Accumulation Of Intracellular Cl Research Articles

Activity‐dependent intracellular chloride accumulation and diffusion controls GABAA receptor‐mediated synaptic transmission

In the CNS, prolonged activation of GABA(A) receptors (GABA(A)Rs) has been shown to evoke biphasic postsynaptic responses, consisting of an initial hyperpolarization followed by a depolarization. A potential mechanism underlying the depolarization is an acute chloride (Cl(-)) accumulation resulting in a shift of the GABA(A) reversal potential (E(GABA)). The amount of GABA-evoked Cl(-) accumulation and accompanying depolarization depends on presynaptic and postsynaptic properties of GABAergic transmission, as well as on cellular morphology and regulation of Cl(-) intracellular concentration ([Cl(-)](i)). To analyze the influence of these factors on the Cl(-) and voltage behavior, we studied spatiotemporal dynamics of activity-dependent [Cl(-)](i) changes in multicompartmental models of hippocampal cells based on realistic morphological data. Simulated Cl(-) influx through GABA(A) Rs was able to exceed physiological Cl(-) extrusion rates thereby evoking HCO(3)(-) -dependent E(GABA) shift and depolarizing responses. Depolarizations were observed in spite of GABA(A) receptor desensitization. The amplitude of the depolarization was frequency-dependent and determined by intracellular Cl(-) accumulation. Changes in the dendritic diameter and in the speed of GABA clearance in the synaptic cleft were significant sources of depolarization variability. In morphologically reconstructed granule cells subjected to an intense GABAergic background activity, dendritic inhibition was more affected by accumulation of intracellular Cl(-) than somatic inhibition. Interestingly, E(GABA) changes induced by activation of a single dendritic synapse propagated beyond the site of Cl(-) influx and affected neighboring synapses. The simulations suggest that E(GABA) may differ even along a single dendrite supporting the idea that it is necessary to assign E(GABA) to a given GABAergic input and not to a given neuron.

ClC-3 and IClswell are Required for Normal Neutrophil Chemotaxis and Shape Change

Polymorphonuclear leukocytes undergo directed movement to sites of infection, a complex process known as chemotaxis. Extension of the polymorphonuclear leukocyte (PMN) leading edge toward a chemoattractant in association with uropod retraction must involve a coordinated increase/decrease in membrane, redistribution of cell volume, or both. Deficits in PMN phagocytosis and trans-endothelial migration, both highly motile PMN functions, suggested that the anion transporters, ClC-3 and ICl(swell), are involved in cell motility and shape change ( Moreland, J. G., Davis, A. P., Bailey, G., Nauseef, W. M., and Lamb, F. S. (2006) J. Biol. Chem. 281, 12277-12288 ). We hypothesized that ClC-3 and ICl(swell) are required for normal PMN chemotaxis through regulation of cell volume and shape change. Using complementary chemotaxis assays, EZ-TAXIScantrade mark and dynamic imaging analysis software, we analyzed the directed cell movement and morphology of PMNs lacking normal anion transporter function. Murine Clcn3(-/-) PMNs and human PMNs treated with anion transporter inhibitors demonstrated impaired chemotaxis in response to formyl peptide. This included decreased cell velocity and failure to undergo normal cycles of elongation and retraction. Impaired chemotaxis was not due to a diminished number of formyl peptide receptors in either murine or human PMNs, as measured by flow cytometry. Murine Clcn3(-/-) and Clcn3(+/+) PMNs demonstrated a similar regulatory volume decrease, indicating that the ICl(swell) response to hypotonic challenge was intact in these cells. We further demonstrated that ICl(swell) is essential for shape change during human PMN chemotaxis. We speculate that ClC-3 and ICl(swell) have unique roles in regulation of PMN chemotaxis; ICl(swell) through direct effects on PMN volume and ClC-3 through regulation of ICl(swell).

