouse jejunum.

What is the central question of this study? The aim was to investigate the roles of extracellular chloride in electrical slow waves and resting membrane potential of mouse jejunal smooth muscle by replacing chloride with the impermeant anions gluconate and isethionate. What is the main finding and its importance? The main finding was that in smooth muscle cells, the resting Cl- conductance is low, whereas transmembrane Cl- movement in interstitial cells of Cajal (ICCs) is a major contributor to the shape of electrical slow waves. Furthermore, the data confirm that ICCs set the smooth muscle membrane potential and that altering Cl- homeostasis in ICCs can alter the smooth muscle membrane potential. Intracellular Cl- homeostasis is regulated by anion-permeable channels and transporters and contributes to excitability of many cell types, including smooth muscle and interstitial cells of Cajal (ICCs). Our aims were to investigate the effects on electrical activity in mouse jejunal muscle strips of replacing extracellular Cl- (Cl-o ) with the impermeant anions gluconate and isethionate. On reducing Cl-o , effects were observed on electrical slow waves, with small effects on smooth muscle membrane voltage (Em ). Restoration of Cl- hyperpolarized smooth muscle Em proportional to the change in Cl-o concentration. Replacement of 90% of Cl-o with gluconate reversibly abolished slow waves in five of nine preparations. Slow waves were maintained in isethionate. Gluconate and isethionate substitution had similar concentration-dependent effects on peak amplitude, frequency, width at half peak amplitude, rise time and decay time of residual slow waves. Gluconate reduced free ionized Ca2+ in Krebs solutions to 0.13mm. In Krebs solutions containing normal Cl- and 0.13mm free Ca2+ , slow wave frequency was lower, width at half peak amplitude was smaller, and decay time was faster. The transient hyperpolarization following restoration of Cl-o was not observed in W/Wv mice, which lack pacemaker ICCs in the small intestine. We conclude that in smooth muscle cells, the resting Cl- conductance is low, whereas transmembrane Cl- movement in ICCs plays a major role in generation or propagation of slow waves. Furthermore, these data support a role for ICCs in setting smooth muscle Em and that altering Cl- homeostasis in ICCs can alter smooth muscle Em .

New selective inhibitors of calcium-activated chloride channels - T16A(inh) -A01, CaCC(inh) -A01 and MONNA - what do they inhibit?

T16A(inh)-A01, CaCC(inh)-A01 and MONNA are identified as selective inhibitors of the TMEM16A calcium-activated chloride channel (CaCC). The aim of this study was to examine the chloride-specificity of these compounds on isolated resistance arteries in the presence and absence (±) of extracellular chloride. Isolated resistance arteries were maintained in a myograph and tension recorded, in some instances combined with microelectrode impalement for membrane potential measurements or intracellular calcium monitoring using fura-2. Voltage-dependent calcium currents (VDCC) were measured in A7r5 cells with voltage-clamp electrophysiology using barium as a charge carrier. Rodent arteries preconstricted with noradrenaline or U46619 were concentration-dependently relaxed by T16A(inh) -A01 (0.1-10 μM): IC50 and maximum relaxation were equivalent in ±chloride (30 min aspartate substitution) and the T16A(inh) -A01-induced vasorelaxation ±chloride were accompanied by membrane hyperpolarization and lowering of intracellular calcium. However, agonist concentration-response curves ±chloride, with 10 μM T16A(inh) -A01 present, achieved similar maximum constrictions although agonist-sensitivity decreased. Contractions induced by elevated extracellular potassium were concentration-dependently relaxed by T16A(inh)-A01 ±chloride. Moreover, T16A(inh) -A01 inhibited VDCCs in A7r5 cells in a concentration-dependent manner. CaCC(inh) -A01 and MONNA (0.1-10 μM) induced vasorelaxation ±chloride and both compounds lowered maximum contractility. MONNA, 10 μM, induced substantial membrane hyperpolarization under resting conditions. T16A(inh) -A01, CaCC(inh) -A01 and MONNA concentration-dependently relax rodent resistance arteries, but an equivalent vasorelaxation occurs when the transmembrane chloride gradient is abolished with an impermeant anion. These compounds therefore display poor selectivity for TMEM16A and inhibition of CaCC in vascular tissue in the concentration range that inhibits the isolated conductance.

