ATP-dependent Cl Research Articles

Ectopic GABAA receptor β3 subunit determines Cl- / -ATPase and chloride transport in HEK 293FT cells.

Neuronal intracellular chloride concentration ([Cl- ]i ) is a crucial determinant of transmission mediated by the γ-aminobutyric acid type A receptor (GABAA R), which subserves synaptic and extrasynaptic inhibition as well as excitation. The Cl- ion is the main carrier of charge through the GABAA R; however, bicarbonate ions ( ) flowing in the opposite direction can also contribute to the net current. The direction of Cl- and fluxes is determined by the underlying electrochemical gradient, which is controlled by Cl- transporters and channels. Accumulating evidence suggests that active mechanisms of chloride transport across the GABAA R pore can underlie the regulation of [Cl- ]i . Measurement of Cl- / -ATPase activity and Cl- transport in HEK 293FT cells expressing homomeric or heteromeric GABAA R ensembles (α2, β3, or γ2) with fluorescent dye for chloride demonstrated that receptor subtypes containing the β3 subunit show enzymatic activity and participate in GABA-mediated or ATP-dependent Cl- transport. GABA-mediated flow of Cl- ions into and out of the cells occurred for a short time period but then rapidly declined. However, Cl- ion flux was stabilized for a long time period in the presence of ions. The reconstituted β3 subunit isoform, purified as a fusion protein, confirmed that β3 is critical for ATPase; however, only the triplet variant showed the full receptor function. The high sensitivity of the enzyme to γ-phosphate inhibitors led us to postulate that the β3 subunit is catalytic. Our discovery of a GABAA R type that requires ATP consumption for chloride movement provides new insight into the molecular mechanisms of inhibitory signaling.

Dual character of GABA action on Cl–-transport by the reconstituted Cl–/-ATPase from rat brain

The Cl–/-ATPase from the plasma membranes of animal brains is an ATP-consuming Cl–-transporting ATPase P-type that is structurally coupled to GABAA receptors. The aim of work was to study the GABA effect on Cl– transport across liposomal membranes by the reconstituted ATPase under various conditions (i.e. in the absence and in the presence of ATP in the incubation medium). We reconstituted the affinity-purified enzyme in the liposomes with a fluorescent dye for Cl–. The Cl–-transport in proteoliposomes was evaluated from variations in fluorescence. Native-PAGE of the purified enzyme preparation, as well as western blot analysis with an antibody against the GABAAR β3 subunit, showed one band with a molecular mass of 300 kDa. The addition of GABA (100 μM) quickly resulted (1–3 s) in an increase in the Cl– influx into proteoliposomes. The application of ATP (3 mM) resulted in a gradual increase in the Cl– inflow into the proteoliposomes at 0.5–4 min. Vanadate (10 μM) did not change the GABA-induced Cl– inflow into the liposomes, but completely inhibited the ATP-dependent Cl– input. Under conditions where Cl– was previously loaded into proteoliposomes by ATP-dependent Cl– input, the addition of GABA to the incubation medium caused a short-lasting Cl– efflux from the proteoliposomes. However, an additional long-time incubation of these proteoliposomes resulted in the restoration of Cl– accumulation. Pentobarbital, propofol, or diazepam elevate and picrotoxin or furosemide eliminate the effect of GABA on the Cl–-transport processes. These findings indicate a dual nature of GABA action on the Cl–/-ATPase.

