Mechanism of action of cholera toxin: Effect of receptor density and multivalent binding on activation of adenylate cyclase

Summary Cl^– influx into cells ofChara corallina is shown to be stimulated by a factor of 2 to 4 by starvation of Cl^–. The time constant for the induction of this effect is about 4.0 ksec and that for its decay when Cl^– is reprovided, 1.7 ksec. Intracellular perfusion of tonoplast-free cells with solutions of varying Cl^– concentration shows that Cl^– influx can be controlled directly by the concentration of Cl^– at the inside of the plasma membrane. Both the time course for the initial stages of induction of the starvation-stimulated flux and its absolute magnitude can be accounted for by assuming cytoplasmic Cl^– concentration to be the only intracellular condition to change during Cl^– starvation. The existence of a feedback loop between cytoplasmic Cl^– and Cl^– influx provides an alternative explanation to observations previously used in support of a Cl^–/OH^– exchange hypothesis (F.A. Smith, 1972,New Phytol. 71:595).     

Mechanism of Cl- sensitivity in internal ion receptors of the leech; an inward current gated off by Cl- in the nephridial nerve cells

Summary The nephridial nerve cells of the leech, Hirudo medicinalis, 34 sensory cells, each associated with one nephridium, are sensitive to changes in extracellular Cl^- concentration, an important factor in ion homeostasis. Using single-electrode current- and voltage clamp and ion substitution techniques, the specificity and mechanism of Cl^- sensitivity of the nephridial nerve cell was studied in isolated preparations. Increase of the normally low external Cl^- concentration leads to immediate and sustained hyperpolarization, decrease of the frequency of bursts and decrease of membrane conductance. The response is halogen specific: Cl^- can be replaced by Br^–, but not by organic mono- or divalent anions or inorganic divalent anions.At physiological Cl^- concentrations (36mM extra-cellular Cl^-), the nephridial nerve cell has a high resting conductance for Cl^- and the membrane potential is governed by Cl^-. In high extracellular Cl^- concentrations (110–130 mM), membrane conductance is low, most likely due to the gating off of Cl^- channels. Under these conditions, membrane potential is dominated by the K⁺ distribution and the nephridial nerve cell hyperpolarizes towards E_K.Abbreviations NNC nephridial nerve cell - V _m membrane potential - E _Cl(k) equilibrium potential for Cl (K) - IV-curve current-voltage relationship     

Na+ and Cl− conductances are controlled by cytosolic Cl− concentration in the intralobular duct cells of mouse mandibular glands

Our previously published whole-cell patch-clamp studies on the cells of the intralobular (granular) ducts of the mandibular glands of male mice revealed the presence of an amiloride-sensitive Na⁺ conductance in the plasma membrane. In this study we demonstrate the presence also of a Cl^– conductance and we show that the sizes of both conductances vary with the Cl^– concentration of the fluid bathing the cytosolic surface of the plasma membrane. As the cytosolic Cl^– concentration rises from 5 to 150 mmol/liter, the size of the inward Na⁺ current declines, the decline being half-maximal when the Cl^– concentration is approximately 50 mmol/liter. In contrast, as cytosolic Cl^– concentration increases, the inward Cl^– current remains at a constant low level until the Cl^– concentration exceeds 80 mmol/liter, when it begins to increase. Studies in which Cl^– in the pipette solution was replaced by other anions indicate that the Na⁺ current is suppressed by intracellular Br^-, Cl^– and NO ₃ ^- but not by intracellular I^-, glutamate or gluconate. Our studies also show that the Cl^– conductance allows passage of Cl^– and Br^- equally well, I-less well, and NO ₃ ^- , glutamate and gluconate poorly, if at all. The findings with NO ₃ ^- are of particular interest because they show that suppression of the Na⁺ current by a high intracellular concentration of a particular anion does not depend on actual passage of that anion through the Cl^– conductance. In mouse granular duct cells there is, thus, a reciprocal regulation of Na⁺ and Cl^– conductances by the cytosolic Cl^– concentration. Since the cytosolic Cl^– concentration is closely correlated with cell volume in many epithelia, this reciprocal regulation of Na⁺ and Cl^– conductances may provide a mechanism by which ductal Na⁺ and Cl transport rates are adjusted so as to maintain a stable cell volume.This project was supported by the National Health and Medical Research Council of Australia. We thank Professor P. Barry (University of New South Wales) for assistance with the junction potential measurements.     

