Plasmodium falciparum activates endogenous Cl(-) channels of human erythrocytes by membrane oxidation.

Intraerythrocytic survival of the malaria parasite Plasmodium falciparum requires that host cells supply nutrients and dispose of waste products. This solute transport is accomplished by infection-induced new permeability pathways (NPP) in the erythrocyte membrane. Here, whole-cell patch-clamp and hemolysis experiments were performed to define properties of the NPP. Parasitized but not control erythrocytes constitutively expressed two types of anion conductances, differing in voltage dependence and sensitivity to inhibitors. In addition, infected but not control cells hemolyzed in isosmotic sorbitol solution. Both conductances and hemolysis of infected cells were inhibited by reducing agents. Conversely, oxidation induced identical conductances and hemolysis in non-infected erythrocytes. In conclusion, P.falciparum activates endogenous erythrocyte channels by applying oxidative stress to the host cell membrane.     

Basolateral Cl channels in primary airway epithelial cultures

Salt and water absorption and secretion across the airway epithelium are important for maintaining the thin film of liquid lining the surface of the airway epithelium. Movement of Cl across the apical membrane involves the CFTR Cl channel; however, conductive pathways for Cl movement across the basolateral membrane have been little studied. Here, we determined the regulation and single-channel properties of the Cl conductance (G(Cl)) in airway surface epithelia using epithelial cultures from human or bovine trachea and freshly isolated ciliated cells from the human nasal epithelium. In Ussing chamber studies, a swelling-activated basolateral G(Cl) was found, which was further stimulated by forskolin and blocked by N-phenylanthranilic acid (DPC) = sucrose > flufenamic acid = niflumic acid = glibenclamide > CdCl(2) = 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) = DIDS = ZnCl(2) > tamoxifen > 4,4'-dinitro-2,2'-stilbene-disulfonate disodium salt (DNDS). In whole cell patch-clamp experiments, three types of G(Cl) were identified: 1) a voltage-activated, DIDS- (but not Cd-) blockable and osmosensitive G(Cl); 2) an inwardly rectifying, hyperpolarization-activated and Cd-sensitive G(Cl); and 3) a forskolin-activated, linear G(Cl), which was insensitive to Cd and DIDS. In cell-attached patch-clamp recordings, the basolateral pole of isolated ciliated cells expressed three types of Cl channels: 1) an outwardly rectifying, swelling-activated Cl channel; 2) a strongly inwardly rectifying Cl channel; and 3) a forskolin-activated, low-conductance channel. We propose that, depending on the driving force for Cl across the apical membrane, basolateral Cl channels confine Cl(-) secretion or support transcellular Cl(-) absorption.     

Volume-dependent ATP-conductive large-conductance anion channel as a pathway for swelling-induced ATP release

In mouse mammary C127i cells, during whole-cell clamp, osmotic cell swelling activated an anion channel current, when the phloretin-sensitive, volume-activated outwardly rectifying Cl(-) channel was eliminated. This current exhibited time-dependent inactivation at positive and negative voltages greater than around +/-25 mV. The whole-cell current was selective for anions and sensitive to Gd(3)+. In on-cell patches, single-channel events appeared with a lag period of approximately 15 min after a hypotonic challenge. Under isotonic conditions, cell-attached patches were silent, but patch excision led to activation of currents that consisted of multiple large-conductance unitary steps. The current displayed voltage- and time-dependent inactivation similar to that of whole-cell current. Voltage-dependent activation profile was bell-shaped with the maximum open probability at -20 to 0 mV. The channel in inside-out patches had the unitary conductance of approximately 400 pS, a linear current-voltage relationship, and anion selectivity. The outward (but not inward) single-channel conductance was suppressed by extracellular ATP with an IC(50) of 12.3 mM and an electric distance (delta) of 0.47, whereas the inward (but not outward) conductance was inhibited by intracellular ATP with an IC(50) of 12.9 mM and delta of 0.40. Despite the open channel block by ATP, the channel was ATP-conductive with P(ATP)/P(Cl) of 0.09. The single-channel activity was sensitive to Gd(3)+, SITS, and NPPB, but insensitive to phloretin, niflumic acid, and glibenclamide. The same pharmacological pattern was found in swelling-induced ATP release. Thus, it is concluded that the volume- and voltage-dependent ATP-conductive large-conductance anion channel serves as a conductive pathway for the swelling-induced ATP release in C127i cells.     

