The Ca2+-inactivated Cl− Channel at Work: Selectivity,Blocker Kinetics and Transport Visualization

Removal of extracellular divalent cations activated a Cl⁻ channel in the plasma membrane of Xenopus laevis oocytes. This so-called Ca²⁺-inactivated Cl⁻ channel (CaIC) was present in every oocyte and was investigated using two-electrode whole-cell voltage clamp and single-channel patch-clamp techniques. Beside other Cl⁻ channel inhibitors, anthracene-9-carboxylic acid (9-AC) and 3′azido-3′deoxythymidine (AZT), a nucleoside analogue commonly used as an antiviral drug, blocked at least partly the CalC-mediated currents. Using the Cl⁻-sensitive dye 6-methoxy-N-(sulfopropyl)quinolinium (SPQ) we could visualize the transport of Cl⁻ from the oocyte cytoplasm to the surrounding medium after activation of the CaIC by Ca²⁺ removal. In the absence of external Cl⁻ and Ca²⁺, the emission intensity of SPQ declined continuously, indicating a quenching of fluorescence by the efflux of Cl⁻ in the millimolar range. In the presence of external Ca²⁺, no emission changes could be observed during the same time period. Chelating external Ca²⁺ in absence of Cl⁻ immediately activated Ca²⁺-inactivated Cl⁻ channels leading to subsequent emission decrease of SPQ. Investigations on the selectivity of the CaIC revealed only poor discrimination between different anions. With single-channel measurements, we found an anion selectivity sequence I⁻ > Br⁻ > Cl⁻≫ gluconate as it is also typical for maxi Cl⁻ channels. Contrary to the majority of all other transport systems of the Xenopus oocyte, which show reduced activity due to membrane depolarization or endocytotic removal of the transport protein from the plasma membrane during oocyte maturation, the CaIC remained active in maturated oocytes. Single-channel measurements on maturated oocytes, also known as eggs, showed the presence of Ca²⁺-inactivated Cl⁻ channels. However, this egg CaIC revealed an altered sensitivity to external Ca²⁺ concentrations. All these data confirm and extend our previous observations on the CaIC and give clear evidence that this channel is peculiar among all Cl⁻ channels described up to now. Received: 16 May 1996/Revised: 4 September 1996     

Ion Channel Phenotype of Melanoma Cell Lines

Melanoma cells are transformed melanocytes of neural crest origin. K⁺ channel blockers have been reported to inhibit melanoma cell proliferation. We used whole-cell recording to characterize ion channels in four different human melanoma cell lines (C8161, C832C, C8146, and SK28). Protocols were used to identify voltage-gated (K_V), Ca²⁺-activated (K_Ca), and inwardly rectifying (K_IR) K⁺ channels; swelling-sensitive Cl⁻ channels (Cl_swell); voltage-gated Ca²⁺ channels (Ca_V) and Ca²⁺ channels activated by depletion of intracellular Ca²⁺ stores (CRAC); and voltage-gated Na⁺ channels (Na_V). The presence of Ca²⁺ channels activated by intracellular store depletion was further tested using thapsigargin to elicit a rise in [Ca²⁺] _i . The expression of K⁺ channels varied widely between different cell lines and was also influenced by culture conditions. K_IR channels were found in all cell lines, but with varying abundance. Whole-cell conductance levels for K_IR differed between C8161 (100 pS/pF) and SK28 (360 pS/pF). K_Ca channels in C8161 cells were blocked by 10 nm apamin, but were unaffected by charybdotoxin (CTX). K_Ca channels in C8146 and SK28 cells were sensitive to CTX (K _d = 4 nm), but were unaffected by apamin. K_V channels, found only in C8146 cells, activated at ∼−20 mV and showed use dependence. All melanoma lines tested expressed CRAC channels and a novel Cl_swell channel. Cl_swell current developed at 30 pS/sec when the cells were bathed in 80% Ringer solution, and was strongly outwardly rectifying (4:1 in symmetrical Cl⁻). We conclude that different melanoma cell lines express a diversity of ion channel types. Received: 2 April 1996/Revised: 22 August 1996     

Purinergic signalling in rat GFSHR-17 granulosa cells: an <Emphasis Type="Italic">in vitro</Emphasis> model of granulosa cells in maturing follicles

