Extravesicular Cl Research Articles

Of Chloride and Quantal Size

The uptake into synaptic vesicles of glutamate—the major excitatory neurotransmitter in brain—depends on the proton electrochemical gradient generated by the vacuolar-type H + -ATPase and is modulated by extravesicular Cl – (see Schweizer). Extravesicular Cl – promotes the acidification of synaptic vesicles, a process that depends on its permeation into the vesicle, and Schenck et al . found that, whereas synaptic vesicles from mice lacking the CLC-3 Cl – channel acidified when exposed to ATP and Cl – , acidification was blunted in vesicles from mice lacking the major vesicular glutamate transporter (VGLUT1). Moreover, introduction of VGLUT1 into liposomes containing an electrogenic bacterial ATP synthase enabled their acidification by extravesicular Cl – . Thus, VGLUT1 itself mediates a major synaptic vesicle Cl – conductance. Like glutamate uptake into synaptic vesicles, glutamate uptake into VGLUT1-containing liposomes showed a biphasic dependence on external Cl – , with uptake greatest at low-mM external Cl – . Furthermore, experiments in which liposomes were preloaded with either 100 mM potassium chloride or 100 mM potassium gluconate revealed that luminal Cl – markedly enhanced ATP-dependent glutamate uptake. The promotion of glutamate uptake by luminal Cl – involved glutamate transport driven by the transmembrane electrical gradient (ΔΨ) generated by proton translocation; however, the transmembrane pH gradient contributed a component to glutamate uptake as well. Noting that the Cl – content of endocytosed synaptic vesicles depends on extracellular Cl – concentration, the authors propose that extracellular Cl – concentration may play a major role in regulating vesicular glutamate uptake and thus the size of individual quanta of neurotransmitter. S. Schenck, S. M. Wojcik, N. Brose, S. Takamori, A chloride conductance in VGLUT1 underlies maximal glutamate loading into synaptic vesicles. Nat. Neurosci. 12 , 156–162 (2009). [PubMed] F. E. Schweizer, Exit chloride, enter glutamate. Nat. Neurosci . 12 , 111–112 (2009). [PubMed]

A chloride conductance in VGLUT1 underlies maximal glutamate loading into synaptic vesicles

Uptake of glutamate into synaptic vesicles is mediated by vesicular glutamate transporters (VGLUTs). Although glutamate uptake has been shown to depend critically on Cl(-), the precise contribution of this ion to the transport process is unclear. We found that VGLUT1, and not ClC-3 as proposed previously, represents the major Cl(-) permeation pathway in synaptic vesicles. Using reconstituted VGLUT1, we found that the biphasic dependence of glutamate transport on extravesicular Cl(-) is a result of the permeation of this anion through VGLUT1 itself. Moreover, we observed that high luminal Cl(-) concentrations markedly enhanced loading of glutamate by facilitation of membrane potential-driven uptake and discovered a hitherto unrecognized transport mode of VGLUT1. Because a steep Cl(-) gradient across the synaptic vesicle membrane exists in endocytosed synaptic vesicles, our results imply that the transport velocity and the final glutamate content are highly influenced, if not determined, by the extracellular Cl(-) concentration.

Characterization of Cl-HCO3 exchange in basolateral membrane of rat distal colon.