Effect of phenol on the GABAAR-coupled Cl−/HCO3−-ATPase from fish brain: An in vitro approach on the enzyme function

Phenol (C6H5OH) has a toxic effect on the central nervous system of animals and humans. The Cl−/HCO3−-ATPase from the plasma membranes of animal brains is the primary active P-type Cl−-transporting system that is coupled to GABAA receptor (GABAAR). In this paper, we used an in vitro approach to assess the effects of phenol (1–500μM) on the functional parameters of the Cl−/HCO3−-ATPase isolated from the fish brain. The enzyme is insensitive to phenol in the presence of Cl− or HCO3− in the incubation medium. By contrast, in the presence of Cl−/HCO3−, phenol inhibits (I50=27μM) both the enzyme activity and its participation in ATP-dependent Cl− transport through the membranes of artificial liposomes. Enriched plasma membranes and purified enzyme preparations were separated using hrCNE-PAGE. The ATPase activity in native gels was detected in the presence of phenol (100μM). Detection of ATPase activity in a purified preparation, showed a native protein of 300kDa, in agreement with western blot analysis with antibodies against GABAAR β3 subunits. SDS-PAGE showed that one subunit with a molecular weight of 56kDa was directly phosphorylated by γ-32P–ATP and dephosphorylated in the presence of phenol. The in vitro approach described in this work allowed the first demonstration that GABAAR-coupled Cl−/HCO3−-ATPase can be a protein marker for assessment of the toxicity of phenolics on the central nervous system.

Cystic fibrosis transmembrane conductance regulator‐mRNA delivery: a novel alternative for cystic fibrosis gene therapy

Cystic fibrosis (CF) is the most frequent lethal genetic disease in the Caucasian population. CF is caused by a defective gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP- and ATP-dependent Cl(-) channel and central regulatory protein in epithelia. CFTR influences the fluid composition of the mucus in the respiratory tract. The most common mutation inducing CF, ΔF508, impairs CFTR processing within the cell and thus prevents functional CFTR expression in the apical membrane. The present study aimed to investigate the functional restoration of CFTR in human CF airway epithelia after transfection with optimized wild-type (wt)CFTR-mRNA. We used primary cultured human nasal epithelial (HNE) cells and the human bronchial epithelial cell line CFBE41o(-) that stably expresses ΔF508-CFTR and carried out transepithelial Ussing chamber measurements after transfection with optimized wtCFTR-mRNA. We confirmed the data obtained using immunofluorescence and protein biochemical approaches. Transfection of the CFBE41o(-) cells with wtCFTR-mRNA restored cAMP-induced CFTR currents similar to the values seen in control cells (16HBE14o(-)). Using immunofluorescence approaches, we demonstrated that a considerable amount of CFTR is located at the apical surface in the CF cells after transfection. Western blot analyses of wtCFTR-mRNA transfected CFBE41o(-) cells confirmed these findings. Furthermore, we demonstrated physiological relevance by using primary cultured HNE cells and showed an almost two-fold increase in the cAMP-stimulated CFTR current after transfection. From these data, we conclude that CFTR-mRNA transfection could comprise a novel alternative for gene therapy to restore impaired CFTR function.

Effect of $$ {\text{HCO}}_3^{-} $$ Ions on the ATP-Dependent GABAA Receptor-Coupled Cl– Channel in Rat Brain Plasma Membranes

We studied the effect of Cl(-) (10-75 mM) and HCO(3)(-) ions (10-25 mM) on the ATP-dependent GABA(A) receptor-coupled Cl(-) channel (Cl(-)-ATPase) in rat brain plasma membranes. The total enzyme activity was detected in the presence of both anions at a Cl(-)/ HCO(3)(-) ratio of 5:1 (Cl(-), HCO(3)(-)-ATPase). Specific inhibitors of P-type transport ATPases (N-ethylmaleimide, o-vanadate, and oligomycin) suppressed Cl(-), HCO(3)(-)-ATPase, while the Cl(-)- and HCO(3)(-)-ATPase activities were low sensitive to these ligands. Bicuculline abolished the activating effect of Cl(-) and HCO(3)(-) ions on the enzyme. HCO(3)(-) ions had no effect on the ATP-dependent Cl(-) transport into proteoliposomes (with the involvement of reconstituted ATPase). In experiment with Cl(-)-preloaded liposomes, addition of HCO(3)(-) ions to the incubation medium caused the reversion of Cl(-) transport (ion efflux from liposomes). Our results suggest that HCO(3)(-) ions play an important role in the modification of properties of the ATP-dependent GABA(A) receptor-coupled Cl(-) channel and GABA(A) receptor-induced Cl(-)/ HCO(3)(-) exchange. These ions are probably involved in GABA(A) receptor-induced Cl(-)/ HCO(3)(-) exchange in neuronal membranes.