Purinergic signalling in rat GFSHR-17 granulosa cells was characterised by Ca²⁺-imaging and perforated patch-clamp. We observed a resting intracellular Ca²⁺-concentration ([Ca²⁺]_i) of 100 nM and a membrane potential of −40 mV. This was consistent with high K⁺− and Cl⁻ permeability and a high intracellular Cl⁻ concentration of 40 mM. Application of ATP for 5–15 s every 3 min induced repeated [Ca²⁺]_i increases and a 30 mV hyperpolarization. The phospholipase C inhibitor U73122 or the IP₃-receptor antagonist 2-aminoethoethyl diphenyl borate suppressed ATP responses. Further biochemical and pharmacological experiments revealed that ATP responses were related to stimulation of P2Y₂ and P2Y₄ receptors and that the [Ca²⁺]_i increase was a prerequisite for hyperpolarization. Inhibitors of Ca²⁺-activated channels or K⁺ channels did not affect the ATP-evoked responses. Conversely, inhibitors of Cl⁻ channels hyperpolarized cells to −70 mV and suppressed further ATP-evoked hyperpolarization. We propose that P2Y₂ and P2Y₄ receptors in granulosa cells modulate Cl⁻ permeability by regulating Ca²⁺-release.     

Regulation of Chloride Channels in Secretory Epithelia

Fluid and electrolyte secretion from secretory epithelia is a highly regulated process. Chloride channel activity at the apical membrane determines the rate and direction of salt and water secretion. Multiple classes of Cl⁻ channels with distinct gating mechanisms are involved in moving ions and water. Secretory agonists that induce intracellular increases in two second messenger systems, cAMP and [Ca²⁺] _i , are generally associated with secretion. However, changes in cell volume and the membrane potential may also play a role in regulating fluid and electrolyte secretion in some tissues. In this review we discuss the regulation of the different types of Cl⁻ channels found in secretory epithelia. Received: 16 September 1997/Revised: 13 November 1997     

Tubular Fluid Secretion in the Seminiferous Epithelium: Ion Transporters and Aquaporins in Sertoli Cells

Sertoli cells play a key role in the establishment of an adequate luminal environment in the seminiferous tubules of the male reproductive tract. Secretion of the seminiferous tubular fluid (STF) is vital for the normal occurrence of spermatogenesis and for providing a means of transport to the developing spermatozoa. However, several studies on this subject have not completely clarified the origin and composition of this fluid. Electrolyte and water are central components of STF. Sertoli cells secrete an iso-osmotic fluid with a higher content of K⁺ than the blood and express various membrane and water transporters (Na⁺/K⁺-ATPase; Ca²⁺-ATPase; V-type ATPase; Cl⁻ channels; CFTR Cl⁻ channels; K⁺ channels; L-, T- and N-type Ca²⁺ channels; Na⁺/H⁺ exchangers; Na⁺-driven HCO₃ ⁻/Cl⁻ exchangers (NDCBEs); Na⁺/HCO₃ ⁻ cotransporters (NBCes); Na⁺–K⁺–2Cl⁻ cotransporter; Na⁺/Ca²⁺ exchanger; and aquaporins 0 and 8) involved in cellular and secretory functions. Studies with knockout mice for some of these transporters showed tubular fluid accumulation and associated infertility, revealing the relevance of these processes for the normal occurrence of spermatogenesis. Nevertheless, the role of the several membrane transporters in the establishment of STF electrolyte composition needs to be further elucidated. This review summarizes the available data on the ionic composition of STF and on the Sertoli cell membrane mechanisms responsible for ion and water movement. Deepening the knowledge on the mechanisms involved in the secretion, composition and regulation of SFT is essential and will be a major step in understanding the infertility associated with some pathological conditions.     

Nifedipine-sensitive low voltage-activated calcium current in neurons of the rat laterodorsal thalamic nucleus

The kinetic and pharmacological properties of low voltage-activated (LVA) Ca²⁺ channels were studied in neurons of the laterodorsal (LD) thalamic nucleus in brain slices from 12-day-old rats. A homogeneous population of LVA Ca²⁺ channels was found in the tested neurons. LVA Ca²⁺ current evoked by a step depolarization from a holding potential more negative than −70 mV was found to be sensitive to nifedipine (K _d=2.6 (M). This current gained its maximum at −55 mV and demonstrated fast monoexponential decay with the time constant of 32.3±4.0 msec (n=15). Lanthanum (1 μM) effectively blocked LVA Ca²⁺ current, while nickel (25 μM) did not affect this current. It is concluded that the channels that, according to their pharmacological properties, provide the studied LVA Ca²⁺ current cannot be regarded as T-type Ca²⁺ channels and belong to some other type of LVA Ca²⁺ channels.     