Plasmodium induces swelling-activated ClC-2 anion channels in the host erythrocyte

Intraerythrocytic growth of the human malaria parasite Plasmodium falciparum depends on delivery of nutrients. Moreover, infection challenges cell volume constancy of the host erythrocyte requiring enhanced activity of cell volume regulatory mechanisms. Patch clamp recording demonstrated inwardly and outwardly rectifying anion channels in infected but not in control erythrocytes. The molecular identity of those channels remained elusive. We show here for one channel type that voltage dependence, cell volume sensitivity, and activation by oxidation are identical to ClC-2. Moreover, Western blots and FACS analysis showed protein and functional ClC-2 expression in human erythrocytes and erythrocytes from wild type (Clcn2(+/+)) but not from Clcn2(-/-) mice. Finally, patch clamp recording revealed activation of volume-sensitive inwardly rectifying channels in Plasmodium berghei-infected Clcn2(+/+) but not Clcn2(-/-) erythrocytes. Erythrocytes from infected mice of both genotypes differed in cell volume and inhibition of ClC-2 by ZnCl(2) (1 mm) induced an increase of cell volume only in parasitized Clcn2(+/+) erythrocytes. Lack of ClC-2 did not inhibit P. berghei development in vivo nor substantially affect the mortality of infected mice. In conclusion, activation of host ClC-2 channels participates in the altered permeability of Plasmodium-infected erythrocytes but is not required for intraerythrocytic parasite survival.     

An outwardly rectifying anionic background current in atrial myocytes from the human heart

This report describes a hitherto unreported anionic background current from human atrial cardiomyocytes. Under whole-cell patch-clamp with anion-selective conditions, an outwardly rectifying anion current (I(ANION)) was observed, which was larger with iodide than nitrate, and with nitrate than chloride as charge carrier. In contrast with a previously identified background anionic current from small mammal cardiomyocytes, I(ANION) was not augmented by the pyrethroid tefluthrin (10 microM); neither was it inhibited by hyperosmolar external solution nor by DIDS (200 microM); thus I(ANION) was not due to basal activity of volume-sensitive anion channels. I(ANION) was partially inhibited by the Cl(-) channel blockers NPPB (50 microM) and Gly H-101 (30 microM). Incorporation of I(ANION) into a human atrial action potential (AP) simulation led to depression of the AP plateau, accompanied by alterations to plateau inward calcium current, and to AP shortening at 50% but not 90% of complete repolarization, demonstrating that I(ANION) can influence the human atrial AP profile.     

Swelling-induced, CFTR-independent ATP release from a human epithelial cell line: lack of correlation with volume-sensitive cl(-) channels.