Sodium-independent Cl movement (i.e., Cl-anion exchange) has not previously been identified in the basolateral membranes of rat colonic epithelial cells. The present study demonstrates Cl-HCO3 exchange as the mechanism for 36Cl uptake in basolateral membrane vesicles (BLMV) prepared in the presence of a protease inhibitor cocktail from rat distal colon. Studies of 36Cl uptake performed with BLMV prepared with different types of protease inhibitors indicate that preventing the cleavage of the COOH-terminal end of AE2 protein by serine-type proteases was responsible for the demonstration of Cl-HCO3 exchange. In the absence of voltage clamping, both outward OH gradient (pHout/pHin: 7.5/5.5) and outward HCO3 gradient stimulated transient 36Cl uptake accumulation. However, voltage clamping with K-ionophore, valinomycin, almost completely (87%) inhibited the OH gradient-driven 36Cl uptake, whereas HCO3 gradient-driven 36Cl uptake was only partially inhibited (38%). Both electroneutral HCO3 and OH gradient-driven 36Cl uptake were 1) completely inhibited by DIDS, an anion exchange inhibitor, with a half-maximal inhibitory constant (Ki) of approximately 26.9 and 30.6 microM, respectively, 2) not inhibited by 5-nitro-2-(3-phenylpropylamino)benzoic acid(NPPB), a Cl channel blocker, 3) saturated by increasing extravesicular Cl concentration with a Km for Cl of approximately 12.6 and 14.2 mM, respectively, and 4) present in both surface and crypt cells. Intracellular pH (pHi) was also determined with 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-acetomethylester (BCECF-AM) in an isolated superfused crypt preparation. Removal of Cl resulted in a DIDS-inhibitable increase in pHi both in HCO3-buffered and in the nominally HCO3-free buffered solutions (0.28 +/- 0.02 and 0.11 +/- 0.02 pH units, respectively). We conclude that a carrier-mediated electroneutral Cl-HCO3 exchange is present in basolateral membranes and that, in the absence of HCO3, Cl-HCO3 exchange can function as a Cl-OH exchange and regulate pHi across basolateral membranes of rat distal colon.

Iodide and bromide inhibit Ca(2+) uptake by cardiac sarcoplasmic reticulum.

Recent studies indicate that the Ca(2+) permeability of the sarcoplasmic reticulum (SR) can be affected by its anionic environment. Additionally, anions could directly modulate the SR Ca(2+) pump or the movement of compensatory charge across the SR membrane during Ca(2+) uptake or release. To examine the effect of anion substitution on cardiac SR Ca(2+) uptake, fluorometric Ca(2+) measurements and spectrophotometric ATPase assays were used. Ca(2+) uptake into SR vesicles was inhibited in a concentration-dependent manner when Br(-) or I(-) replaced extravesicular Cl(-) (when Br(-) completely replaced Cl(-), uptake velocity was approximately 70% of control; when I(-) completely replaced Cl(-), uptake velocity was approximately 39% of control). Replacement of Cl(-) with SO(2)(-4) had no effect on SR uptake. Although both I(-) and Br(-) inhibited net Ca(2+) uptake, neither anion directly inhibited the SR Ca(2+) pump nor did they increase the permeability of the SR membrane to Ca(2+). Our results support the hypothesis that an anionic current that occurs during SR Ca(2+) uptake is reduced by the substitution of Br(-) or I(-) for Cl(-).

Effect of chronic inflammation on ileal short-chain fatty acid/bicarbonate exchange.

Short-chain fatty acids (SCFA) have been demonstrated to at least partially ameliorate chronic intestinal inflammation. However, whether and how intestinal SCFA absorption may be altered during chronic intestinal inflammation is unknown. A rabbit model of chronic ileitis produced by coccidia was used to determine the effect of chronic inflammation on ileal SCFA/HCO(-)(3) exchange. SCFA/HCO(-)(3) exchange was present in the brush-border membrane (BBM) of villus but not crypt cells from normal rabbit ileum. An anion-exchange inhibitor, DIDS, significantly inhibited SCFA/HCO(-)(3) exchange. Extravesicular Cl(-) did not alter the uptake of SCFA, suggesting that SCFA/HCO(-)(3) exchange is a transport process distinct from Cl(-)/HCO(-)(3) exchange. In chronically inflamed ileum, SCFA/HCO(-)(3) exchange was also present only in BBM of villus cells. The exchanger was sensitive to DIDS and was unaffected by extravesicular Cl(-). However, SCFA/HCO(-)(3) exchange was significantly reduced in villus cell BBM vesicles (BBMV) from chronically inflamed ileum. Kinetic studies demonstrated that the maximal rate of uptake of SCFA, but not the affinity for SCFA, was reduced in chronically inflamed rabbit ileum. These data demonstrate that a distinct SCFA/HCO(-)(3) exchange is present on BBMV of villus but not crypt cells in normal rabbit ileum. SCFA/HCO(-)(3) exchange is inhibited in chronically inflamed rabbit ileum. The mechanism of inhibition is most likely secondary to a reduction in transporter numbers rather than altered affinity for SCFA.