Ion Channels in Cell Proliferation and Apoptotic Cell Death

Cell proliferation and apoptosis are paralleled by altered regulation of ion channels that play an active part in the signaling of those fundamental cellular mechanisms. Cell proliferation must - at some time point - increase cell volume and apoptosis is typically paralleled by cell shrinkage. Cell volume changes require the participation of ion transport across the cell membrane, including appropriate activity of Cl⁻ and K⁺ channels. Besides regulating cytosolic Cl⁻ activity, osmolyte flux and, thus, cell volume, most Cl⁻ channels allow HCO₃⁻ exit and cytosolic acidification, which inhibits cell proliferation and favors apoptosis. K⁺ exit through K⁺ channels may decrease intracellular K⁺ concentration, which in turn favors apoptotic cell death. K⁺ channel activity further maintains the cell membrane potential, a critical determinant of Ca²⁺ entry through Ca²⁺ channels. Cytosolic Ca²⁺ may trigger mechanisms required for cell proliferation and stimulate enzymes executing apoptosis. The switch between cell proliferation and apoptosis apparently depends on the magnitude and temporal organization of Ca²⁺ entry and on the functional state of the cell. Due to complex interaction with other signaling pathways, a given ion channel may play a dual role in both cell proliferation and apoptosis. Thus, specific ion channel blockers may abrogate both fundamental cellular mechanisms, depending on cell type, regulatory environment and condition of the cell. Clearly, considerable further experimental effort is required to fully understand the complex interplay between ion channels, cell proliferation and apoptosis.     

BK Ca Channels Activating at Resting Potential without Calcium in LNCaP Prostate Cancer Cells

Large-conductance Ca²⁺-dependent K⁺ (BK_Ca) channels are activated by intracellular Ca²⁺ and membrane depolarization in an allosteric manner. We investigated the pharmacological and biophysical characteristics of a BK_Ca-type K⁺ channel in androgen-dependent LNCaP (lymph node carcinoma of the prostate) cells with novel functional properties, here termed BK_L. K⁺ selectivity, high conductance, activation by Mg²⁺ or NS1619, and inhibition by paxilline and penitrem A largely resembled the properties of recombinant BK_Ca channels. However, unlike conventional BK_Ca channels, BK_L channels activated in the absence of free cytosolic Ca²⁺ at physiological membrane potentials; the half-maximal activation voltage was shifted by about −100 mV compared with BK_Ca channels. Half-maximal Ca²⁺-dependent activation was observed at 0.4 μM for BK_L (at −20 mV) and at 4.1 μM for BK_Ca channels (at +50 mV). Heterologous expression of hSlo1 in LNCaP cells increased the BK_L conductance. Expression of hSlo-β1 in LNCaP cells shifted voltage-dependent activation to values between that of BK_L and BK_Ca channels and reduced the slope of the P_open (open probability)-voltage curve. We propose that LNCaP cells harbor a so far unknown type of BK_Ca subunit, which is responsible for the BK_L phenotype in a dominant manner. BK_L-like channels are also expressed in the human breast cancer cell line T47D. In addition, functional expression of BK_L in LNCaP cells is regulated by serum-derived factors, however not by androgens.     

A Bicarbonate- and Weak Acid-permeable Chloride Conductance Controlled by Cytosolic Ca2+ and ATP in Rat Submandibular Acinar Cells

A Ca²⁺-activated Cl⁻ conductance in rat submandibular acinar cells was identified and characterized using whole-cell patch-clamp technique. When the cells were dialyzed with Cs-glutamate-rich pipette solutions containing 2 mm ATP and 1 μm free Ca²⁺ and bathed in N-methyl-d-glucamine chloride (NMDG-Cl) or Choline-Cl-rich solutions, they mainly exhibited slowly activating currents. Dialysis of the cells with pipette solutions containing 300 nm or less than 1 nm free Ca²⁺ strongly reduced the Cl⁻ currents, indicating the currents were Ca²⁺-dependent. Relaxation analysis of the ``on' currents of slowly activating currents suggested that the channels were voltage-dependent. The anion permeability sequence of the Cl⁻ channels was: NO⁻ ₃ (2.00) > I⁻ (1.85) ≥ Br⁻ (1.69) > Cl⁻ (1.00) > bicarbonate (0.77) ≥ acetate (0.70) > propionate (0.41) ≫ glutamate (0.09). When the ATP concentration in the pipette solutions was increased from 0 to 10 mm, the Ca²⁺-dependency of the Cl⁻ current amplitude shifted to lower free Ca²⁺ concentrations by about two orders of magnitude. Cells dialyzed with a pipette solution (pCa = 6) containing ATP-γS (2 mm) exhibited currents of similar magnitude to those observed with the solution containing ATP (2 mm). The addition of the calmodulin inhibitors trifluoperazine (100 μm) or calmidazolium (25 μm) to the bath solution and the inclusion of KN-62 (1 μm), a specific inhibitor of calmodulin kinase, or staurosporin (10 nm), an inhibitor of protein kinase C to the pipette solution had little, if any, effect on the Ca²⁺-activated Cl⁻ currents. This suggests that Ca²⁺/Calmodulin or calmodulin kinase II and protein kinase C are not involved in Ca²⁺-activated Cl⁻ currents. The outward Cl⁻ currents at +69 mV were inhibited by NPPB (100 μm), IAA-94 (100 μm), DIDS (0.03–1 mm), 9-AC (300 μm and 1 mm) and DPC (1 mm), whereas the inward currents at −101 mV were not. These results demonstrate the presence of a bicarbonate- and weak acid-permeable Cl⁻ conductance controlled by cytosolic Ca²⁺ and ATP levels in rat submandibular acinar cells. Received: 9 January 1996/Revised: 20 May 1996     