Regulation of ClC-1 and KATP channels in action potential–firing fast-twitch muscle fibers

Action potential (AP) excitation requires a transient dominance of depolarizing membrane currents over the repolarizing membrane currents that stabilize the resting membrane potential. Such stabilizing currents, in turn, depend on passive membrane conductance (G_m), which in skeletal muscle fibers covers membrane conductances for K⁺ (G_K) and Cl⁻ (G_Cl). Myotonic disorders and studies with metabolically poisoned muscle have revealed capacities of G_K and G_Cl to inversely interfere with muscle excitability. However, whether regulation of G_K and G_Cl occur in AP-firing muscle under normal physiological conditions is unknown. This study establishes a technique that allows the determination of G_Cl and G_K with a temporal resolution of seconds in AP-firing muscle fibers. With this approach, we have identified and quantified a biphasic regulation of G_m in active fast-twitch extensor digitorum longus fibers of the rat. Thus, at the onset of AP firing, a reduction in G_Cl of ∼70% caused G_m to decline by ∼55% in a manner that is well described by a single exponential function characterized by a time constant of ∼200 APs (phase 1). When stimulation was continued beyond ∼1,800 APs, synchronized elevations in G_K (∼14-fold) and G_Cl (∼3-fold) caused G_m to rise sigmoidally to ∼400% of its level before AP firing (phase 2). Phase 2 was often associated with a failure to excite APs. When AP firing was ceased during phase 2, G_m recovered to its level before AP firing in ∼1 min. Experiments with glibenclamide (K_ATP channel inhibitor) and 9-anthracene carboxylic acid (ClC-1 Cl⁻ channel inhibitor) revealed that the decreased G_m during phase 1 reflected ClC-1 channel inhibition, whereas the massively elevated G_m during phase 2 reflected synchronized openings of ClC-1 and K_ATP channels. In conclusion, G_Cl and G_K are acutely regulated in AP-firing fast-twitch muscle fibers. Such regulation may contribute to the physiological control of excitability in active muscle.     

Structural evidence for the order of preference of inorganic substrates in mammalian heme peroxidases: crystal structure of the complex of lactoperoxidase with four inorganic substrates,SCN-, I-, Br– and Cl-

Lactoperoxidase (LPO) is a member of the family of mammalian heme peroxidases. It catalyzes the oxidation of halides and pseudohalides in presence of hydrogen peroxide. LPO has been co-crystallized with inorganic substrates, SCN^-, I^-, Br^- and Cl^-. The structure determination of the complex of LPO with above four substrates showed that all of them occupied distinct positions in the substrate binding site on the distal heme side. The bound substrate ions were separated from each other by one or more water molecules. The heme iron is coordinated to His-351 N^ϵ2 on the proximal side while it is coordinated to conserved water molecule W-1 on the distal heme side. W-1 is hydrogen bonded to Br^- ion which is followed by Cl^- ion with a hydrogen bonded water molecule W-5′ between them. Next to Cl^- ion is a hydrogen bonded water molecule W-7′ which in turn is hydrogen bonded to W-8′ and N atom of SCN^-. W-80 is hydrogen bonded to W-9′ which is hydrogen bonded to I^-. SCN^- ion also interacts directly with Asn-230 and through water molecules with Ser-235 and Phe-254. Therefore, according to the locations of four substrate anions, the order of preference for binding to lactoperoxidase is observed as Br^- > Cl^- > SCN^- > I^-. The positions of anions are further defined in terms of subsites where Br^- is located in subsite 1, Cl^- in subsite 2, SCN^- in subsite 3 and I^- in subsite 4.     