Vesicular L-Glutamate Transporter in Microvesicles from Bovine Pineal Glands: DRIVING FORCE, MECHANISM OF CHLORIDE ANION ACTIVATION, AND SUBSTRATE SPECIFICITY

Pinealocytes, endocrine cells that synthesize and secrete melatonin, possess a large number of synaptic-like microvesicles (MVs) containing the L-glutamate transporter (Moriyama, Y., and Yamamoto, A. (1995) FEBS Lett., 367, 233-236). In this study, the L-glutamate transporter in MVs isolated from bovine pineal glands was characterized as to its driving force, requirement of anions, and substrate specificity. Upon the addition of ATP, the MVs accumulated L-glutamate. The uptake was significantly dependent on the extravesicular Cl- concentration, being negligible in the absence of Cl- and maximum at 2-5 mM and decreasing gradually at 20-100 mM. The membrane potential (inside positive) was maximum at 0-10 mM Cl- and then decreased gradually depending on the Cl- concentration, whereas a pH gradient was practically absent without Cl- and increased gradually up to 100 mM Cl-. Ammonium acetate or nigericin plus K+, a dissipator of a pH gradient, had little effect on or was slightly stimulatory toward the uptake, whereas valinomycin plus K+ inhibited both formation of the membrane potential and the glutamate uptake to similar extents. The ATP- and Cl(-)-dependent glutamate uptake was inhibited by fluoride, iodide, or thiocyanate, without vacuolar H(+)-ATPase being affected. An anion channel blocker, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, similarly inhibited the glutamate uptake in a Cl- protectable manner. Furthermore, ATP- and glutamate-dependent acidification of MVs was observed when 4 mM Cl- was present. Among more than 50 kinds of glutamate analogues tested, only a few compounds, including 1-aminocyclohexane-trans-1,3-dicarboxylic acid, caused similar acidification. A good correlation was observed between the acidification and the inhibition of glutamate uptake by glutamate analogues. These results indicated that 1) the major driving force of the glutamate uptake is the membrane potential, 2) Cl- regulates the glutamate uptake, probably via anion-binding site(s) on the transporter, and 3) the transporter shows strict substrate specificity. Hence, the overall properties of the vesicular glutamate transporter in the MVs well matched those of the synaptic vesicle glutamate transporter. We concluded that the vesicular glutamate transporter, being similar if not identical to the neuronal counterpart, operates in endocrine cells.

Apical membrane Cl-butyrate exchange: Mechanism of short chain fatty acid stimulation of active chloride absorption in rat distal colon

The cellular model of short chain fatty acid stimulation of electroneutral Na-Cl absorption in large intestine proposes that SCFA, following its uptake across the apical membrane, recycles and is coupled to functional Na-H and Cl-short chain fatty acid exchanges. To establish the presence of a Cl-butyrate exchange (used as a model short chain fatty acid), studies of 36Cl and 14C-butyrate uptake across apical membrane vesicles of rat distal colon were performed. An outward butyrate-gradient stimulated transient accumulation of 36Cl uptake that was not inhibited by pH clamping with valinomycin (a K ionophore) and FCCP (a proton ionophore). Outward butyrate-gradient-stimulated 36Cl uptake was inhibited by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) with a half-maximal inhibitory concentration (IC50) of 68.4 microM, and was saturated by both increasing extravesicular Cl concentration (Km for Cl of 26.8 +/- 3.4 mM and a Vmax of 12.4 +/- 0.6 nmol/mg protein x 9 sec) and increasing intravesicular butyrate concentration (Km for butyrate of 5.9 mM and a Vmax for Cl of 5.9 nmol/mg protein x 9 sec). 36Cl uptake was also stimulated by outward gradients of other short chain fatty acids (e.g., propionate, acetate and formate). In contrast, an outward Cl gradient failed to enhance 14C-butyrate uptake. Extravesicular Cl more than extravesicular butyrate enhanced 36Cl efflux from apical membrane vesicles. These studies provide compelling evidence for the presence of an electroneutral, pH-activated, Cl-butyrate exchange which in concert with Na-H exchange is the mechanism by which butyrate stimulates electroneutral Na-Cl absorption.

