Volume-activated taurine permeability in cells of the human erythroleukemic cell line K562

The effects of hypotonic shock on cell volume, taurine influx and efflux were examined in the human erythroleukemic cell line K562. Cells exposed to hypotonic solutions exhibited a regulatory volume decrease (RVD) following rapid increases in cell volume. Cell swelling was associated with a increased taurine influx and efflux. The volume-activated taurine pathway was Na⁺-independent, and increased in parallel with increasing cell volume. The chloride channel blocker, 2,5-dichlorodiphenylamine-2-carboxylic acid (DCDPC), completely blocked the volume-activated taurine influx and efflux, while [dihydroin-denyl]oxy]alkanoic acids (DIOA) and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), an anion exchanger and anion channel blocker, respectively, also inhibited significantly. These results suggest that taurine transport is increased in response to hypotonic stress, which may be mediated via a volume-activated, DCDPC-sensitive anion channel. © 1996 Wiley-Liss, Inc.     

Volume-sensitive taurine efflux from mammary tissue is not obliged to utilize volume-activated anion channels

Cell-swelling, induced by a hyposmotic shock, activates the release of taurine from lactating rat mammary tissue expiants. The degree of stimulation of taurine efflux was dependent upon the extent of cell-swelling. Volume-sensitive taurine release was attenuated by the anion transport inhibitors NPPB, DIOA, DIDS, niflumate, flufenamate, mefenamate and diiodosalicylate but not by salicylate. Cell-swelling, following a hyposmotic challenge, did not increase the unidirectional efflux of radiolabelled I^– or D-asparate from mammary tissue expiants. The results suggest that although mammary tissue expresses a volume-sensitive amino acid transport system which is inhibited by anion transport blockers the pathway has no identity with volume-activated anion channels.     

Osmoregulation of taurine efflux from cultured human breast cancer cells: comparison with volume activated Cl- efflux and regulation by extracellular ATP.

The properties and regulation of volume-activated taurine efflux from MDA-MB-231 and MCF-7 cells have been investigated. Volume-activated taurine release from both cell lines was almost completely inhibited by diidosalicylate. DIDS , was more effective at inhibiting swelling-induced taurine release from MCF-7 than from MDA-MB-231 cells. On the basis of comparing taurine, Cl(-) and I(-) efflux time courses, it appears that volume-activated taurine efflux does not utilize volume-sensitive anion channels in MDA-MB- 231 and MCF-7 cells. Extracellular ATP stimulated volume-activated taurine release from MDA-MB-231 cells but not from MCF-7 cells. The effect of ATP was mimicked by UTP and was dependent upon external calcium and inhibited by suramin. However, suramin inhibited volume-activated taurine efflux from both MDA-MB-231 and MCF-7 cells even in the absence of exogenously added ATP suggesting that it acts directly on the taurine efflux pathway and/or is inhibiting the effect of ATP released from the cells. Volume-activated taurine efflux from MDA-MB-231 cells was stimulated by ionomycin. In contrast, ionomycin had no effect on taurine release from MCF-7 cells. Adenosine also stimulated volume-activated taurine efflux from MDA-MB-231 cells. The results suggest that purines regulate taurine transport in MDA-MB- 231 cells via more than one type of receptor.     

Volume-activated trimethylamine oxide efflux in red blood cells of spiny dogfish (Squalus acanthias)

The aims of this study were to determine the pathway of swelling-activated trimethylamine oxide (TMAO) efflux and its regulation in spiny dogfish (Squalus acanthias) red blood cells and compare the characteristics of this efflux pathway with the volume-activated osmolyte (taurine) channel present in erythrocytes of fishes. The characteristics of the TMAO efflux pathway were similar to those of the taurine efflux pathway. The swelling-activated effluxes of both TMAO and taurine were significantly inhibited by known anion transport inhibitors (DIDS and niflumic acid) and by the general channel inhibitor quinine. Volume expansion by hypotonicity, ethylene glycol, and diethyl urea activated both TMAO and taurine effluxes similarly. Volume expansion by hypotonicity, ethylene glycol, and diethyl urea also stimulated the activity of tyrosine kinases p72syk and p56lyn, although the stimulations by the latter two treatments were less than by hypotonicity. The volume activations of both TMAO and taurine effluxes were inhibited by tyrosine kinase inhibitors, suggesting that activation of tyrosine kinases may play a role in activating the osmolyte effluxes. These results indicate that the volume-activated TMAO efflux occurs via the organic osmolyte (taurine) channel and may be regulated by the volume activation of tyrosine kinases.     