Effects of GABAA-ergic ligands on Cl− transport induced by the Cl−, HCO−3-ATPase from carp (Cyprinus carpio L.) brain reconstituted in proteoliposomes

A Cl−, HCO3−-ATPase isolated from the plasma membranes of fish brain was reconstituted in artificial proteoliposomes. The original sensitivity to GABAA-ergic ligands was preserved in the reconstituted enzyme. GABA was shown to activate the liposome-embedded enzyme in a concentration range from 10 to 100 μM while picrotoxin (100 μM) removed this activating affect. A Cl−-sensitive fluorescent probe (MEQ) was used to study the ATP-dependent Cl− transport through the membrane of the proteoliposomes. ATP (in concentrations of 1.5 mM and higher) was found to induce Cl−-transport into the proteoliposomes. This ATP-dependent transport was increased in the presence of GABA (100 μM), while the latter effect could also be blocked by picrotoxin (100 μM). It was concluded that the Cl−, HCO3−-ATPase could be involved in ATP-dependent Cl−-transport and regulated by activators and blockers of GABAA receptors.

Properties of Cl--Stimulated Mg2+-ATPase Sensitive to Ligands of Inhibitory Receptors in Brain Plasma Membranes of the Bream Abramis brama

Properties of Cl−-stimulated Mg2+-ATPase in the brain plasma membranes of the bream Abramis brama L. were studied; this enzyme is composed of “basal” Mg2+-ATPase activity that can be stimulated by 40–80% by Cl− ions (Cl−-ATPase). These anions stimulate the basal Mg2+-ATPase starting with 8 mM concentration, their maximal effect being observed at a concentration of 30–100 mM. The Cl−-ATPase activity was found at a low molarity of HEPES-Tris buffer ( 30 mM). The “basal” Mg2+-ATPase activity was detected in the whole studied pH range (5.5–9.0), with maximum at pH 7.2–7.8 values, whereas optimum to reveal Cl−-ATPase was at high and low H+ concentrations (pH 6.0 and 8.5, respectively). At physiological pH values (7.2–7.5) the Cl−-ATPase activity was not revealed, but was detected after preincubation of the enzyme with 10 µM GABA. The “basal” Mg2+-ATPase, like Cl−-ATPase, hydrolyzed ATP with a maximal rate, while CTP, ITP, and ADP only slightly, and did not hydrolyze GTP and AMP. The Cl−-ATPase activity decreased in the presence of divalent cations in the following order: Mg2+ > Co2+ > Mn2+ = Cd2+ > Al3+ = Cu2+, and it was not found in the presence of Ca2+ and Zn2+. Anions of halogen series activated the “basal” Mg2+-ATPase in the descending order: Cl− > Br− > J− > F−. Among other monovalent anions, HCO3− activated the enzyme, NO3− practically had no effect, and SCN− inhibited its activity. Blockers of Cl− transport (ethacrinic acid, furosemide, and SITS) and GABA-receptor ligands (pentobarbital, diazepam, and picrotoxin) suppressed the enzyme activity. Out of SH-reagents, PCMB inhibited the enzyme, while NEM did not affect it. The H+-ATPase blocker oligomycin inhibited the enzyme, while the blocker of Na+,K+-ATPase ouabain and the blocker of Ca2+,Mg2+-ATPase ruthenium red had no effect. The properties of the Cl−-stimulated Mg2+-ATPase of fish brain are discussed in comparison with those of the rat brain Cl−-ATPase. The conclusion is made that the bream brain enzyme differs markedly from Cl−-ATPase (the ATP-dependent Cl−-pump) of mammalian brain.

Electrophysiological approach to studying CFTR.