Inhibitory Effects of Chloride on the Activation of Caspase-1, IL-1β Secretion, and Cytolysis by the P2X7 Receptor

The P2X7 receptor (P2X7R) is an ATP-gated cation channel that activates caspase-1 leading to the maturation and secretion of IL-1beta. Because previous studies indicated that extracellular Cl- exerts a negative allosteric effect on ATP-gating of P2X7R channels, we tested whether Cl- attenuates the P2X7R-->caspase-1-->IL-1beta signaling cascade in murine and human macrophages. In Bac1 murine macrophages, substitution of extracellular Cl- with gluconate produced a 10-fold increase in the rate and extent of ATP-induced IL-1beta processing and secretion, while reducing the EC50 for ATP by 5-fold. Replacement of Cl- with gluconate also increased the potency of ATP as an inducer of mature IL-1beta secretion in primary mouse bone marrow-derived macrophages and in THP-1 human monocytes/macrophages. Our observations were consistent with actions of Cl- at three levels: 1) a negative allosteric effect of Cl-, which limits the ability of ATP to gate the P2X7R-mediated cation fluxes that trigger caspase-1 activation; 2) an intracellular accumulation of Cl- via nonselective pores induced by P2X7R with consequential repression of caspase-1-mediated processing of IL-1beta; and 3) a facilitative effect of Cl- substitution on the cytolytic release of unprocessed pro-IL-1beta that occurs with sustained activation of P2X7R. This cytolysis was repressed by the cytoprotectant glycine, permitting dissociation of P2X7R-regulated secretion of mature IL-1beta from the lytic release of pro-IL-1beta. These results suggest that under physiological conditions P2X7R are maintained in a conformationally restrained state that limits channel gating and coupling of the receptor to signaling pathways that regulate caspase-1.

Tetanic stimulation induces short-term potentiation of inhibitory synaptic activity in the rostral nucleus of the solitary tract.

Whole cell recordings from neurons in the rostral nucleus of the solitary tract (rNST) were made to explore the effect of high-frequency tetanic stimulation on inhibitory postsynaptic potentials (IPSPs). IPSPs were elicited in the rNST by local electrical stimulation after pharmacological blockade of excitatory synaptic transmission. Tetanic stimulation at frequencies of 10-30 Hz resulted in sustained hyperpolarizing IPSPs that had a mean amplitude of -68 mV. The hyperpolarization resulted in a decrease in neuronal input resistance and was blocked by the gamma-aminobutyric acid-A (GABAA) antagonist bicuculline. For most of the neurons (n = 87/102), tetanic stimulation resulted in a maximum hyperpolarization immediately after initiation of the tetanic stimulation, but for some neurons the maximum was achieved after three or more consecutive shock stimuli in the tetanic train of stimuli. When the extracellular Ca2+ concentration was reduced, the maximum IPSP amplitude was reached after several consecutive shock stimuli in the tetanic train for all neurons. Tetanic stimulation at frequencies of 30 Hz and higher resulted in IPSPs that were not sustained but decayed to a more positive level of hyperpolarization. In some neurons the decay was sufficient to become depolarizing and resulted in a biphasic IPSP. It was possible to evoke this biphasic IPSP in all the neurons tested if the cells were hyperpolarized to -75 to -85 mV. The ionic mechanism of the depolarizing IPSPs was examined and was found to be due to an elevation of the extracellular K+ concentration and accumulation of intracellular Cl-. Tetanic stimulation increased the mean 80-ms decay time constant of a single shock-evoked IPSP up to 8 s. The length of the IPSP decay time constant was dependent on the duration and frequency of the tetanic stimulation as well as the extracellular Ca2+ concentration. Afferent sensory input to the rNST consists of trains of relatively high-frequency spike discharges similar to the tetanic stimulation frequencies used to elicit the IPSPs in the brain slices. Thus the short-term changes in inhibitory synaptic activity in the slice preparation probably occur in vivo and may play a key role in taste processing by facilitating synaptic integration.

Rapid co‐transport of sodium and chloride ions in giant salivary gland cells of the leech Haementeria ghilianii.