Specificity of the volume-activated amino acid efflux pathway in cultured human breast cancer cells

It has been shown that cell swelling stimulates the efflux of taurine from MCF-7 and MDA-MB-231 cells via a pathway which has channel-like properties. The purpose of this study was to examine the specificity of the volume-activated taurine efflux pathway in both cell lines. A hyposmotic shock increased the efflux of glycine, L-alanine, AIB (α-aminoisobutyric acid), D-aspartate but not L-leucine from MDA-MB-231 and MCF-7 cells. It was evident that the time course of activation/inactivation of those amino acids whose efflux was affected by cell swelling was similar to that of volume-activated taurine efflux. The effect of exogenous ATP on swelling-induced glycine, AIB and D-aspartate efflux from MDA-MB-231 cells was similar to that found on taurine efflux. In addition, volume-activated AIB efflux from MDA-MB-231 cells, like that of swelling-induced taurine efflux, was inhibited by diiodosalicylate. Tamoxifen inhibited volume-activated taurine release from both MDA-MB-231 and MCF-7 cells. The results suggest that neutral and anionic α-amino acids are able to utilize the volume-activated taurine efflux pathway in both cell lines. The effect of tamoxifen on breast cancer growth may, in part, be related to perturbations in cell volume regulation.     

Transport of organic substrates via a volume-activated channel.

We have investigated the volume-activated transport of organic solutes in flounder erythrocytes. Osmotic swelling of cells suspended in a Na(+)-free medium led to increased membrane transport of taurine, glucose, and uridine. For each compound there was a significant lag period (1-2 min at 10 degrees C) between cell swelling and activation of the flux. The volume-activated fluxes of each of the substrates increased in parallel with increasing cell volume, and those of taurine and uridine increased linearly with concentration (up to 19 mM). The volume-activated fluxes of each of the three compounds showed similar sensitivities to a number of anion-selective channel blockers (5-nitro-2-(3-phenylpropylamino)benzoic acid > 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid approximately MK-196 > niflumic acid > furosemide); the IC50 for the inhibition of the volume-activated fluxes by NPPB was around 12 microM. The results are consistent with the hypothesis that the volume-activated transport of organic osmolytes is via a pathway with the characteristics of a volume-activated "chloride channel." This raises the question of whether the transport of organic substrates might represent a physiological role for such channels in other cell types.     

Amino Acids as Osmolytes in the Retina

Amino acids play a role as osmolytes during the regulatory volume decrease subsequent to hyposmotic swelling, but less is known about its role when swelling occurs in isosmotic conditions. In this work we examined the efflux of labelled GABA, taurine and glutamate (traced as D-aspartate) from the chick retina, after isosmotic swelling evoked by KCl-containing solutions, and compared its features to those in hyposmotic swelling. In both conditions, GABA and taurine efflux were more sensitive to swelling than glutamate, as assessed by the activation threshold and the amount released. The amino acid efflux in hyposmotic media was decreased by DIDS, tamoxifen and NPPB, agents acting as Cl channels blockers, which also inhibit the osmosensitive Cl efflux. The component associated with swelling in the KCl-stimulated efflux was assessed by the reduction observed when Cl is replaced by an impermeant anion, or by the influence of hyperosmotic media. GABA and taurine efflux exhibited a large swelling-dependent component, which was lower for D-aspartate. This component was markedly decreased by NPPB, but this was due to an effect of the blocker preventing swelling. These results suggest that the influx of Cl, acting as K counterion, which is responsible for cell swelling, occurs through a pathway sensitive to NPPB, similarly to that activated by hyposmolarity. This finding may be of interest in studies aiming at preventing the cell edema which occurs in a number of pathologies.     

Red cell volume regulation: the pivotal role of ionic strength in controlling swelling-dependent transport systems.