Cystic fibrosis transmembrane conductance regulator (CFTR) is aphosphorylation and ATP-dependent Cl∼ channel. It is predominantly expressed in the apical membrane of epithelial cells. The presence and function of CFTR in a cell is sensitively measured using electrophysiological techniques. The patch clamp technique is the most frequently used method to measure CFTR in single cells or in isolated patches of cell membrane. A key step for the successful recording of CFTR is its identification and distinction from other Cl∼ channels. In patch clamp recordings this is done by probing for physiological, pharmacological, or biophysical characteristics of the channel. This chapter describes first, how to successfully record CFTR currents using the patch clamp technique. Second, it focuses on current noise analysis as a useful tool to investigate the regulation of CFTR activity.

The Aplysia californica Cl- pump is a P-type ATPase: evidence through inhibition studies

Utilizing a proteoliposomal preparation containing Cl(-)-ATPase from Aplysia californica foregut, it was shown that orthovanodate inhibited Cl(-)-ATPase activity, ATP-dependent Cl- transport, ATP-dependent membrane potential change and ATP-dependent phosphorylation. N-ethylmalemide and p-chloromercurobenzoate also inhibited the Cl- pump biochemical and physiological transport characteristics. However, bafilomycin, azide, N, N'-dicyclohexylcarboiimide (DCCD), and efrapeptin had no effect on the Cl- pump biochemical or physiological characteristics, suggesting that this Cl- pump was a P-type ATPase. It was concluded that this P-type ATPase Cl- pump is the mechanism that is responsible for the net absorptive flux of Cl- in the A. californica foregut.

Inhibition of a Cl−- Transporting P-type ATPase in Aplysia Gut

Abstract Utilizing a basolateral membrane vesicle preparation containing Cl−-ATPase from Aplysia foregut, it was shown that orthovanodate inhibited Cl−-ATPase activity, ATP-dependent Cl− transport and ATP-dependent membrane potential change. N-ethylmalemide (NEM) and p-chloromercurobenzoate (PCMBS) also inhibited the Cl− pump biochemical and transport characteristics. However, bafilomycin, azide, DCCD or efrapeptin had no effect on the Cl− pump characteristics suggesting that this Cl− pump was a P-type ATPase.

Cl(-)-ATPase in rat brain and kidney.

Cl(-)-stimulated ATPase/ATP-dependent Cl(-) pump (Cl(-)-ATPase/pump) has been found as a candidate for an active outwardly directed Cl(-) transporter in brain neurons. (1) A 520-kDa protein complex with Cl(-)-ATPase/pump activity was isolated from rat brain. It consisted of four protein subunits (51, 55, 60, and 62 kDa proteins), the 51-kDa protein being a covalent phosphorylenzyme subunit. (2) An antiserum against the 51-kDa protein inhibited Cl(-)-ATPase/pump activity. Western blot analysis showed an immunoreactive 51-kDa protein in the brain, spinal cord, and kidney. By enzyme histochemistry and immunohistochemistry, Cl(-)-ATPase-like activity or immunoreactivity was observed on the plasma membranes of brain neurons, and on the baso-lateral membranes of type A intercalated cells of renal collecting ducts. (3) Reconstituted Cl(-)-ATPase/pump activity was highest in liposomes with phosphatidylinositol-4-monophosphate. LiCl, an inhibitor of inositolphosphatase, reduced Cl(-)-ATPase activity and increased intracellular Cl(-) concentrations in cultured rat hippocampal neurons with increased phosphatidylinositol turnover. (4) In the brains of patients with Alzheimer's disease (AD), where phosphatidylinositol 4-kinase activity is reduced, Cl(-)-ATPase activity was also reduced. Thus, Cl(-)-ATPase is likely an outwardly directed ATP-dependent Cl(-) transporter that consists of four subunits and is regulated by phosphatidylinositol-4-monophosphate. Changes in Cl(-)-ATPase activity may be related to the pathophysiology of human neurodegenerative diseases. J. Exp. Zool. 289:224-231, 2001.