Sodium, Potassium, and Chloride Fluxes in Intercostal Muscle from Normal Goats and Goats with Hereditary Myotonia

In isolated bundles of external intercostal muscle from normal goats and goats with hereditary myotonia the following were determined: concentrations and unidirectional fluxes of Na⁺, K⁺, and Cl^-, extracellular volume, water content, fiber geometry, and core-conductor constants. No significant difference between the two groups of preparations was found with respect to distribution of fiber size, intracellular concentrations of Na⁺ or Cl^-, fiber water, resting membrane potential, or overshoot of action potential. The intracellular Cl^- concentration in both groups of preparations was 4 to 7 times that expected if Cl^- were distributed passively between intracellular and extracellular water. The membrane permeability to K (P_K) calculated from efflux data was (a) at 38°C, 0.365 x 10^-6 cm sec^-1 for normal and 0.492 x 10^-6 for myotonic muscle, and (b) at 25°C, 0.219 x 10^-6 for normal and 0.199 x 10^-6 for myotonic muscle. From Cl^- washout curves of normal muscle usually only three exponential functions could be extracted, but in every experiment with myotonic muscle there was an additional, intermediate component. From these data PP_cl could be calculated; it was 0.413 x 10^-6 cm sec^-1 for myotonic fibers and was 0.815 x 10^-6 cm sec^-1 for normal fibers. The resting membrane resistance of myotonic fibers was 4 to 6 times greater than that of normal fibers.     

Regulation of conductance by the number of fixed positive charges in the intracellular vestibule of the CFTR chloride channel pore

Rapid chloride permeation through the cystic fibrosis transmembrane conductance regulator (CFTR) Cl⁻ channel is dependent on the presence of fixed positive charges in the permeation pathway. Here, we use site-directed mutagenesis and patch clamp recording to show that the functional role played by one such positive charge (K95) in the inner vestibule of the pore can be “transplanted” to a residue in a different transmembrane (TM) region (S1141). Thus, the mutant channel K95S/S1141K showed Cl⁻ conductance and open-channel blocker interactions similar to those of wild-type CFTR, thereby “rescuing” the effects of the charge-neutralizing K95S mutation. Furthermore, the function of K95C/S1141C, but not K95C or S1141C, was inhibited by the oxidizing agent copper(II)-o-phenanthroline, and this inhibition was reversed by the reducing agent dithiothreitol, suggesting disulfide bond formation between these two introduced cysteine side chains. These results suggest that the amino acid side chains of K95 (in TM1) and S1141 (in TM12) are functionally interchangeable and located closely together in the inner vestibule of the pore. This allowed us to investigate the functional effects of increasing the number of fixed positive charges in this vestibule from one (in wild type) to two (in the S1141K mutant). The S1141K mutant had similar Cl⁻ conductance as wild type, but increased susceptibility to channel block by cytoplasmic anions including adenosine triphosphate, pyrophosphate, 5-nitro-2-(3-phenylpropylamino)benzoic acid, and Pt(NO₂)₄²⁻ in inside-out membrane patches. Furthermore, in cell-attached patch recordings, apparent voltage-dependent channel block by cytosolic anions was strengthened by the S1141K mutation. Thus, the Cl⁻ channel function of CFTR is maximal with a single fixed positive charge in this part of the inner vestibule of the pore, and increasing the number of such charges to two causes a net decrease in overall Cl⁻ transport through a combination of failure to increase Cl⁻ conductance and increased susceptibility to channel block by cytosolic substances.     

Electrical properties of chloride transport across theNecturus proximal tubule

Summary The chloride conductance of the basolateral cell membrane of theNecturus proximal tubule was studied using conventional and chloride-sensitive liquid ion exchange microelectrodes. Individual apical and basolateral cell membrane and shunt resistances, transepithelial and basolateral, cell membrane potential differences, and electromotive forces were determined in control and after reductions in extracellular Cl^–. When extracellular Cl^– activity is reduced in both apical and basolateral solutions the resistance of the shunt increases about 2.8 times over control without any significant change in cell membrane resistances. This suggests a high Cl^– conductance of the paracellular shunt but a low Cl^– conductance of the cell membranes. Reduction of Cl^– in both bathing solutions or only on the basolateral side hyperpolarizes both the basolateral cell membrane potential difference and electromotive force. Hyperpolarization of the basolateral cell membrane potential difference after low Cl^– perfusion was abolished by exposure to HCO ₃ ^– -free solutions and SITS treatment. In control conditions, intracellular Cl^– activity was significantly higher than predicted from the equilibrium distribution across both the apical and basolateral cell membranes. Reducing Cl^– in only the basolateral solution caused a decrease in intracellular Cl^–. From an estimate of the net Cl^– flux across the basolateral cell membrane and the electrochemical driving force, a Cl^– conductance of the basolateral cell membrane was predicted and compared to measured values. It was concluded that the Cl^– conductance of the basolateral cell membrane was not large enough to account for the measured flux of Cl^– by electrodiffusion alone. Therefore these results suggest the presence of an electroneutral mechanism for Cl^– transport across the basolateral cell membrane of theNecturus proximal tubule cell.     