A volume increase of trout erythrocytes can be induced either by beta-adrenergic stimulation of a Na+/H+ antiport in an isotonic medium (isotonic swelling) or by suspending red cells in an hypotonic medium (hypotonic swelling). In both cases cells regulate their volume by a loss of osmolytes via specific pathways. After hypotonic swelling several volume-dependent pathways were activated allowing K+, Na+, taurine and choline to diffuse. All these pathways were fully inhibited by furosemide and inhibitors of the anion exchanger (DIDS, niflumic acid), and the K+ loss was mediated essentially via a 'Cl(-)-independent' pathway. After isotonic swelling, the taurine, choline and Na+ pathways were practically not activated and the K+ loss was strictly 'Cl(-)-dependent'. Thus cellular swelling is a prerequisite for activation of these pathways but, for a given volume increase, the degree of activation and the degree of anion-dependence of the K+ pathway depend on the nature of the stimulus, whether hormonal or by reduction of osmolality. It appears that the pattern of the response induced by hormonal stimulation is not triggered by either cellular cAMP (since it can be reproduced in the absence of hormone by isotonic swelling in an ammonium-containing saline) or by the tonicity of the medium in which swelling occurs since after swelling in an isotonic medium containing urea, the cells adopt the regulatory pattern normally observed after hypotonic swelling. We demonstrated that the stimulus is the change in cellular ionic strength induced by swelling: when ionic strength drops, the cells adopt the hypotonic swelling pattern; when ionic strength increases, the isotonic swelling pattern is activated. To explain this modulating effect of ionic strength a speculative model is proposed, which also allows the integration of two further sets of experimental results: (i) all the volume-activated transport systems are blocked by inhibitors of the anion exchanger and (ii) a Cl(-)-dependent, DIDS-sensitive K+ pathway can be activated in static volume trout red cells (i.e., in the absence of volume increase) by the conformational change of hemoglobin induced by the binding of O2 or CO to the heme.     

Reduction of phospholemman expression decreases osmosensitive taurine efflux in astrocytes

The role of phospholemman (PLM) in taurine and Cl(-) efflux elicited by 30% hyposmotic solution was studied in cultured cerebellar astrocytes with reduced PLM expression by antisense oligonucleotide (AO) treatment. PLM, a substrate for protein kinases (PK) C and A, is a protein that increases an anion current in Xenopus oocytes and forms taurine-selective channels in lipid bilayers. Taurine contributes as an osmolyte to regulatory volume decrease (RVD) and is highly permeable through PLM channels in bilayers. Two antisense oligonucleotides (AO1 and AO2) effectively decreased the expression of the PLM protein by 40% and 30%, respectively, and markedly reduced [(3)H]taurine efflux by 67% and 62%. AO treatment also decreased the osmosensitive release of Cl(-), followed as (125)I. The inhibition of Cl(-) efflux (23% for AO1 and 13% for AO2) was notably lower than for [(3)H]taurine. The contribution of PKC and PKA in the function of PLM was also evaluated in astrocytes. Pharmacological activation or inhibition of PKC and PKA revealed that the osmosensitive taurine efflux is essentially PKC-independent while (125)I efflux is reduced by the PKC blockers H-7 (21%) and G?6983 (41%). The PKA activator forskolin and dbcAMP increased taurine efflux by 66-70% and (125)I efflux by 21-45%. Norepinephrine increased the osmosensitive taurine efflux at about the same extent as dbcAMP and forskolin, and this was reduced by PKA blockers. These results suggest that PLM plays a role in RVD in astrocytes by predominantly influencing taurine fluxes, which are modulated by PKA but not PKC.     

Osmotic swelling activates two pathways for K+ efflux in a rat hepatoma cell line.

The pathways for the efflux of K(+) from osmotically-swollen HTC rat hepatoma cells were investigated using (86)Rb(+) as a tracer for K(+). Exposure of HTC cells to a hypotonic solution (<250 mOsm kg(-1)) resulted in a transient efflux of (86)Rb(+) that reached a maximal value after approximately 1 min, and inactivated within 3 min. This initial (86)Rb(+) efflux was inhibited by charybdotoxin, clotrimazole and Ba(2+), but not by apamin or paxilline, consistent with it being via an intermediate-conductance Ca(2+)-activated K(+) channel. For cells exposed to an extracellular osmolality < 180 mOsm kg(-1) there was an additional (86)Rb(+) efflux component which was slower to activate, taking 4 - 6 min to reach a maximum, and remaining active for > 20 min. The second (86)Rb(+) efflux component was not inhibited by K(+) channel blockers but was inhibited by the anion channel blockers, tamoxifen, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and niflumate. The time-courses for its activation and inactivation, as well as its dependence on the extracellular osmolality, were very similar to those observed for the hypotonically-activated efflux of the organic osmolyte, taurine. The data are consistent with the second component of (86)Rb(+) efflux and the efflux of taurine from osmotically-swollen cells occurring via a common pathway having a marked selectivity for taurine over (86)Rb(+).     