To examine a possible relation between the swelling-induced ATP release pathway and the volume-sensitive Cl(-) channel, we measured the extracellular concentration of ATP released upon osmotic swelling and whole-cell volume-sensitive Cl(-) currents in a human epithelial cell line, Intestine 407, which lacks expression of cystic fibrosis transmembrane conductance regulator (CFTR). Significant release of ATP was observed within several minutes after a hypotonic challenge (56-80% osmolality) by the luciferin/luciferase assay. A carboxylate analogue Cl(-) channel blocker, 5-nitro-2-(3-phenylpropylamino)-benzoate, suppressed ATP release in a concentration-dependent manner with a half-maximal inhibition concentration of 6.3 microM. However, swelling-induced ATP release was not affected by a stilbene-derivative Cl(-) channel blocker, 4-acetamido-4'-isothiocyanostilbene at 100 microM. Glibenclamide (500 microM) and arachidonic acid (100 microM), which are known to block volume-sensitive outwardly rectifying (VSOR) Cl(-) channels, were also ineffective in inhibiting the swelling-induced ATP release. Gd(3+), a putative blocker of stretch-activated channels, inhibited swelling-induced ATP release in a concentration-dependent manner, whereas the trivalent lanthanide failed to inhibit VSOR Cl(-) currents. Upon osmotic swelling, the local ATP concentration in the immediate vicinity of the cell surface was found to reach approximately 13 microM by a biosensor technique using P2X(2) receptors expressed in PC12 cells. We have raised antibodies that inhibit swelling-induced ATP release from Intestine 407 cells. Earlier treatment with the antibodies almost completely suppressed swelling-induced ATP release, whereas the activity of VSOR Cl(-) channel was not affected by pretreatment with the antibodies. Taking the above results together, the following conclusions were reached: first, in a CFTR-lacking human epithelial cell line, osmotic swelling induces ATP release and increases the cell surface ATP concentration over 10 microM, which is high enough to stimulate purinergic receptors; second, the pathway of ATP release is distinct from the pore of the volume-sensitive outwardly rectifying Cl(-) channel; and third, the ATP release is not a prerequisite to activation of the Cl(-) channel.     

Low-conductance chloride channel activated by cAMP in the epithelial cell line T84

We have studied the modulation and pharmacological properties of two anion channels in T₈₄ cells by recording single channel and transepithelial currents. One channel had an outwardly rectifying current-voltage I/V curve, was rarely active in cell-attached patches, and was unaffected by cAMP. The other channel had lower conductance (8.7 pS at 37°C) and a more ohmic I/V relationship. Exposure to cAMP increased the probability of observing low-conductance channel activity in cell-attached patches> 6-fold. Extracellular DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid) or IAA-94 (an indanyloxyacetic acid) inhibited the outward rectifier but did not affect the low-conductance channel or cAMP-stimulated transepithelial current. These results suggest the low-conductance Cl channel may contribute to apical membrane conductance during cAMP-stimulated secretion.     

An inwardly rectifying potassium channel in the basolateral membrane of sheep parotid secretory cells

Summary Using whole-cell patch-clamp techniques, we demonstrate that sheep parotid secretory cells have both inwardly and outwardly rectifying currents. The outwardly rectifying current, which is blocked by 10 mmol/liter tetraethylammonium (TEA) applied extracellularly, is probably carried by the 250 pS Ca²⁺-and voltage-activated K⁺ (BK) channel which has been described in previous studies. In contrast, the inwardly rectifying current, which is also carried by K⁺ ions, is not sensitive to TEA. It is similar to the inwardly rectifying currents observed in many excitable tissues in that (i) its conductance is dependent on the square root of the extracellular K⁺, (ii) the voltage range over which it is activated is influenced by the extracellular K⁺ concentration and (iii) it is blocked by the addition of Cs⁺ ions (670 µmol/liter) to the bathing solution. Our previously published cell-attached patch studies have shown that the channel type most commonly observed in the basolateral membrane of unstimulated sheep parotid secretory cells is a K⁺ channel with a conductance of 30 pS and, in this study, we find that its conductance also depends on the square root of the extracellular K⁺ concentration. It thus seems likely that it carries the inwardly rectifying K⁺ current seen in the whole-cell studies.     

Effects of NPPB (5-nitro-2-(3-phenylpropylamino)benzoic acid) on chloride transport in intestinal tissues and the T84 cell line.