CORRELATED MORPHOLOGICAL AND PHYSIOLOGICAL STUDIES ON ISOLATED SINGLE MUSCLE FIBERS : I. Fine Structure of the Crayfish Muscle Fiber

Single fibers isolated from walking leg muscles of crayfish have 8- to 10-µ sarcomeres which are divided into A, I, and Z bands. The H zone is poorly defined and no M band is distinguishable. Changes in the width of the I band, accompanied by change in the overlap between thick and thin myofilaments, occur when the length of the sarcomere is changed by stretching or by shortening the fiber. The thick myofilaments (ca. 200 A in diameter) are confined to the A band. The thin myofilaments (ca. 50 A in diameter) are difficult to resolve except in swollen fibers, when they clearly lie between the thick filaments and run to the Z disc. The sarcolemma invaginates at 50 to 200 sites in each sarcomere. The sarcolemmal invaginations (SI) form tubes about 0.2 µ in diameter which run radially into the fiber and have longitudinal side branches. Tubules about 150 A in diameter arise from the SI and from the sarcolemma. The invaginations and tubules are all derived from and are continuous with the plasma membrane, forming the transverse tubular system (TTS), which is analogous with the T system of vertebrate muscle. In the A band region each myofibril is enveloped by a fenestrated membranous covering of sarcoplasmic reticulum (SR). Sacculations of the SR extend over the A-I junctions of the myofibrils, where they make specialized contacts (diads) with the TTS. At the diads the opposing membranes of the TTS and SR are spaced 150 A apart, with a 35-A plate centrally located in the gap. It appears likely that the anion-permselective membrane of the TTS which was described previously is located at the diads, and that this property of the diadic structures therefore may function in excitation-contraction coupling.     

Chloride movement across the basolateral membrane of proximal tubule cells

Summary Electrophysiologic and tracer experiments have shown that Cl^– entersNecturus proximal tubule cells from the tubule lumen by a process coupled to the flow of Na⁺, and that Cl^– entry is electrically silent. The mechanism of Cl^– exit from the cell across the basolateral membrane has not been directly studied. To evaluate the importance of the movement of Cl^– ions across the basolateral membrane, the relative conductance of Cl^– to K⁺ was determined by a new method. Single-barrel ion-selective microelectrodes were used to measure intracellular Cl^– and K⁺ as a function of basolateral membrane PD as it varied normally from tubule to tubule. Basolateral membrane Cl^– conductance was about 10% of K⁺ conductance by this method. A second approach was to voltage clamp the basolateral PD to 20 mV above and below the spontaneous PD, while sensing intracellular Cl^– activity with the second barrel of a double-barrel microelectrode. An axial wire electrode in the tubule lumen was used to pass current across the tubular wall and thereby vary the basolateral membrane PD. Cell Cl^– activity was virtually unaffected by the PD changes. We conclude that Cl^– leavesNecturus proximal tubule cells by a neutral mechanism, possibly coupled to the efflux of Na⁺ or K⁺.     