Transport energetics of the Cl − pump in Aplysia gut

Basolateral membranes of Aplysia foregut epithelia contain an ATP-dependent Cl − transporter (Cl − pump). Increased activity of the Cl − pump, coupled to apical and basolateral membrane depolarization, changed the Cl − transport energetics across the apical membrane but did not change the vectorially-opposite Cl − transport energetics across the basolateral membrane.

Reconstituted Cl- pump protein: a novel ion(Cl-)-motive ATPase.

Cl- absorption by the Aplysia californica foregut is effected through an active Cl- transport mechanism located in the basolateral membrane of the epithelial absorptive cells. These basolateral membranes contain both Cl(-)-stimulated ATPase and ATP-dependent Cl- transport activities which can be incorporated into liposomes via reconstitution. Utilizing the proteoliposomal preparation, it was demonstrated that ATP, and its subsequent hydrolysis, Mg2+, Cl-, and a pH optimum of 7.8 were required to generate maximal intraliposomal Cl- accumulation, electrical negativity, and ATPase activity. Additionally, an inwardly-directed valinomycin-induced K+ diffusion potential, making the liposome interior electrically positive, enhanced both ATP-driven Cl- accumulation and electrical potential while an outwardly-directed valinomycin-induced K+ diffusion potential, making the liposome interior electrically negative, decreased both ATP-driven Cl- accumulation and electrical potential compared with proteoliposomes lacking the ionophore. Either orthovanadate or p-chloromercurobenzene sulfonate inhibited both the ATP-dependent intraliposomal Cl- accumulation, intraliposomal negative potential difference, and also Cl(-)-stimulated ATPase activity. Both aspects of Cl- pump transport kinetics and its associated catalytic component kinetics were the first obtained utilizing a reconstituted transporter protein. These results strongly support the hypothesis that Cl(-)-ATPase actively transports Cl- by an electrogenic process.

A Cl- pump in rat brain neurons.

Cl(-)-stimulated and ethacrynic acid-sensitive ATPase (Cl(-)-ATPase) of plasma membrane origin in the rat brain is a candidate for an active outwardly directed Cl- translocating system. Biochemistry of Cl(-)-ATPase and ATP-dependent Cl- transport (Km values for ATP and Cl-, nucleotide specificity, pH dependency, and sensitivity to ethacrynic acid) suggested that Cl(-)-ATPase is an ATP-driven Cl- pump. Activity of the reconstituted Cl(-)-ATPase/pump increased in the presence of phosphatidylinositol-4-monophosphate, and this pump activity further increased at an inside-positive membrane potential or in the presence of a protonophore, suggesting that the Cl(-)-ATPase/pump is an electrogenic Cl- transporter, probably regulated by phosphoinositide turnover in vivo. In cultured hippocampal pyramidal cell-like neurons from embryonic rat brain, ethacrynic acid and ATP-consuming treatment increased, but furosemide, an inhibitor of Na+/K+/Cl- cotransporter, decreased, [Cl-]i when monitored using Cl(-)-sensitive fluorescent probes. The stationary levels of [Cl-]i were lower and the effects of ethacrynic acid were more prominent in perikarya than in dendrites, while the effects of furosemide were more obvious in dendrites than in perikarya. The lower perikaryonic [Cl-]i and the marked effects of ethacrynic acid were observed in the later stage rather than in the early stage of culture. Thus, region-specific localization and developmental changes in the activities of Cl- transporters probably result in uneven and age-dependent distribution of Cl- in the neurons.

P-glycoprotein regulates a volume-activated chloride current in bovine non-pigmented ciliary epithelial cells.