NPPB (5-nitro-2-(3-phenylpropylamino)benzoic acid) has been reported to block Cl- channels in isolated rabbit nephrons with high potency (IC50 = 80 nM). The effects of this compound on Cl(-)-mediated transport processes in intestinal tissues have been studied using agonist-stimulated short-circuit current (T84) in Ussing chamber experiments and 36Cl- fluxes in monolayers of a colonic cell line (T84). NPPB inhibited PGE1-stimulated Isc in rabbit distal colon and ileum at concentrations in the range 20 to 100 microM. However, NPPB at the same concentrations also inhibited glucose-stimulated Isc in rabbit ileum, suggesting that its effects were not restricted to those on Cl- transport. Consistent with this, exposure of rabbit distal colon to 100 microM NPPB was found to reduce endogenous ATP levels by 69%, implying that, at these concentrations, NPPB could impair active transport processes by an effect on cellular energy metabolism. Clear evidence for a direct effect of NPPB on epithelial chloride channels was found in studies on Cl- fluxes in T84 cell monolayers. NPPB inhibited VIP-stimulated Cl- uptake into T84 cells with an IC50 of 414 microM. NPPB (1 mM) also inhibited Cl- efflux from pre-loaded cells confirming its effect as a weak Cl- channel blocker in this system.     

Citrate-permeable channels in the plasma membrane of cluster roots from white lupin

White lupin (Lupinus albus) is well adapted to phosphorus deficiency by developing cluster roots that release large amounts of citrate into the rhizosphere to mobilize the sparingly soluble phosphorus. To determine the mechanism underlying citrate release from cluster roots, we isolated protoplasts from different types of roots of white lupin plants grown in phosphorus-replete (+P) and phosphorus-deficient (-P) conditions and used the patch-clamp technique to measure the whole-cell currents flowing across plasma membrane of these protoplasts. Two main types of anion conductance were observed in protoplasts prepared from cluster root tissue: (1) an inwardly rectifying anion conductance (IRAC) activated by membrane hyperpolarization, and (2) an outwardly rectifying anion conductance (ORAC) that became more activated with membrane depolarization. Although ORAC was an outward rectifier, it did allow substantial inward current (anion efflux) to occur. Both conductances showed citrate permeability, with IRAC being more selective for citrate3- than Cl- (PCit/PCl = 26.3), while ORAC was selective for Cl- over citrate (PCl/PCit = 3.7). Both IRAC and ORAC were sensitive to the anion channel blocker anthracene-9-carboxylic acid. These currents were also detected in protoplasts derived from noncluster roots of -P plants, as well as from normal (noncluster) roots of plants grown with 25 microm phosphorus (+P). No differences were observed in the magnitude or frequency of IRAC and ORAC currents between the cluster roots and noncluster roots of -P plants. However, the IRAC current from +P plants occurred less frequently than in the -P plants. IRAC was unaffected by external phosphate, but ORAC had reduced inward current (anion efflux) when phosphate was present in the external medium. Our data suggest that IRAC is the main pathway for citrate efflux from white lupin roots, but ORAC may also contribute to citrate efflux.     

Tefluthrin modulates a novel anionic background conductance (I(AB)) in guinea-pig ventricular myocytes

This report describes for the first time a novel anionic background current (I(AB)) identified in guinea-pig isolated ventricular myocytes. It also shows that I(AB) has both novel and differential pharmacology from other (cardiac) chloride currents. Using the whole-cell patch-clamp technique and external anion substitution, I(AB) was found to be outwardly rectifying and highly permeable to NO(-)(3), with a relative permeability sequence of NO(-)(3) > I(-) > Cl(-). I(AB) was not blocked by 50 microM DIDS, by hypertonic external solution, or by the nonselective protein kinase inhibitor H7-DHC. Exposure to the pyrethroid agent tefluthrin (10 microM) increased the current density of I(AB) significantly at positive voltages (P < 0.05), but had no significant effect on other cardiac chloride currents. We conclude that I(AB) possesses a distinct pharmacology and does not fall into the three major classes of cardiac chloride conductance commonly reported.     