Inhibitory Miniature Potentials in the Stretch Receptor Neurons of Crayfish

Intracellular microelectrodes inserted into the soma of crayfish stretch receptor neurons record frequent fluctuations of the membrane potential. Time course, amplitude, and interval distribution indicate that they are miniature potentials. At the average resting potential the polarity of the miniature potentials depends on the anion used in the microelectrode: KCl electrodes record depolarizing, K citrate or K₂SO₄ electrodes, hyperpolarizing miniature potentials. The inhibitory postsynaptic potentials (i.p.s.p.'s) show a similar polarity change. The reversal potentials of i.p.s.p.'s and miniature potentials are equal and within 10 mv of the resting potential, more negative with K citrate (or K₂SO₄), less negative with KCl electrodes. Reversal can be accomplished by changing the membrane potential by stretching or by current passing. Injection of Cl^- into the soma or replacement of external Cl by propionate results in an abrupt increase of the amplitude of the miniature potentials lasting for several minutes. The miniature potentials like the i.p.s.p.'s are reversibly abolished by the application of picrotoxin and γ-aminobutyric acid. They are not affected by tetrodotoxin, nor by acetylocholine, eserine, or atropine. It is concluded that the miniature potentials represent a spontaneous quantal release of transmitter substance from inhibitory nerve terminals, and that the transmitter substance predominantly increases the Cl^- permeability of the postsynaptic membrane. The effect of the spontaneously released transmitter on the behavior of the receptor neuron is considerable. The membrane conductance is increased by up to 36% and the excitability is correspondingly depressed.     

The relation between the chloride status of the photosynthetic water splitting complex and the inhibitory effectiveness of amines

The protective role of chloride ions (Cl^–) against inhibition of the photosynthetic water splitting complex by amines was investigated with purified photosystem II membrane particles from tobacco chloroplasts. Seemingly competitive interactions occurred between Cl^– (except at low concentrations) and Tris, but not between Cl^– and NH₃. The rate of Cl^– release was not increased by the amines but, instead, may have been limited by a labilization under the experimental conditions of the extrinsic 23 kDa polypeptide. An additional detachment of the 18 kDa polypeptide was seen when SO₄ ^2– ions were present. Tris induced changes of the thermoluminescence patterns of flash illuminated photosystem II particles were found to be different from those caused by either Cl^– deficiency or high pH. It is concluded that the protective functions of Cl^– are brough about not because it is bound to the target site of the inhibitory actions of Lewis bases like amines and hydroxyl ions. Instead, this effect of Cl^– may be due to its influence on the tertiary and quaternary structures of the water oxidizing protein complex.     

Mechanism of Cl− transport at the plasma membrane ofChara corallina: II. Transinhibition and the determination of H+/Cl− binding order from a reaction kinetic model

Summary Internal Cl^– and low internal pH are strong inhibitors of Cl^– influx at the plasma membrane ofChara. The present investigation seeks to understand the mechanism by which this is achieved. Since both Cl^– and H⁺ are transported by the same system, one possible mechanism is simply through a change in the electrochemical gradients of these ions. However, it is found that transport is more sensitive to theinternal concentrations of the two ions than to their respective gradients. It is demonstrated that Cl^– influx, which shows Michaelis-Menten kinetics with respect to external concentration, is affected only in itsV _max by internal Cl^– and pH; the apparentK _m of the transport system for external Cl^– is unchanged. In addition, it is found that there is an apparent interaction between internal Cl^– and pH in their effects on Cl^– influx, both in intact cells and those that have been perfused internally. A kinetic model is proposed which can account quantitatively for all these observations simply through the effects of substrate concentration on the apparent rate constants of a recycling carrier. The model predicts (i) strictly ordered binding of Cl^– and H⁺ to the carrier at both internal and external surfaces, with Cl^– first on and first off (ii) movement of charge through the membrane on the loaded, rather than the unloaded, carrier. The present model is expected to account for similar kinetic observations from a variety of other cotransport systems.     