Characterisation of three pathways for osmolyte efflux in human erythroleukemia cells

Cell volume decrease is a key step during differentiation of erythroid cells. This could arise from membrane transporter activation leading to a loss of cell osmolytes; however, the pathways involved are poorly understood. We have characterised Cl(-)-independent K(+) and (3)H-taurine efflux from the erythroleukemia cell line, K562. K(+) efflux (measured using (86)Rb(+)) from pre-loaded cells subjected to hypo-osmotic challenge demonstrated two phases, a rapid increase in K(+) efflux followed by a smaller slower increase. Swelling-activated taurine efflux only demonstrated a single phase. Both phases of K(+) efflux were significantly (P<0.05) blocked by anion channel inhibitor 5-nitro-2-(3-phenypropylamino)-benzoic acid (NPPB). However the antiestrogen, tamoxifen, only inhibited the slow late phase. The initial rapid phase had a higher IC(50) for NPPB inhibition than the slow phase, and was insensitive to protein kinases inhibitors KN-62, wortmannin and PD98059. For the slow K(+) efflux phase, the IC(50) for NPPB inhibition and the inhibition by KN-62, wortmannin, genistein or PD98059, were very similar to those measured for the hypo-osmotically-activated taurine efflux. With NPPB (100 microM) present, the slow K(+) efflux phase was further significantly decreased by the Ca(2+) chelator BAPTA-AM or by the Ca(2+)-activated K(+) channel blockers clotrimazole and charybdotoxin but not by apamin. Thus, at least 3 Cl(-)-independent pathways are involved: (a) a tamoxifen-sensitive and taurine-permeable anion channel; (b) a tamoxifen-insensitive and taurine-impermeable K(+) efflux pathway; and (c) a subtype of Ca(2+)-activated K(+) channel. Any or all of these could be involved in the cell volume decrease associated with differentiation in K562 cells.     

High affinity transport of taurine by the Drosophila aspartate transporter dEAAT2

Excitatory amino acid transporters (EAATs) are structurally related plasma membrane proteins known to mediate the Na(+)/K(+)-dependent uptake of the amino acids l-glutamate and dl-aspartate. In the nervous system, these proteins contribute to the clearance of glutamate from the synaptic cleft and maintain excitatory amino acid concentrations below excitotoxic levels. Two homologues exist in Drosophila melanogaster, dEAAT1 and dEAAT2, which are specifically expressed in the nervous tissue. We previously reported that dEAAT2 shows unique substrate discrimination as it mediates high affinity transport of aspartate but not glutamate. We now show that dEAAT2 can also transport the amino acid taurine with high affinity, a property that is not shared by two other transporters of the same family, Drosophila dEAAT1 and human hEAAT2. Taurine transport by dEAAT2 was efficiently blocked by an EAAT antagonist but not by inhibitors of the structurally unrelated mammalian taurine transporters. Taurine and aspartate are transported with similar K(m) and relative efficacy and behave as mutually competitive inhibitors. dEAAT2 can mediate either net uptake or the heteroexchange of its two substrates, both being dependent on the presence of Na(+) ions in the external medium. Interestingly, heteroexchange only occurs in one preferred substrate orientation, i.e. with taurine transported inwards and aspartate outwards, suggesting a mechanism of transinhibition of aspartate uptake by intracellular taurine. Therefore, dEAAT2 is actually an aspartate/taurine transporter. Further studies of this protein are expected to shed light on the role of taurine as a candidate neuromodulator and cell survival factor in the Drosophila nervous system.     

Activation of ion transport pathways by changes in cell volume.