Cl-HCO3 and Cl-OH exchanges mediate Cl uptake in apical membrane vesicles of rat distal colon

This study describes Cl-HCO3 and Cl-OH exchanges as the mechanism for Cl uptake by apical membrane vesicles (AMV) of rat distal colon. Although HCO3 gradient-stimulated 36Cl uptake was additionally stimulated by the additional presence of a pH gradient, pH gradient-stimulated 36Cl uptake was not further enhanced by a HCO3 gradient. HCO3 gradient-stimulated and OH gradient-stimulated 36Cl uptake was not inhibited by voltage clamping, with K and its ionophore valinomycin, but was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, an anion exchange inhibitor, with an apparent inhibitory constant of 7.8 and 106.0 microM, respectively. Increasing intravesicular OH concentration in the absence of HCO3 (with fixed extravesicular Cl concentration) yielded a sigmoidal curve for 36Cl uptake. In contrast, increasing intravesicular OH concentration in the presence of equimolar intra- and extravesicular HCO3 (25 mM) yielded a saturable hyperbolic curve. Increasing extravesicular Cl concentration saturated both HCO3 gradient-stimulated and OH gradient-stimulated 36Cl uptake, with a kinetic constant for Cl of approximately 11.9 and 22.6 mM, respectively. We conclude that Cl uptake in AMV of rat distal colon occurs via two separate anion (Cl-HCO3 and Cl-OH) exchange processes. We speculate that one of these two anion exchanges may be responsible for transcellular Cl movement, while the other may be important in the regulation of intracellular pH homeostasis.

Ion movements accompanying glutamate uptake into Muller cells

Microsomes formed from rabbit kidney medulla and reconstituted proteoliposomes formed from these microsomes were capable of amiloride-inhibited Na+ transport that was insensitive to valinomycin either with or without K+. This indicated that the Na+ transport process was electroneutral. This Na+ transport process was insensitive to extravesicular Cl− or HCO3− and not stimulated by high intravesicular gradients of K+, Ca2+ or Mg2+, which indicated that the process did not require NaCl or NaHCO3 co-transport or Na+/K+, Na+/Ca2+ or Na+/Mg2+ counter-transport. Na+ uptake into microsomes or proteoliposomes was inhibited by extravesicular K+, Ca2+, Mg2+ or La3+, which indicated that these ions interacted with the Na+-binding site on the transport protein. Na+ uptake into microsomes was stimulated by intravesicular protons and inhibited by extravesicular protons. This suggested that microsomes were capable of Na+-H+ exchange and this was confirmed when Na+ was shown to stimulate H+ efflux from microsomes. The amiloride-inhibited Na+ transporter from medulla microsomes which has been reconstituted into proteoliposomes is most likely a Na+-H+ exchanger. This Na+ transporter was totally insensitive to the uncoupler 1799, either in the presence or absence of valinomycin plus K+ and less sensitive to NH3 than to amiloride. This indicated that amiloride inhibited Na+ transport not merely by acting as a weak-base uncoupler but by directly interacting with the protein responsible for Na+-H+ exchange.

Co2+ as a shift reagent for 35Cl NMR of chloride with vesicles and cells.

Applications of high-resolution 35Cl NMR to the study of chloride in vivo and in vesicles have hitherto been limited by problems of NMR detectability and of resolving internal from external signals. We have characterized the effects of Co2+ on the 35Cl resonance of Cl- in solution and have shown that when added to suspensions of lipid vesicles, Co2+ shifts the 35Cl signal of the extravesicular Cl-, allowing clear resolution and quantitation of two peaks. We have assigned these signals to chloride inside and outside the vesicles. The spectra do not change over a 90-min period, demonstrating the stability of the vesicles in the presence of Co2+. This technique is shown to be applicable to red blood cell ghosts, where intravesicular and extravesicular chloride signals were separated and measured and chloride/sulfate exchange through the band 3 anion transport protein A was followed. In two plant species (an alga and a higher plant), an intracellular Cl- signal can be observed and resolved from the extracellular signal. The intracellular transportable chloride was found to be fully NMR-visible (+/- 5%) in the algal cells. The high steady-state levels of Cl- seen in the alga were consistent with previous work using 36Cl- labeling on a related species [Doblinger, R., & Tromballa, H.W. (1982) Planta 156, 10-15]. Successive spectra acquired after adding Co2+ to Chlorella cells under deenergizing conditions allow us to follow the time course of movement of Cl- out of the cells.