Chloride Transport in Porous Lipid Bilayer Membranes

This paper describes dissipative Cl_- transport in "porous" lipid bilayer membranes, i.e., cholesterol-containing membranes exposed to 1–3 x 10^-7 M amphotericin B. P_DCl (cm·s^-1), the diffusional permeability coefficient for Cl^-, estimated from unidirectional ³⁶Cl^- fluxes at zero volume flow, varied linearly with the membrane conductance (Gm, Ω^-1·cm^-2) when the contributions of unstirred layers to the resistance to tracer diffusion were relatively small with respect to the membranes; in 0.05 M NaCl, P_DCl was 1.36 x 10^-4 cm·s^-1 when Gm was 0.02 Ω^-1·cm^-2. Net chloride fluxes were measured either in the presence of imposed concentration gradients or electrical potential differences. Under both sets of conditions: the values of P_DCl computed from zero volume flow experiments described net chloride fluxes; the net chloride fluxes accounted for ~90–95% of the membrane current density; and, the chloride flux ratio conformed to the Ussing independence relationship. Thus, it is likely that Cl^- traversed aqueous pores in these anion-permselective membranes via a simple diffusion process. The zero current membrane potentials measured when the aqueous phases contained asymmetrical NaCl solutions could be expressed in terms of the Goldman-Hodgkin-Katz constant field equation, assuming that the P_DNa/P_DCl ratio was 0.05. In symmetrical salt solutions, the current-voltage properties of these membranes were linear; in asymmetrical NaCl solutions, the membranes exhibited electrical rectification consistent with constant-field theory. It seems likely that the space charge density in these porous membranes is sufficiently low that the potential gradient within the membranes is approximately linear; and, that the pores are not electrically neutral, presumably because the Debye length within the membrane phase approximates the membrane thickness.     

Plasmalemma Chloride Transport in Chara corallina: Inhibition by 4,4'-Diisothiocyano-2,2'-Disulfonic Acid Stilbene

Chloride transport, presumably via a Cl⁻-2H⁺ co-transport system, was investigated in Chara corallina. At pH 6.5, the control influx (3.1 picomoles per centimeter² per second) was stimulated 4-fold by an 18-hour Cl⁻ starvation. The stimulated influx was inhibited to 4.7 picomoles per centimeter² per second after a 60-minute pre-exposure to 0.5 millimolar 4,4′-diisothiocyano-2,2′-disulfonic acid stilbene (DIDS). This compares with a nonsignificant inhibition of the control under similar conditions. At 2 millimolar DIDS, both stimulated and control influx were inhibited to values of 1.1 and 2.2 picomoles per centimeter² per second, respectively; in all cases, DIDS inhibition was reversible. Over the pH range 4.8 to 8.5, the control and DIDS-inhibited influx showed only slight pH sensitivity; in contrast, the stimulated flux was strongly pH dependent (pH 6.5 optimum). Inasmuch as changes in pH alter membrane potential, N-ethylmaleimide was used to depolarize the membrane; this had no effect on Cl⁻ influx. A transient depolarization of the membrane (about 20 millivolts) was observed on restoration of Cl⁻ to starved cells. The membrane also depolarized transiently when starved cells were exposed to 0.5 millimolar DIDS, but the depolarization associated with Cl⁻ restoration was inhibited by a 40-minute pretreatment with DIDS. Exposure of control cells to DIDS caused only a small hyperpolarization (about 7 millivolts). DIDS may have blocked Cl⁻ influx by inhibiting the putative plasmalemma H⁺-translocating ATPase. Histochemical studies on intact cells revealed no observable effect of DIDS on plasmalemma ATPase activity. However, DIDS application after fixation resulted in complete inhibition of ATPase activity.

The differential sensitivity of the stimulated and control flux to inhibition by DIDS may reflect an alteration of transport upon stimulation, but could also result from differences in pretreatment. The stimulated cells were pretreated with DIDS in the absence of Cl⁻, in contrast to the presence of Cl⁻ during pretreatment of controls. The differential effect could result from competition between Cl⁻ and DIDS for a common binding site. Our histochemical ATPase results indicate that Cl⁻ transport and membrane ATPase are separate systems, and the latter is only inhibited by DIDS from the inside of the cell.