1. Double-barrelled Cl(-)-selective microelectrodes were used to measure the apparent intracellular Cl- activity (aiCl) and membrane potential (Em) of leech salivary gland cells. In standard physiological solution buffered with HEPES (10 mM), intracellular Cl- activity (corrected for interference) was 38 +/- 8 mM (n = 11) compared to a value of 12.8 mM expected for passive Cl- distribution. The mean Em was -49.4 +/- 8.2 mV (n = 21) which was about 27 mV negative to the Cl- equilibrium potential. 2. Removal of external Cl- led to a slow fall in aiCl until a steady-state level of 4-11 mM was reached in 30-60 min. Recovery of aiCl on readdition of external Cl- took only 2-3 min. The uptake followed an exponential time course having a single rate constant of 1.73 +/- 0.1 min-1 (n = 5) whereas the loss appeared to occur in two phases. Changes in external Cl- produced immediate changes in Em which were the opposite of those expected for a high Cl- permeability, i.e. Cl- removal produced an immediate hyperpolarization (3-18 mV) and readdition of Cl- produced a transient depolarization (5-22 mV). 3. The intracellular accumulation of Cl- was dependent on the external Cl- activity. Even when the external Cl- concentration was reduced to 3%, the cells accumulated Cl- against an electrochemical gradient. 4. Cl- accumulation was temperature sensitive (Q10 approximately 2). 5. On removal of external Na+, aiCl fell to a level which was close to that expected for passive distribution. The active reaccumulation of Cl-, after intracellular Cl- depletion, was abolished in the absence of external Na+; aiCl slowly increased to its passive level. Steady-state aiCl or its recovery by Cl(-)-depleted cells was not affected by the absence of K+ in the bathing solution. 6. The reaccumulation of Cl- was not affected by furosemide (1-5 mM), bumetanide (10(-4) M), amiloride (10(-3) M) or 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid (SITS, 10(-4) M). 7. Removal of external Cl- caused a fall in intracellular Na+ activity (aiNa, measured with Na(+)-selective microelectrodes) from 15.9 +/- 6.8 mM (n = 9) to 2.5 +/- 1.3 mM (n = 3). When external Cl- was readded, aiNa rose to 46.5 +/- 6.6 mM (n = 3) before slowly recovering towards its original value. The maximal change in aiNa was 41.7 +/- 4.5 mM (n = 3) and the rate constant for Na+ uptake was 1.8 +/- 0.4 min-1 (n = 3).(ABSTRACT TRUNCATED AT 400 WORDS)

Determinants of the transmembrane distribution of chloride in rat lymphocytes: role of Cl(-)-HCO3- exchange

The purpose of the present experiments was to establish the factors that determine the intracellular Cl- concentration ([Cl-]i) of lymphocytes. Coulometric and isotope equilibration determinations indicated that [Cl-]i was in the range of 70-85 mmol/l cells. Since the membrane potential (Em) of these cells approximates -55 mV, [Cl-]i is severalfold higher than the level expected at electrochemical equilibrium (approximately 16 mM). This suggests that conductive pathways contribute marginally to the distribution of Cl-. Accordingly, altering the force driving conductive Cl- fluxes by manipulating Em had little effect on [Cl-]i. The possible role of electroneutral cation-Cl- cotransport in the accumulation of intracellular Cl- was also assessed. 36Cl- uptake was largely unaffected by omission of extracellular Na+ and K+ or by addition of bumetanide, a potent cotransport inhibitor. Moreover, [Cl-]i remained unaltered for at least 1 h in cells incubated without Na+ or K+ or in the presence of loop diuretics. Thus it appears unlikely that Cl(-)-anion cotransport plays a major role in maintaining [Cl-]i. A vigorous stilbene disulfonate-sensitive anion exchanger was detected in thymocytes. This system constitutes a large fraction of the Cl- flux pathways and is possibly a major contributor to the establishment of [Cl-]i. Accordingly, modifying the force driving Cl- through the exchanger, by altering pH at constant PCO2, resulted in changes in cellular Cl- content and associated changes in cell volume. These effects were markedly reduced in the nominal absence of HCO3- or in the presence of disulfonic stilbene derivatives, suggesting that they are mediated by Cl(-)-HCO3- exchange.(ABSTRACT TRUNCATED AT 250 WORDS)

Intracellular chloride regulation in amphibian dorsal root ganglion neurones studied with ion-selective microelectrodes.