1. The whole-cell patch clamp technique was used to investigate the swelling-activated currents in bovine non-pigmented ciliary epithelial (NPCE) cells. 2. Exposure to hypotonic solution activated a current that was blocked by 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). The I-V relationship was shifted in the direction expected for a Cl- current when the external Cl- was replaced by gluconate (permeability ratio P(gluconate)/PCl = 0.17). The inhibition of the current evoked by voltage clamp steps of +80 mV yielded an IC50 for NPPB of 13.4 microM. 3. The current was found to be dependent on ATP. With ATP in the patch pipette the current could be repeatedly activated by exposure to hypotonic solution but when ATP was omitted the current ran down with time. 4. The development of this current was associated with visible cell swelling and inhibitors of regulatory volume decrease in these cells, e.g. tamoxifen, 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid (SITS) and 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS), also inhibited this current. 5. The volume-activated current was additionally blocked by NPPB, verapamil, quinidine and dideoxyforskolin. 6. The current was independent of external calcium and exhibited slight outward rectification and time-dependent inactivation at strong depolarizing potentials. 7. Disrupting the cytoskeleton and microtubules with cytochalasin B and colchicine had no effect on the activation of the Cl- current. 8. An antibody (C219) to the MDR1 gene product, P-glycoprotein, caused a functional block of the swelling-activated Cl- current when added to the patch pipette. 9. Immunofluorescence studies using the monoclonal antibodies C219 and JSB-1 demonstrated the presence of P-glycoprotein in the ciliary epithelial cells. The immunofluorescence was stronger on the non-pigmented than on the pigmented cells. 10. It is concluded that swelling in NPCE cells activates a Ca(2+)-independent, ATP-dependent Cl- current and that the activity of this current is associated with P-glycoprotein. 11. It is suggested that this Cl- current contributes to regulatory volume decrease and may participate in the secretory activity of these cells.

γ-Aminobutyric acid increases intracellular Ca 2+ concentration in cultured cortical neurons: role of Cl − transport

The effect of γ-aminobutyric acid (GABA) on intracellular Ca 2+ concentration ([Ca 2+] i) in cultured prenatal rat cortical neurons was investigated using fluorescence imaging. GABA or muscimol, but not baclofen, increased [Ca 2+] i in a dose-dependent manner. The GABA A receptor antagonists, bicuculline and picrotoxin, inhibited the GABA response. Furosemide, an inhibitor of the Na +/K +/2Cl − cotransporter, inhibited the GABA response in a noncompetitive manner. Ethacrynic acid, an inhibitor of an ATP-dependent Cl − pump, also inhibited the GABA-induced increased in [Ca 2+] i. These results suggest a role for Cl − transport processes in the GABA response. The coapplication of GABA and high K + led to a non-additive increase in the GABA response. The GABA response was also inhibited by nifedipine, a voltage-gated Ca 2+ channel blocker, and abolished by the absence of extracellular Ca 2+. Results indicate that the GABA response shares a common pathway of Ca 2+ movement with the high K +-induced response. These observations suggest that the stimulation with GABA results in Ca 2+ influx through voltage-gated Ca 2+ channels, and that these effects are dependent on Cl − transport systems.

Stimulation of Cl- secretion by extracellular ATP does not depend on increased cytosolic Ca2+ in HT-29.cl16E

Extracellular ATP and elevated cytosolic Ca2+ concentration ([Ca2+]i) are major secretagogues for Cl- in the goblet cell-like clone cl.16E derived from colonic HT-29 cells. The involvement of [Ca2+]i as a messenger for the purinergically stimulated Cl- secretion was investigated using whole cell patch-clamp and Ussing chamber techniques, as well as [Ca2+]i measurements using fura 2-loaded cells. Under voltage-clamp conditions, the whole cell current at +50 mV was 3 +/- 1 pA/pF under unstimulated conditions. Stimulation of purinergic receptors with 200 microM extracellular ATP increased the current at +50 mV to 41 +/- 10 pA/pF, with a half-maximal effective dose (ED50) of approximately 3 microM. The current was transient, usually lasting 1-2 min, and the current-voltage relationship was approximately linear between -70 and +50 mV. Evidence that the ATP-stimulated current was carried by Cl- included 1) the reversal potential of the current closely followed the Cl- equilibrium potential, and 2) the stimulated current was absent when Cl- was removed from both bath and pipette solutions. Exposure to ATP also increased [Ca2+]i, with an ED50 of approximately 1 microM and maximal changes (at 200 microM) from baseline (71 +/- 3 nM) to 459 +/- 50 nM. The ATP-dependent Cl- conductance increase was not diminished when [Ca2+]i was clamped at 100 nM using a Ca(2+)-1,2-bis(2- aminophenoxy)ethane-N,N,N',N'-tetraacetic acid or Ca(2+)-ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid buffering system. However, the ATP effect did require some basal level of Ca2+ because clamping [Ca2+]i at < 10 nM abolished activation of the Cl- conductance. The presence of the protein kinase A inhibitor H-89 or the protein kinase C inhibitor staurosprine did not change the ATP-activated Cl-conductance. These data demonstrate that the ATP-stimulated increase in Cl- current does not require an increase in [Ca2+]i, suggesting the involvement of either another signaling pathway or direct activation of Cl- channels by purinergic receptors.