Sodium-dependent chloride transport in basolateral membrane vesicles isolated from rabbit proximal tubule

The mechanisms for Cl transport across basolateral membrane vesicles (BLMV) isolated from rabbit renal cortex were examined by using the Cl-sensitive fluorescent indicator 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ). The transporters studied included Cl/base exchange, Cl/base/Na cotransport, K/Cl cotransport, and Cl conductance. Initial rates of chloride influx (JCl) were determined from the measured time course of SPQ fluorescence in BLMV following inwardly directed gradients of Cl and gradients of other ions and/or pH. For a 50 mM inwardly directed Cl gradient in BLMV which were voltage and pH clamped (7.0) using K/valinomycin and nigericin, JCl was 0.80 +/- 0.14 nmol S-1 (mg of vesicle protein)-1 (mean +/- SD, n = 8 separate preparations). In the absence of Na and CO2/HCO3 in voltage-clamped BLMV, JCl increased 56% +/- 5% in response to a 1.9 pH unit inwardly directed H gradient; the increase was further enhanced by 40% +/- 3% in the presence of CO2/HCO3 and inhibited 30% +/- 8% by 100 microM dihydro-4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. Na gradients did not increase JCl in the absence of CO2/HCO3; however, an outwardly directed Na gradient in the presence of CO2/HCO3 increased JCl by 31% +/- 8% with a Na KD of 7 +/- 2 mM. These results indicate the presence of Cl/OH and Cl/HCO3 exchange, and Cl/HCO3 exchange trans-stimulated by Na. There was no significant effect of K gradients in the presence or absence of valinomycin, suggesting lack of significant K/Cl cotransport and Cl conductance under experimental conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Development of a colorimetric method for functional chloride channel assay

Anion channels play significant physiological roles in humans and animals. However, the effort of screening for anion channel modulators was limited by the available assay technologies. This report discusses the development of a cell-based functional chloride channel assay using iodine as the chloride channel functional indicator. Iodine concentrations were measured with modified Sandell-Kolthoff reaction using colorimetric detection. The assay was rapid and quantitative. When WSS-1 cells were activated by gamma-aminobutyric acid (GABA) in the condition that gamma-aminobutyric acid type A receptor (GABAA receptor) conducted outwardly rectifying chloride channel function, the EC50 of GABA was 7.69 microM. IC50s were 0.53 microM for bicuculline and 3.1 microM for picrotoxin, respectively, in the presence of 10 microM GABA. When Capan-1 cells were activated by forskolin, the EC50 was 0.14 microM. The assay can also be applied to inwardly rectifying anion channels as exemplified by GABAA channel with an EC50 of 294 microM. Thus, the assay is universal and reliable and can be used for anion channel high-throughput screening.     

Heterotrimeric G-proteins activate Cl- channels through stimulation of a cyclooxygenase-dependent pathway in a model liver cell line.

Circulating hormones produce rapid changes in the Cl(-) permeability of liver cells through activation of plasma membrane receptors coupled to heterotrimeric G-proteins. The resulting effects on intracellular pH, membrane potential, and Cl(-) content are important contributors to the overall metabolic response. Consequently, the purpose of these studies was to evaluate the mechanisms responsible for G-protein-mediated changes in membrane Cl(-) permeability using HTC hepatoma cells as a model. Using patch clamp techniques, intracellular dialysis with 0.3 mm guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) increased membrane conductance from 10 to 260 picosiemens/picofarads due to activation of Ca(2+)-dependent Cl(-) currents that were outwardly rectifying and exhibited slow activation at depolarizing potentials. These effects were mimicked by intracellular AlF(4)(-) (0.03 mm) and inhibited by pertussis toxin (PTX), consistent with current activation through Galpha(i). Studies using defined agonists and inhibitors indicate that Cl(-) channel activation by GTPgammaS occurs through an indomethacin-sensitive pathway involving sequential activation of phospholipase C, mobilization of Ca(2+) from inositol 1,4,5-trisphosphate-sensitive stores, and stimulation of phospholipase A(2) and cyclooxygenase (COX). Accordingly, the conductance responses to GTPgammaS or to intracellular Ca(2+) were inhibited by COX inhibitors. These results indicate that PTX-sensitive G-proteins regulate the Cl(-) permeability of HTC cells through Ca(2+)-dependent stimulation of COX activity. Thus, receptor-mediated activation of Galpha(i) may be essential for hormonal regulation of liver transport and metabolism through COX-dependent opening of a distinct population of plasma membrane Cl(-) channels.     