Swelling-activated K+ and Cl- channels, which mediate RVD, are found in most cell types. Prominent exceptions to this rule include red cells, which together with some types of epithelia, utilize electroneutral [K(+)-Cl-] cotransport for down-regulation of volume. Shrinkage-activated Na+/H+ exchange and [Na(+)-K(+)-2 Cl-] cotransport mediate RVI in many cell types, although the activation of these systems may require special conditions, such as previous RVD. Swelling-activated K+/H+ exchange and Ca2+/Na+ exchange seem to be restricted to certain species of red cells. Swelling-activated calcium channels, although not carrying sufficient ion flux to contribute to volume changes may play an important role in the activation of transport pathways. In this review of volume-activated ion transport pathways we have concentrated on regulatory phenomena. We have listed known secondary messenger pathways that modulate volume-activated transporters, although the evidence that volume signals are transduced via these systems is preliminary. We have focused on several mechanisms that might function as volume sensors. In our view, the most important candidates for this role are the structures which detect deformation or stretching of the membrane and the skeletal filaments attached to it, and the extraordinary effects that small changes in concentration of cytoplasmic macromolecules may exert on the activities of cytoplasmic and membrane enzymes (macromolecular crowding). It is noteworthy that volume-activated ion transporters are intercalated into the cellular signaling network as receptors, messengers and effectors. Stretch-activated ion channels may serve as receptors for cell volume itself. Cell swelling or shrinkage may serve a messenger function in the communication between opposing surfaces of epithelia, or in the regulation of metabolic pathways in the liver. Finally, these transporters may act as effector systems when they perform regulatory volume increase or decrease. This review discusses several examples in which relatively simple methods of examining volume regulation led to the discovery of transporters ultimately found to play key roles in the transmission of information within the cell. So, why volume? Because it's functionally important, it's relatively cheap (if you happened to have everything else, you only need some distilled water or concentrated salt solution), and since it involves many disciplines of experimental biology, it's fun to do.     

Ionic events during the volume response of human peripheral blood lymphocytes to hypotonic media. II. Volume- and time-dependent activation and inactivation of ion transport pathways

Hypotonic dilution of human peripheral blood lymphocytes (PBL) induces large conductive permeabilities for K+ and Cl-, associated with the capacity of the cells to regulate their volumes. When rapid cation leakage is assured by the addition of the ionophore gramicidin, the behavior of the anion conductance pathway can be independently examined. Using this technique it is demonstrated that the volume- induced activation of Cl- transport is triggered at a threshold of approximately 1.15 X isotonic cell volume. If the volume of a cell is increased to this level or above, the Cl- transport system is activated, whereas if the volume of a swollen cell is decreased below the threshold value, the Cl- transport is inactivated. Activation and inactivation are independent of the relative volume changes and of the actual cellular Na+, K+, or Cl- concentrations, as well as of the changes in membrane potential in PBL. When net salt movement and thus volume change are inhibited by specific blockers of K+ transport (e.g., quinine, or Ca2+ depletion), volume-induced Cl- conductance shows a time-dependent inactivation, with a half-time of 5-8 min. The Cl- conductance, when activated, appears to involve an all-or-none response. In contrast, volume-induced K+ conductance is a graded response, with the increase in K+ flux being roughly proportional to the hypotonicity-induced increase in cell volume. The data indicate that during lymphocyte volume response in hypotonic media, anion conductance increases by orders of magnitude, exceeding the K+ conductance, so that the rate of the volume decrease (KCl efflux) is determined by a graded alteration in K+ conductance. When the cell volume approaches the isotonic value, it is stabilized by the inactivation of the anion conductance pathway.     

Regulation of taurine transport in rat hippocampal neurons by hypo-osmotic swelling

Taurine, an important mediator of cellular volume regulation in the central nervous system, is accumulated into neurons and glia by means of a highly specific sodium-dependent membrane transporter. During hyperosmotic cell shrinkage, net cellular taurine content increases as taurine transporter activity is enhanced via elevated gene expression of the transporter protein. In hypo-osmotic conditions, taurine is rapidly lost from cells by means of taurine-conducting membrane channels. We reasoned that changes in taurine transporter activity also might accompany cell swelling to minimize re-accumulation of taurine from the extracellular space. Thus, we determined the kinetic and pharmacological characteristics of neuronal taurine transport and the response to osmotic swelling. Accumulation of radioactive taurine is strongly temperature dependent and occurs via saturable and non-saturable pathways. At concentrations of taurine expected in extracellular fluid in vivo, 98% of taurine accumulation would occur via the saturable pathway. This pathway obeys Michaelis-Menten kinetics with a Km of 30.0 +/- 8.8 microm (mean +/- SE) and Jmax of 2.1 +/- 0.2 nmol/mg protein min. The saturable pathway is dependent on extracellular sodium with an effective binding constant of 80.0 +/- 3.1 mm and a Hill coefficient of 2.1 +/- 0.1. This pathway is inhibited by structural analogues of taurine and by the anion channel inhibitors, 4,4'-diisothiocyanostilbene-2, 2'-disulfonic acid (DIDS) and 5-nitro-2-(3 phenylpropylamino) benzoic acid (NPPB). NPPB, but not DIDS, also reduces the ATP content of the cell cultures. Osmotic swelling at constant extracellular sodium concentration reduces the Jmax of the saturable transport pathway by approximately 48%, increases Kdiff for the non-saturable pathway by 77%, but has no effect on cellular ATP content. These changes in taurine transport occurring in swollen neurons in vivo would contribute to net reduction of taurine content and resulting volume regulation.     