Electrogenic and phosphatidylinositol-4-monophosphate-stimulated Cl − transport by Cl − pump in the rat brain

Rat brain Cl −-ATPase was solubilized and reconstituted in asolectin liposomes. Phosphatidylinositol-4-monophosphate increased Cl −-ATPase and ATP-dependent Cl − uptake activities in proteoliposomes. The ATP-dependent Cl − uptake was inhibited by a Cl −-ATPase inhibitor, ethacrynic acid, and increased at an inside-positive membrane potential or in the presence of a protonophore. These findings suggest that Cl −-ATPase is an electrogenic Cl − transporter, or a primary Cl − pump, probably regulated by phosphoinositide turnover in vivo.

Membrane chloride conductance and capacitance in Jurkat T lymphocytes during osmotic swelling

Video microscopy and whole-cell patch-clamp recording were used to monitor changes in relative cell volume (V/Vo), chloride conductance (gCl), and membrane capacitance (Cm) during osmotically induced swelling in Jurkat T lymphocytes. Cellular swelling was initiated with hyperosmotic pipette solutions. Simultaneous evaluation of V/Vo and gCl revealed a 59-s delay between the inception of swelling and the activation of outwardly rectifying, ATP-dependent Cl- channels. Following the delay, increases in V/Vo and gCl progressed in parallel. In contrast, Cm, a measure of cell surface area, fell gradually at a rate of approximately 150 fF/min after whole-cell access was achieved. The decline in Cm lasted 200 s and was followed by a rapid rise (approximately 750 fF/min). The rise in Cm coincided with a variable increase in "leak" current, gCl increased at a slower rate and reached lower peak values in experiments performed without ATP; ATP had no effect on the biphasic Cm time course. The temporal separation of conductance and capacitance during swelling suggests that gCl and Cm vary independently, supporting the hypothesis that a large portion, if not all, of the whole-cell Cl- conductance activated during swelling is provided by volume-sensitive Cl- channels preexisting in the plasma membrane.

Reaction sequence and molecular mass of a Cl(-)-translocating P-type ATPase.

The basolateral membranes of Aplysia californica foregut absorptive cells contain both Cl(-)-stimulated ATPase and ATP-dependent Cl- transport activities, and each was inhibited by orthovanadate. Both of these orthovanadate-sensitive activities were reconstituted into proteoliposomes. The reaction sequence kinetics were determined by [gamma-32P]ATP-induced phosphorylation of the reconstituted Cl- pump. Rapid phosphorylation and dephosphorylation kinetics of acyl phosphate bonding were confirmed by destabilization of the phosphoprotein by either hydroxylamine or high pH. Mg2+ caused phosphorylation of the enzyme; Cl- caused dephosphorylation. Orthovanadate almost completely inhibited the Mg(2+)-driven phosphorylation reaction. The molecular mass of the catalytic unit (subunit) of the enzyme appeared to be 110 kDa, which is in agreement with molecular masses of all other catalytic units (subunits) of P-type ATPases.