Plasmodium falciparum-activated chloride channels are defective in erythrocytes from cystic fibrosis patients

An inwardly rectifying anion channel in malaria-infected red blood cells has been proposed to function as the "new permeation pathway" for parasite nutrient acquisition. As the channel shares several properties with the cystic fibrosis transmembrane conductance regulator (CFTR), we tested their interrelationship by whole-cell current measurements in Plasmodium falciparum-infected and uninfected red blood cells from control and cystic fibrosis (CF) patients. A CFTR-like linear chloride conductance as well as a malaria parasite-induced and a shrinkage-activated endogenous inwardly rectifying chloride conductance with properties identical to the malaria-induced channel were all found to be defective in CF erythrocytes. Surprisingly, the absence of the inwardly rectifying chloride conductance in CF erythrocytes had no gross effect on in vitro parasite growth or new permeation pathway activity, supporting an argument against a close association between the Plasmodium-activated chloride channel and the new permeation pathway. The functional expression of CFTR in red blood cells opens new perspectives to exploit the erythrocyte as a readily available cell type in electrophysiological, diagnostic, and therapeutic studies of CF.     

G-protein activators induce a potassium conductance in murine macrophages

The whole-cell patch clamp technique was used to test whether intracellular application of G-protein activators affect ionic currents in murine macrophages. Both the J774.1 macrophage-like cell line and primary bone marrow derived macrophages were used. Cells were bathed in Na Hanks' solution and intracellularly dialyzed (via the patch pipette) with K Hanks (145 mM KCl, < 100 nM Ca) plus or minus the G-protein activators GTP gamma S (10 microM), GppNHp (10 microM), or AIF4- (200 microM AlCl3 + 5 mM KF). In the absence of G-protein activators, only two K currents, an inwardly rectifying K current (Kir) and an outward, inactivating K current (Ko) were observed. In the presence of protein activators, two effects were observed: (i) the Kir conductance, which is stable for up to 30 min under control conditions, decayed twice as fast and (ii) an outwardly rectifying, noninactivating current appeared. The induced outward current appeared < 2 min after attaining the whole-cell patch clamp configuration. The current could be distinguished from the Kir and Ko currents on the basis of its direction of rectification (outward), barium sensitivity (> 1 mM), and kinetics (no time-dependent inactivation). Intracellular application of GTP (500 microM), GDP (500 microM), cAMP (100 microM + 0.5 mM ATP), or IP3 (20 microM) did not induce the current; 100 microM ATP gamma S activated a half-maximal amount of current. Induction of outward current by 10 microM GTP gamma S could be prevented by pre-exposing cells to pertussis toxin but not cholera toxin. This current is K selective since (i) its induction was accompanied by hyperpolarization of the cell toward EK, even after Kir had "washed out", (ii) it was present after > 90% of both intracellular and extracellular Cl were replaced by isethionate, and (iii) the induced outward conductance was absent when Ki was completely replaced by Cs, and was reduced by approximately 1/3 when [K]i was reduced by 1/3. Quinidine (1 mM) and 4-aminopyridine (10 mM) inhibited the current, but apamin (1 microM) and charybdotoxin (1 microM) did not.     

Electrophysiological response of rat atrial myocytes to acidosis

The effect of acidosis on the electrical activity of isolated rat atrial myocytes was investigated using the patch-clamp technique. Reducing the pH of the bathing solution from 7.4 to 6.5 shortened the action potential. Acidosis had no significant effect on transient outward or inward rectifier currents but increased steady-state outward current. This increase was still present, although reduced, when intracellular Ca(2+) was buffered by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA); BAPTA also inhibited acidosis-induced shortening of the action potential. Ni(2+) (5 mM) had no significant effect on the acidosis-induced shortening of the action potential. Acidosis also increased inward current at -80 mV and depolarized the resting membrane potential. Acidosis activated an inwardly rectifying Cl(-) current that was blocked by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), which also inhibited the acidosis-induced depolarization of the resting membrane potential. It is concluded that an acidosis-induced increase in steady-state outward K(+) current underlies the shortening of the action potential and that an acidosis-induced increase in inwardly rectifying Cl(-) current underlies the depolarization of the resting membrane potential during acidosis.     