Muscarinic receptor regulation of osmosensitive taurine transport in human SH-SY5Y neuroblastoma cells

The ability of G protein‐coupled receptors to regulate osmosensitive uptake of the organic osmolyte, taurine, into human SH‐SY5Y neuroblastoma cells has been examined. When monitored under isotonic conditions and in the presence of physiologically relevant taurine concentrations (1–100 μM), taurine influx was mediated exclusively by a Na⁺‐dependent, high‐affinity (K_m = 2.5 μM) saturable transport mechanism (V_max = 0.087 nmol/mg protein/min). Reductions in osmolarity of > 20% (attained under conditions of a constant NaCl concentration) resulted in an inhibition of taurine influx (> 30%) that could be attributed to a reduction in V_max, whereas the K_m for uptake remained unchanged. Inclusion of the muscarinic cholinergic agonist, oxotremorine‐M (Oxo‐M), also resulted in an attenuation of taurine influx (EC₅₀～0.7 μM). Although Oxo‐M‐mediated inhibition of taurine uptake could be observed under isotonic conditions (～25–30%), the magnitude of inhibition was significantly enhanced by hypotonicity (～55–60%), a result that also reflected a reduction in the V_max, but not the K_m, for taurine transport. Oxo‐M‐mediated inhibition of taurine uptake was dependent upon the availability of extracellular Ca²⁺ but was independent of protein kinase C activity. In addition to Oxo‐M, inclusion of either thrombin or sphingosine 1‐phosphate also attenuated volume‐dependent taurine uptake. The ability of Oxo‐M to inhibit the influx of taurine was attenuated by 4‐[(2‐butyl‐6,7‐dichloro‐2‐cyclopentyl‐2,3‐dihydro‐1‐oxo‐1H‐inden‐5‐yl)oxy]butanoic acid, an inhibitor of the volume‐sensitive organic osmolyte and anion channel. 4‐[(2‐Butyl‐6,7‐dichloro‐2‐cyclopentyl‐2,3‐dihydro‐1‐oxo‐1H‐inden‐5‐yl)oxy]butanoic acid also prevented receptor‐mediated changes in the efflux and influx of K⁺ under hypoosmotic conditions. The results suggest that muscarinic receptor activation can regulate both the volume‐dependent efflux and uptake of taurine and that these events may be functionally coupled.     

Expression of band 3 anion exchanger induces chloride current and taurine transport: structure-function analysis.

Most, but not all, cell types release intracellular organic solutes (e.g. taurine) in response to cell swelling to achieve cell volume regulation. Although this efflux is blocked by classical inhibitors of the electroneutral anion exchanger band 3 (AE1), it is thought to involve an anion channel. The role of band 3 in volume-dependent taurine transport was determined by expressing, in Xenopus oocytes, band 3 from erythrocytes which do (trout) or do not (mouse) release taurine when swollen. AE1 of both species elicited anion exchange activity, but only trout band 3 showed chloride channel activity and taurine transport. Chimeras constructed from trout and mouse band 3 allowed the identification of some protein domains critically associated with channel activity and taurine transport. The data provide evidence that swelling-induced taurine movements occur via an anion channel which is dependent on, or controlled by, band 3. They suggest the involvement of proteins of the band 3 (AE) family in cell volume regulation.     

Effects of calcium, calmodulin, protein kinase C and protein tyrosine kinases on volume-activated taurine efflux in human erythroleukemia cells.