Impact of Cyclin-Dependent Kinase CDK4 Inhibition on Eryptosis

Background/Aims: The cyclin-dependent kinase 4 (CDK4) participates in the regulation of apoptosis of nucleated cells by altering transcriptional regulation of genes governing cell proliferation and cell death. Similar to apoptosis of nucleated cells, erythrocytes may enter eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine (PS) exposure at the cell surface. As mature erythrocytes lack nuclei, acute stimulation of eryptosis cannot result from altered gene expression. Eryptosis is triggered by isotonic cell shrinkage following Cl^- removal (replacement with the impermeant organic anion gluconate) or by oxidative stress (exposure to 0.3 mM tertbutyl-hydroperoxide [tBOOH]). The present study explored whether CDK4 is expressed in erythrocytes and whether the CDK4 inhibitors II (NSC625987) and III (ryuvidine) influence eryptosis. Methods: Western blotting was utilized for determination of the presence of CDK4 protein in human erythrocytes, and FACS analysis to determine Fluo3 fluorescence (reflecting cytosolic Ca²⁺), annexin-V-binding (reflecting PS-exposure) and forward scatter (reflecting cell volume). Results: CDK4 protein was present in human erythrocytes. Cl^- removal was followed by decrease of forward scatter and increase of both annexin-V-binding and Fluo3 fluorescence, an effect significantly curtailed by CDK4 inhibitors II and III. Furthermore, CDK4 inhibition blunted enhanced PS-exposure elicited by tBOOH treatment. Conclusions: The present observations disclose the presence of CDK4 protein in human erythrocytes and the suppression of suicidal erythrocyte death by pharmacological inhibition of CDK4.

Comment on "Local impermeant anions establish the neuronal chloride concentration".

Glykys et al. (Reports, 7 February 2014, p. 670) proposed that cytoplasmic impermeant anions and polyanionic extracellular matrix glycoproteins establish the local neuronal intracellular chloride concentration, [Cl(-)]i, and thereby the polarity of γ-aminobutyric acid type A (GABAA) receptor signaling. The experimental procedures and results in this study are insufficient to support these conclusions. Contradictory results previously published by these authors and other laboratories are not referred to.

Comment on "Local impermeant anions establish the neuronal chloride concentration".

Glykys et al. (Reports, 7 February 2014, p. 670) conclude that, rather than ion transporters, "local impermeant anions establish the neuronal chloride concentration" and thereby determine "the magnitude and direction of GABAAR currents at individual synapses." If this were possible, perpetual ion-motion machines could be constructed. The authors' conclusions conflict with basic thermodynamic principles.

Response to comments on "Local impermeant anions establish the neuronal chloride concentration".

We appreciate the interest in our paper and the opportunity to clarify theoretical and technical aspects describing the influence of Donnan equilibria on neuronal chloride ion (Cl(-)) distributions.

Star players sidelined in chloride homeostasis in neurons.

Star players sidelined in chloride homeostasis in neurons.

Causing Chloride Changes

Imaging of a fluorescent chloride indicator reveals a role for impermeant anions in setting intraneuronal chloride levels.

Local Impermeant Anions Establish the Neuronal Chloride Concentration

Neuronal intracellular chloride concentration [Cl(-)](i) is an important determinant of γ-aminobutyric acid type A (GABA(A)) receptor (GABA(A)R)-mediated inhibition and cytoplasmic volume regulation. Equilibrative cation-chloride cotransporters (CCCs) move Cl(-) across the membrane, but accumulating evidence suggests factors other than the bulk concentrations of transported ions determine [Cl(-)](i). Measurement of [Cl(-)](i) in murine brain slice preparations expressing the transgenic fluorophore Clomeleon demonstrated that cytoplasmic impermeant anions ([A](i)) and polyanionic extracellular matrix glycoproteins ([A](o)) constrain the local [Cl(-)]. CCC inhibition had modest effects on [Cl(-)](i) and neuronal volume, but substantial changes were produced by alterations of the balance between [A](i) and [A](o). Therefore, CCCs are important elements of Cl(-) homeostasis, but local impermeant anions determine the homeostatic set point for [Cl(-)], and hence, neuronal volume and the polarity of local GABA(A)R signaling.

Gibbs-Donnan Potential as a Tool for Membrane Vesicles Polarization

It has been theoretically predicted that under conditions leading to Gibbs-Donnan equilibrium in case when size of one compartment is very different from another (as in system “membrane vesicle/liposomes—incubation medium”) stable transmembrane potential can be formed, which value is sufficient to fit requirement of real transmembrane potential. Four partial cases were considered with different location and charge of impermeable ion and it was concluded that locations of impermeable ions in medium provide stable transmembrane potential with sufficient value of 60 - 70 mV. Potential-sensitive probe, such as DiOC6(3) and oxonol VI, were used to confirm the calculated potential. According to the change in fluorescence level and emission/excitation shift, a stable and relatively high transmembrane potential can be formed if salt of impermeable ion is located in incubation medium. Impermeable cations and anions may be used to create positive and negative transmembrane potential respectively.