     

The nature of the neutral Na+−Cl−-coupled entry at the apical membrane of rabbit gallbladder epithelium: II. Na+−Cl− symport is independent of K+

Summary In the epithelium of rabbit gallbladder, in the nominal absence of bicarbonate, intracellular Cl^– activity is about 25mm, about 4 times higher than intracellular Cl^– activity at the electrochemical equilibrium. It is essentially not affected by 10^–4 m acetazolamide and 10^–4 m 4-acetamido-4-isothiocyanostilbene-2,2-disulfonate (SITS) even during prolonged exposures; it falls to the equilibrium value by removal of Na⁺ from the lumen without significant changes of the apical membrane potential difference. Both intracellular Cl^– and Na⁺ activities are decreased by luminal treatment with 25mm SCN^–; the initial rates of change are not significantly different. In addition, the initial rates of change of intracellular Cl^– activity are not significantly different upon Na⁺ or Cl^– entry block by the appropriate reduction of the concentration of either ion in the luminal solution. Luminal K⁺ removal or 10^–5 m bumetanide do not affect intracellular Cl^– and Na⁺ activities or Cl^– influx through the apical membrane. It is concluded that in the absence of bicarbonate NaCl entry is entirely due to a Na⁺–Cl^– symport on a single carrier which, at least under the conditions tested, does not cotransport K⁺.     

Is there a Cl−−OH− exchange (Cl−−H+ cotransport) mechanism in the brush-border membrane of the intestine of the fresh water trout (Salmo gairdneri,R.)?

Summary Experiments were performed to determine the presence of a Cl^––OH^– exchange (Cl^––H⁺ cotransport) in the brush-border membranes isolated from the intestinal epithelium of freshwater trout. Determinations of alkaline phosphatase activities have shown that vesicle suspensions had an enrichment factor of about 17 in this enzyme indicating a high degree of purification of the brush-border membrane preparation. Cl^– uptake by vesicles in the presence of a proton gradient occurs against a concentration gradient with an overshoot ratio of about 2 and is inhibited by SITS. Several lines of evidence suggest that the mechanism involved is electrical in nature: (i) Cl^– uptake is increased when the proton gradient is increased, but there is a linear relationship between the Cl^– uptake and the Nernst potential of protons. (ii) Cl^– uptake is increased when a proton ionophore is added at low concentration and inhibited at high concentration, suggesting that a proton conductance is involved in the Cl^– uptake. (iii) there is a linear relationship between the initial speed of the uptake of increasing Cl^– concentrations and the Cl^– concentration. (iv) Cl^– uptake can be modulated by different potassium gradients with or without valinomycin. It is concluded that the enterocyte of the freshwater trout is not equipped with a Cl^––OH^– exchange and the Cl^– uptake by vesicles is realized by a Cl^– conductance.     

Biphasic increase of apical Cl− conductance by muscarinic stimulation of ht-29cl.19a human colon carcinoma cell line: Evidence for activation of different cl− conductances by carbachol and forskolin

Summary The modulation of ion transport pathways in filtergrown monolayers of the Cl^–-secreting subclone (19A) of the human colon carcinoma cell line HT-29 by muscarinic stimulation was studied by combined Ussing chamber and microimpalement experiments.Basolateral addition of 10^–4 m carbachol induced a complex poly-phasic change of the cell potential consisting of (i) a fast and short (30-sec) depolarization of 15±1 mV from a resting value of –52±1 mV and an increase of the fractional resistance of the apical membrane (first phase), (ii) a repolarization of 22±1 mV leading to a hyperpolarization of the cell (second phase), (iii) a depolarization of 11±1 mV and a decrease of the fractional resistance of the apical membrane (the third phase), (iv) and sometimes, a hyperpolarization of 6±1 mV and an increase of the fractional resistance of the apical membrane (fourth phase). The transepithelial potential increased with a peak value of 2.4±0.3 mV (basolateral side positive). The transepithelial PD started to increase (serosa positive), coinciding with the start of the second phase of the intracellular potential change, and continued to increase during the third phase. Ion replacements and electrical circuit analyses indicate that the first phase is caused by increase of the Cl^– conductance in the apical and basolateral membrane, the second phase by increased K⁺ conductance of the basolateral membrane, and the third phase and the fourth phase by increase and decrease, respectively, of an apical Cl^– conductance. The first and second phase of the carbachol effect could be elicited also by ionomycin. They were strongly reduced by EGTA. Phorbol dibutyrate (PDB) induced a sustained depolarization of the cell and a decrease of the apical fractional resistance. The results suggest that two different types of Cl^– channels are involved in the carbachol response: one Ca²⁺ dependent and a second which may be PKC sensitive.In the presence of a supramaximal concentration of forskolin, carbachol evoked a further increase of the apical Cl^– conductance.It is concluded that the short-lasting carbachol/Ca²⁺-dependent Cl^– conductance is different from the forskolin-activated conductance. The increase of the Cl^– conductance in the presence of forskolin by carbachol may be due to activation of different Cl^– channels or to modulation of the PKA-activated Cl^– channels by activated PKC.The authors are grateful to Drs. Laboisse and Augeron for providing the cell clone, and we thank Prof. Dr. F.H. Lopes da Silva for his comments. This work was supported by a grant from the Dutch Organization for Scientific Research, NWO.     