Characterization of Cl− transport in vacuolar membrane vesicles using a Cl−-sensitive fluorescent probe: Reaction kinetic models for voltageand concentration-dependence of Cl− flux

The effects of Cl- concentration and membrane potential (delta psi) on Cl- influx in isolated vesicles of vacuolar membrane (tonoplast) from red beet (Beta vulgaris L.) storage tissue have been characterized using the Cl(-)-sensitive fluorescent probe, 6-methoxy-1-(3-sulfonatopropyl)quinolinium (SPQ). The initial rate of Cl- transport into the vesicles was enhanced both by the imposition of a positive delta psi and by increases in extravesicular Cl- concentration. The kinetic mechanism underlying these responses was investigated by examining the accuracy with which the data could be described by several transport models. A model based on constant field theory yielded a poor description of the data, but satisfactory fits were generated by pseudo-two-state reaction kinetic models based on classical carrier schemes. Fits were equally good when it was assumed that charge translocation accompanied Cl- entry, or when charge was carried by the unloaded transport system, as long as only a single charge is translocated in each carrier cycle. Expansion of the models to three states enabled description of the Cl- concentration dependence of transport by changes in a single, voltage insensitive rate constant which is tentatively identified with Cl- binding at the external surface of the membrane. The derived value of the dissociation constant between Cl- and the transport system is estimated at between 30 and 52 mM.

Na-dependent L-glutamate transport by eel intestinal BBMV: role of K+ and Cl-

L-[3H]glutamate uptake into eel (Anguilla anguilla) intestinal brush-border membrane vesicles (BBMV) was a sigmoidal function of extravesicular Na, suggesting that two or more cations accompanied the amino acid during transport. L-[3H]glutamate influx illustrated the following kinetic constants: apparent membrane binding affinity (Kapp) = 0.80 +/- 0.12 mM; influx velocity (Jmax) = 2.61 +/- 0.31 nmol.mg protein-1.min-1; and permeability coefficient (P) = 0.65 +/- 0.10 microliters.mg protein-1. min-1. Results from the imposition of diffusion potentials across vesicle membranes using K-valinomycin or H-carbonyl-cyanide p-chloromethoxyphenylhydrazone suggested that Na-dependent L-glutamate transport was sensitive to transmembrane electrical potential difference. Extravesicular aspartate was a competitive inhibitor of L-[3H]glutamate influx [inhibitory constant (Ki) = 0.28 +/- 0.04 mM]. Intravesicular K and extravesicular Cl ions enhanced maximal amino acid influx and transient L-glutamate accumulation against a concentration gradient (overshoot). Intravesicular K reduced the Kapp of the membrane to L-glutamate, whereas extravesicular Cl increased L-glutamate Jmax. A model for L-[3H]glutamate transport is suggested involving the cotransport of at least two Na and one L-glutamate that is activated by one intravesicular K ion and at least two extravesicular Cl ions.