Identification of a nonselective cation channel in isolated lens fiber cells that is activated by cell shrinkage

The initiation of lens cataract has long been associated with the development of a membrane "leak" in lens fiber cells that depolarizes the lens intracellular potential and elevates intracellular Na(+) and Ca(2+) concentrations. It has been proposed that the leak observed in cataractous lenses is due to the activation of a nonselective cation (NSC) conductance in the normal electrically tight fiber cells. Studies of the membrane properties of isolated fiber cells using the patch-clamp technique have demonstrated a differentiation-dependent shift in membrane permeability from K(+)-dominated in epithelial and short fiber cells toward larger contributions from anion and NSC conductances as fiber cells elongate. In this study, the NSC conductances in elongating lens fiber cells are demonstrated to be due to at least two distinct classes: a Gd(3+)-sensitive, mechanosensitive channel whose blockade is essential for obtaining viable isolated fiber cells, and a second Gd(3+)-insensitive, La(3+)-sensitive conductance that appears to be activated by cell shrinkage. This second conductance was eliminated by the replacement of extracellular Na(+) with the impermeant cation N-methyl-d-glucamine and was potentiated by both hypertonic stress and isosmotic cell shrinkage evoked by the replacement of extracellular Cl(-) with the impermeant anion gluconate. This additional cation conductance may play a role in normal lens physiology by mediating regulatory volume increase under osmotic or other physiological challenges. Since the inappropriate activation of NSC channels is implicated in the initiation of lens cataract, they represent potential targets for the development of novel anticataract therapies.

Monovalent ions control proliferation of Ehrlich Lettre ascites cells

Channels and transporters of monovalent ions are increasingly suggested as putative anticarcinogenic targets. However, the mechanisms involved in modulation of proliferation by monovalent ions are poorly understood. Here, we investigated the role of K+, Na+, and Cl(-) ions for the proliferation of Ehrlich Lettre ascites (ELA) cells. We measured the intracellular concentration of each ion in G(0), G(1), and S phases of the cell cycle following synchronization by serum starvation and release. We show that intracellular concentrations and content of Na+ and Cl(-) were reduced in the G(0)-G(1) phase transition, followed by an increased content of both ions in S phase concomitant with water uptake. The effect of substituting extracellular monovalent ions was investigated by bromodeoxyuridine incorporation and showed marked reduction after Na+ and Cl(-) substitution. In spectrofluorometric measurements with the pH-sensitive dye BCECF, substitution of Na+ was observed to upregulate the activity of the Na+/H+ exchanger NHE1 as well as of Na+-independent acid extrusion mechanisms, facilitating intracellular pH (pH(i)) recovery after acid loading and increasing pH(i). Results using the potential sensitive dye DiBaC4(3) showed a reduced Cl(-) conductance in S compared with G(1) followed by transmembrane potential (E(m)) hyperpolarization in S. Cl(-) substitution by impermeable anions strongly inhibited proliferation and increased free, intracellular Ca2+ ([Ca2+]i), whereas a more permeable anion had little effect. Western blots showed reduced chloride intracellular channel CLIC1 and chloride channel ClC-2 expression in the plasma membrane in S compared with G(1). Our results suggest that Na+ regulates ELA cell proliferation by regulating intracellular pH while Cl(-) may regulate proliferation by fine-tuning of E(m) in S phase and altered Ca2+ signaling.

A cation counterflux supports lysosomal acidification.

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.

An Acid-loading Chloride Transport Pathway in the Intraerythrocytic Malaria Parasite, Plasmodium falciparum

The intraerythrocytic malaria parasite exerts tight control over its ionic composition. In this study, a combination of fluorescent ion indicators and (36)Cl(-) flux measurements was used to investigate the transport of Cl(-) and the Cl(-)-dependent transport of "H(+)-equivalents" in mature (trophozoite stage) parasites, isolated from their host erythrocytes. Removal of extracellular Cl(-), resulting in an outward [Cl(-)] gradient, gave rise to a cytosolic alkalinization (i.e. a net efflux of H(+)-equivalents). This was reversed on restoration of extracellular Cl(-). The flux of H(+)-equivalents was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and, when measured in ATP-depleted parasites, showed a pronounced dependence on the pH of the parasite cytosol; the flux was low at cytosolic pH values < 7.2 but increased steeply with cytosolic pH at values > 7.2. (36)Cl(-) influx measurements revealed the presence of a Cl(-) uptake mechanism with characteristics similar to those of the Cl(-)-dependent H(+)-equivalent flux. The intracellular concentration of Cl(-) in the parasite was estimated to be approximately 48 mm in situ. The data are consistent with the intraerythrocytic parasite having in its plasma membrane a 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid-sensitive transporter that, under physiological conditions, imports Cl(-) together with H(+)-equivalents, resulting in an intracellular Cl(-) concentration well above that which would occur if Cl(-) ions were distributed passively in accordance with the parasite's large, inwardly negative membrane potential.