The effects of calcium, calmodulin, protein kinase C (PKC) and protein tyrosine kinase (PTK) modulators were examined on the volume-activated taurine efflux in the erythroleukemia cell line K562. Exposure to hypoosmotic solution significantly increased taurine efflux and intracellular calcium concentration ([Ca2+]i). The Ca2+ channel blockers La3+ (1 mM), verapamil (200 microM) and nifedipine (100 microM) inhibited the hypoosmotically-induced [Ca2+]i increase by more than 90%, while the volume-activated taurine efflux was inhibited by 61.3 +/- 9.5, 74.1 +/- 9.3 and 38.0 +/- 1.5%, respectively. Furthermore, the calmodulin inhibitors W7 (50 microM) and trifluoperazine (10 microM) and the Ca2+/calmodulin-dependent protein kinase II inhibitor KN-62 (2 microM) significantly blocked the volume-activated taurine efflux by 93.4 +/- 2.7, 77.9 +/- 3.5 and 61.3 +/- 15.8%, respectively. In contrast, the PKC inhibitor staurosporine (200 nM) or the PKC activator phorbol 12-myristate 13-acetate (100 nM) did not have significant effects on the volume-activated taurine efflux. However, pretreatment with PTK inhibitors genistein, tyrphostin A25, and tyrphostin A47 blocked the volume-activated taurine efflux. These results suggest that the volume-activated taurine efflux in K562 cells may not directly involve Ca2+, but may require the presence of calmodulin and/or PTK.     

Characterization of regulatory volume decrease in the THP-1 and HL-60 human myelocytic cell linesn

Exposure to hypotonic stress produces a transient increase in cell volume followed by a regulatory volume decrease (RVD) in both THP-1 and HL-60 cells. In contrast, cells exposed to hypotonic stress in a high K/low Na Hanks' solution not only failed to volume regulate, but displayed a secondary swelling. Thus, while an outward K gradient was required ful KVD, the secondary swelling indicated that hypotonic stress increased permeability in the absence of a negative membrane potential. The K channel blocker quinine (1–4 mM) blocked RVD in both cell types. Gramicidin's ability to overcome the quinine block of RVD indicated that RVD is mediated by a quinine-sensitive cation transport mechanism that is independent of the swelling-induced anion transport mechanism. Barium (1–4 mM), another K channel blocker, slowed the rate of RVD, while 4-aminopyridine, charybdotoxin, tetraethylammonium chloride, tetrabutylammonium chloride, and gadolinium had no effect on RVD. Furthermore, RVD was not mediated by calcium-activated conductances, since it occurred normally in Ca-free medium, in medium containing cadmium, and in BAPTA-loaded cells. Gramicidin produced little or no volume change in isotonic medium, suggesting that basal C1 permeability of both THP-1 and HL-60 cells is low. However, swelling induced an anion efflux pathway that is permeable to both chloride and bromide, but is impermeable to methanesulfonate and glutamate. The anion channel blocker 3,5-diiodosalicylic acid (DISA) antagonized RVD in both cell types. In conclusion, RVD in THP-1 and HL-60 cells is mediated by independent anion and cation transport mechanisms that involve both a DISA-sensitive anion pathway and a quinine-inhibitable K efflux pathway, neither of which requires increases in intra-cellular calcium to be activated. © 1994 wiley-Liss, Inc.     

Glutamate modifies ion conduction and voltage-dependent gating of excitatory amino acid transporter-associated anion channels

Excitatory amino acid transporters (EAATs) mediate two distinct transport processes, a stoichiometrically coupled transport of glutamate, Na+, K+, and H+, and a pore-mediated anion conductance. We studied the anion conductance associated with two mammalian EAAT isoforms, hEAAT2 and rEAAT4, using whole-cell patch clamp recording on transfected mammalian cells. Both isoforms exhibited constitutively active, multiply occupied anion pores that were functionally modified by various steps of the Glu/Na+/H+/K+ transport cycle. Permeability and conductivity ratios were distinct for cells dialyzed with Na(+)- or K(+)-based internal solution, and application of external glutamate altered anion permeability ratios and the concentration dependence of the anion influx. EAAT4 but not EAAT2 anion channels displayed voltage-dependent gating that was modified by glutamate. These results are incompatible with the notion that glutamate only increases the open probability of the anion pore associated with glutamate transporters and demonstrate unique gating mechanisms of EAAT-associated anion channels.