The electrophysiology and pharmacology of lobster neuromuscular synapses

Effects of drugs on resting potential, membrane resistance, and excitatory and inhibitory postsynaptic potentials (e.p.s.p.'s and i.p.s.p.'s) of lobster muscle fibers were studied using intracellular microelectrodes Acetylcholine, d-tubocurarine, strychnine, and other drugs of respectively related actions on vertebrate synapses were without effects even in 1 per cent solutions (10^- w/v). Gamma-aminobutyric acid (GABA) acted powerfully and nearly maximally at 10^-7 to 10^-6 w/v. Membrane resistance fell two- to tenfold, the resting potential usually increasing slightly. This combination of effects, which indicates activation of inhibitory synaptic membrane, was also produced by other short chain ω-amino acids and related compounds that inactivate depolarizing axodendritic synapses of cat. The conductance change, involving increased permeability to Cl^-, by its clamping action on membrane potential shortened as well as decreased individual e.p.s.p.'s. Picrotoxin in low concentration (ca. 10^-7 w/v) and guanidine in higher (ca. 10^-3 w/v) specifically inactivate inhibitory synapses. GABA and picrotoxin are competitive antagonists. The longer chain ω-amino acids which inactivate hyperpolarizing axodendritic synapses of cat are without effect on lobster neuromuscular synapse. However, one member of this group, carnitine (β-OH-GABA betaine), activated the excitatory synapses, a decreased membrane resistance being associated with depolarzation. The pharmacological properties of lobster neuromuscular synapses and probably also of other crustacean inhibitory synapses appear to stand in a doubly inverted relation to axodendritic synapses of cat.     

Properties of the chloride-ATPase fromLimonium salt glands: Activation by,and binding to,specific sugars

Summary Attempts to separate membrane fractions enriched in Cl^–-ATPase activity fromLimonium leaf microsomes were hampered because, it seemed, the microsomal membranes were aggregated in clumps. We found hemagglutination activity, specific for N-acetylgalactosamine and to a lesser extent galactose, in the soluble phase of the homogenate, and we were able to prevent membrane aggregation by adding galactose to the microsomes. We discovered that the Cl^–-ATPase activity of the microsomes was increased by galactose and to an even greater extent by N-acetylgalactosamine. We report that the Cl^–-ATPase binds to galactosamine-sepharose, from which it can be eluted in 0.1m galactose, i.e., the enzyme is associated with a saccharide-binding site similar to that of the hemagglutinins. This procedure results in a 100-fold enrichment of the Cl^–-ATPase activity and represents a new way of purifying a membrane-bound enzyme from a heterogeneous membrane preparation in one step. Enzyme isolated by affinity chromatography of Triton-solubilized membranes was essentially free of other ATPase and accounted for a substantial proportion (sometimes all) of the Cl^–-ATPase of the microsomes. This purified preparation of the enzyme shows N-acetylgalactosamine-specific hemagglutination activity. However, we can show that the Cl^–-ATPase and the hemagglutinins are different entities. Thus, material isolated in the same way from salt-free plants showed hemagglutination but not Cl^–-ATPase activity. Also, the hemagglutinins, but not the Cl^–-ATPase, will bind to galactosaminesepharose in the absence of ATP.This is the first report of enzyme activity associated with a carbohydrate receptorspecific protein. Possible roles for saccharide-binding in the control, assembly, and orientation of the chloride-pump are discussed.