Inwardly rectifying chloride channel activity in intestinal pacemaker cells

Cl(-) channels are proposed to play a role in gut pacemaker activity, but little is known about the characteristics of Cl(-) channels in interstitial cells of Cajal (ICC), the intestinal pacemaker cells. The objective of the present study was to identify whole cell Cl(-) currents in ICC associated with previously observed single-channel activity and to characterize its inward rectification. Whole cell patch-clamp studies showed that ICC express an inwardly rectifying Cl(-) current that was not sensitive to changes in cation composition of the extracellular solutions. Currents were not affected by replacing all cations with N-methyl-d-glucamine (NMDG(+)). Whole cell currents followed the Cl(-) equilibrium potential and were inhibited by DIDS and 9-anthracene carboxylic acid. Ramp protocols of single-channel activity showed that inward rectification was due to reduction in single-channel open probability, not a reduction in single-channel conductance. Single-channel data led to the hypothesis that strong cooperation exists between 30-pS channels that show less cooperation at potentials positive to the reversal potential. Hence, an inwardly rectifying Cl(-) channel plays a prominent role in determining pacemaker activity in the gut.     

Two ATP-activated conductances in bullfrog atrial cells

Currents activated by extracellular ATP were studied in single voltage-clamped bullfrog atrial cells. Rapid application of ATP elicited currents carried through two different conductance pathways: a rapidly desensitizing conductance reversing near -10 mV, and a maintained, inwardly rectifying conductance reversing near -85 mV. ATP activated the desensitizing component of current with a K 1/2 of approximately 50 microM and the maintained component with a K 1/2 of approximately 10 microM. Both types of current were activated by ATP but not by adenosine, AMP, or ADP. The desensitizing current was selectively inhibited by alpha, beta-methylene ATP, and the maintained, inwardly rectifying current was selectively suppressed by extracellular Cs. The desensitizing component of current was greatly reduced when extracellular Na was replaced by N-methylglucamine, but was slightly augmented when Na was replaced by Cs. GTP, ITP, and UTP were all ineffective in activating the desensitizing current, and of a variety of ATP analogues, only ATP-gamma-S was effective. Addition of EGTA or BAPTA to the intracellular solution did not obviously affect the desensitizing current. Fluctuation analysis of currents through the desensitizing conductance suggested that current is carried through ionic channels with a small (less than pS) unitary conductance.     

Synthetic mammalian C-type natriuretic peptide forms large cation channels

We report the first evidence that synthetic human C-type natriuretic peptide-22 and the OaC-type natriuretic peptide-39(18-39), a 22 amino acid fragment of the OaC-type natriuretic peptide-39 from platypus venom, can function directly by forming a novel voltage-gated weakly cation-selective channel in negatively charged artificial lipid bilayer membranes. The channel activity is characterized by a tendency for inactivation at negative voltages, e.g. -60 and -70 mV, whereas at positive voltages the channel is fully open. The channel has a maximal cord conductance of 546+/-23 pS (n = 16) and shows weak outward rectification. The sequence and the permeability ratios were P(K)+: P(Cs)+: P(Na)+: P(choline)+ 1:0.88:0.76:0.13, respectively. The addition of 50 mM TEA+ cis (a blocker of outwardly rectifying K+ channels), 20 mM Cs+ cis (a blocker of inwardly rectifying K+ channels) or 0.5 mM glibenclamide cis (a blocker of ATP-sensitive K+ channels) to the cis chamber did not affect the conductance or the kinetics of the OaC-type natriuretic peptide-39(18-39)-formed channels (n = 2-5). It is concluded that the weak cation selectivity, large conductance and high open probability as well as their voltage dependency are consistent with the ability of these peptides to cause that loss of compartmentation of the membrane, which is a characteristic feature of adverse conditions that cause C-type natriuretic peptide-related pathologies.