1. Intracellular Cl- activity (aiCl) and membrane potential (Em) were measured in frog dorsal root ganglion neurones (DRG neurones) using double-barrelled Cl- -selective microelectrodes. In standard Ringer solution buffered with HEPES (5 mM), equilibrated with air or 100% O2, the resting membrane potential was -57.7 +/- 1.0 mV and aiCl was 23.6 +/- 1.0 mM (n = 53). The value of aiCl was 2.6 times the activity expected for an equilibrium distribution and the difference between Em and ECl was 25 mV. 2. Removal of external Cl- led to a reversible fall in aiCl. Initial rates of decay and recovery of aiCl were 4.1 and 3.3 mM min-1, respectively. During the recovery of aiCl following return to standard Ringer solution, most of the movement of Cl- occurred against the driving force for a passive distribution. Changes in aiCl were not associated with changes in Em. Chloride fluxes estimated from initial rates of change in aiCl when external Cl- was removed were too high to be accounted for by electrodiffusion. 3. The intracellular accumulation of Cl- was dependent on the extracellular Cl- activity (aoCl). The relationship between aiCl and aoCl had a sigmoidal shape with a half-maximal activation of about 50 mM-external Cl-. 4. The steady-state aiCl depended on the simultaneous presence of extracellular Na+ and K+. Similarly, the active reaccumulation of Cl- after intracellular Cl- depletion was abolished in the absence of either Na+ or K+ in the bathing solution. 5. The reaccumulation of Cl- was inhibited by furosemide (0.5-1 x 10(-3) M) or bumetanide (10(-5) M). The decrease in aiCl observed in Cl- -free solutions was also inhibited by bumetanide. 6. Cell volume changes were calculated from the observed changes in aiCl. Cells were estimated to shrink in Cl- -free solutions to about 75% their initial volume, at an initial rate of 6% min-1. 7. The present results provide direct evidence for the active accumulation of Cl- in DRG neurones. The mechanism of Cl- transport is electrically silent, dependent on the simultaneous presence of external Cl-, Na+ and K+ and inhibited by loop diuretics. It is suggested that a Na+:K+:Cl- co-transport system mediates the active transport of Cl- across the cell membrane of DRG neurones.

GABA A receptors on the cytoplasmic side of the Deiters' neurone plasma membrane: mechanism and functional implications.

A micromethod has been used for studying the passage of 36Cl- ions between two microchambers across the GABA acceptive plasma membrane of the rabbit Deiters' neurone, under various different conditions. The presence of 3.3 mM GABA, steady state intracellular concentration in situ (Okada & Shimada, 1976), on the cytoplasmic side of those membranes prolongs remarkably the time for the achievement of 36Cl- equilibrium across the membranes (from 4 minutes to 30 minutes). The effect appears to be mediated by "intracellularly oriented" GABAA receptor complexes as studied with GABAA antagonists such as bicuculline and picrotoxin. Intracellular GABA is still active in this effect at 10(-6) M. This phenomenon is discussed as, when coupled with the intracellular ATP producing machinery, being of importance for synaptically released GABA hyperpolarizing action on these neurones. The action of intracellular GABA on "internally oriented" receptors appears to involve the exposure of positively charged loci at the cytoplasmic side of the membrane. The consequent accumulation of intracellular Cl- at the membrane inner side would account for the higher overall permeability of those ions in the in----out direction. This circumstance was assessed by blocking the intracellular GABA effect either working at a relatively basic pH (8.4) inside or increasing the ionic strength in the intracellular compartment.

Intracellular chloride activities in the isolated perfused shark rectal gland.

The isolated, perfused shark rectal gland secretes Cl when stimulated with adenosine 3',5'-cyclic monophosphate (cAMP). To investigate the mechanism of secretion, we used Cl-selective and conventional (KCl-filled) microelectrodes to measure the intracellular Cl activity (aClc). Under nonsecreting conditions, the electrical potential difference across the basolateral membrane (psi b) was -78 m V and aClc was 57 mM, a value seven times greater than predicted for electrochemical equilibrium across the basolateral membrane. When theophylline and 8-bromo-cAMP were added to the perfusate, the transglandular electrical potential difference doubled and the rate of fluid secretion increased 20-fold; however, neither psi b nor aClc changed. During both nonsecreting and secreting conditions the intracellular accumulation of Cl results in an electrochemical potential difference favoring Cl exit across the apical cell membrane. The constancy of aClc despite the variation in secretion rate suggests that stimulation is associated with an equivalent enhancement of net Cl movement across both the apical and basolateral membranes. When stimulated glands were perfused with Na-free (choline) Ringer, secretion was abolished and aClc fell toward the value predicted for electrochemical equilibrium. These findings suggest that the "uphill" step in Cl secretion lies at the basolateral membrane, where cellular Cl accumulation probably involves secondary active transport; i.e., Cl entry is driven by an inwardly directed electrochemical potential difference for Na.