Na+-glycine cotransport in canalicular liver plasma membrane vesicles

By use of purified rat canalicular liver plasma membrane (cLPM) vesicles, the present study determined the driving forces for glycine transport across this membrane domain. Initial rates of [3H]glycine uptake (10 microM) in cLPM vesicles were stimulated by an inwardly directed Na+ gradient but not by a K+ gradient. Na+ gradient-dependent uptake of glycine demonstrated cation specificity for Na+, dependence on extravesicular Cl-, stimulation by an intravesicular-negative membrane potential, and inhibition by dissipation of the Na+ gradient with gramicidin D. Na+ gradient-dependent glycine cotransport also demonstrated greater sensitivity to inhibition by sarcosine than 2-(methylamino)-isobutyric acid. Accelerated exchange diffusion of [3H]glycine was demonstrated in the presence of Na+ when cLPM vesicles were preloaded with glycine but not with L-alanine or L-proline. Substrate velocity analysis of net Na+-dependent [3H]glycine uptake over the range of amino acid concentrations from 5 microM to 5 mM demonstrated two saturable transport systems, one of high capacity (2.2 +/- 0.2 nmol.mg protein-1.15 s-1) and low affinity (11.2 +/- 1.7 mM) and one of low capacity (51 +/- 14 pmol.mg protein.15 s-1) and comparatively high affinity (66 +/- 12 microM). These results indicate that, in addition to previously described neutral and anionic amino acid transport systems, Na+ gradient-dependent glycine transport mechanisms are present on the canalicular domain of the liver plasma membrane. These canalicular reabsorptive mechanisms may serve to reclaim some of the glycine generated within the canalicular lumen from the intrabiliary hydrolysis of glutathione.

Reconstituted amiloride-inhibited sodium transporter from rabbit kidney medulla is responsible for Na +-H + exchange

Microsomes formed from rabbit kidney medulla and reconstituted proteoliposomes formed from these microsomes were capable of amiloride-inhibited Na + transport that was insensitive to valinomycin either with or without K +. This indicated that the Na + transport process was electroneutral. This Na + transport process was insensitive to extravesicular Cl − or HCO 3 − and not stimulated by high intravesicular gradients of K +, Ca 2+ or Mg 2+, which indicated that the process did not require NaCl or NaHCO 3 co-transport or Na +/K +, Na +/Ca 2+ or Na +/Mg 2+ counter-transport. Na + uptake into microsomes or proteoliposomes was inhibited by extravesicular K +, Ca 2+, Mg 2+ or La 3+, which indicated that these ions interacted with the Na +-binding site on the transport protein. Na + uptake into microsomes was stimulated by intravesicular protons and inhibited by extravesicular protons. This suggested that microsomes were capable of Na +-H + exchange and this was confirmed when Na + was shown to stimulate H + efflux from microsomes. The amiloride-inhibited Na + transporter from medulla microsomes which has been reconstituted into proteoliposomes is most likely a Na +-H + exchanger. This Na + transporter was totally insensitive to the uncoupler 1799, either in the presence or absence of valinomycin plus K + and less sensitive to NH 3 than to amiloride. This indicated that amiloride inhibited Na + transport not merely by acting as a weak-base uncoupler but by directly interacting with the protein responsible for Na +-H + exchange.

Kinetic analysis of the inhibition of anion transport in sarcoplasmic reticulum vesicles by a disulfonic stilbene derivative. Measurement of the change in chloride-diffusion potential by using a fluorescent cyanine dye.

The characterization of the anion transport system in sarcoplasmic reticulum (SR) vesicles was performed by kinetic analysis of the reversible inhibition of gluconate efflux by a disulfonic stilbene derivative; 4-acetamido-4'-isothiocyano-stilbene-2,2'-disulfonate (SITS). The gluconate efflux in SR vesicles was measured by following the change in Cl- ion-diffusion potential due to the mutual diffusion of Cl- and gluconate- using a potential probe; 3,3'-dipropylthiadicarbocyanine iodide (diS-C3-(5)). The main results are as follows. (1) Gluconate efflux was increased by intravesicular gluconate- and was decreased by both extravesicular gluconate- and extravesicular Cl-. (2) Gluconate efflux was depressed when membrane potential became inside-negative. (3) SITS reacted with the site of the anion transport system from the outside of the vesicles and one molecule of SITS inhibited each transport unit. (4) Extravesicular Cl- competed with SITS at the common site of the anion transport system. The dissociation constants for Cl- and SITS are 70 mM and 0.048 microM, respectively. (5) The inhibition of gluconate efflux by SITS was noncompetitive with both intravesicular gluconate and extravesicular gluconate. These results suggest that the anion transport system in SR vesicles can be described by a carrier-mediated transport model in which anions are transported by a mobile transport site accompanied by a net negative charge.