Characterization of the Stretch-activated Chloride Channel in Isolated Human Atrial Myocytes

Macroscopic and unitary currents through stretch-activated Cl⁻ channels were examined in isolated human atrial myocytes using whole-cell, excised outside-out and inside-out configurations of the patch-clamp technique. When K⁺ and Ca²⁺ conductances were blocked and the intracellular Ca²⁺ concentration ([Ca²⁺] _i ) was reduced, application of positive pressure via the pipette activated membrane currents under whole-cell voltage-clamp conditions. The reversal potential of the current shifted by 60 mV per 10-fold change in the external Cl⁻ concentration, indicating that the current was Cl⁻ selective. The current was inhibited by bath application of 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) and 9-anthracenecarboxylic acid (9-AC). β-Adrenergic stimulation failed to activate a Cl⁻ current. In single channel recordings from outside-out patches, positive pressure in the pipette activated the unitary current with half-maximal activation of 14.7 mm Hg at +40 mV. The current-voltage relationship of single channel activity obtained in inside-out patches was linear in symmetrical Cl⁻ solution with the averaged slope conductance of 8.6 ± 0.7 pS (mean ±sd, n= 10). The reversal potential shift of the channel by changing Cl⁻ concentration was consistent with a Cl⁻ selective channel. The open time distribution was best described by a single exponential function with mean open lifetime of 80.4 ± 9.6 msec (n= 9), while at least two exponentials were required to fit the closed time distributions with a time constant for the fast component of 11.5 ± 2.2 msec (n= 9) and that for the slow component of 170.2 ± 21.8 msec (n= 9). Major changes in the single channel activity in response to pressure were caused by changes in the interburst interval. Single channel activity was inhibited by DIDS and 9-AC in a manner similar to whole-cell configuration. These results suggest that membrane stretch induced by applying pressure via the pipette activated a Cl⁻ current in human atrial myocytes. The current was sensitive to Cl⁻ channel blockers and exhibited membrane voltage-independent bursting opening without sensitive to β-adrenergic stimulation. Received: 21 October 1996/Revised: 17 December 1997     

Single anion channels reconstituted from cardiac mitoplasts

Ion channels from sheep cardiac mitoplast (inverted inner mitochondrial membrane vesicle) preparations were incorporated into voltage-clamped planar lipid bilayers. The appearance of anion rather than cation channels could be promoted by exposing the bilayers to osmotic gradients formed by Cl⁻ salts of large, relatively imperment, cations at a pH of 8.8. Two distinct activities were identified. These comprised a multisubstate anion channel of intermediate conductance (∼60 pS in 300vs. 50mm choline Cl, ∼100 pS in symmetric 150mm KCl), and a lower-conductance anion channel (∼25 or ∼50 pS in similar conditions), which only displayed two well-defined substates, at ∼25 and ∼50% of the fully open state. The larger channels were not simple multiples of the lower-conductance channels, but both discriminated poorly, and to a similar extent, between anions and cations (P_Cl ⁻/P_choline ⁺ ∼12, P_Cl ⁻/P_K ⁺∼8). The lower-conductance channel was only minimally selective between different anions (P_{NO 3} ⁻ (1.0)=P_Cl ⁻>P_Br ⁻>P_I ⁻>P_SCN ⁻(0.8)), and its conductance failed to saturate even in high (>1.0 M) activities of KCl. The channels were not obviously voltage dependent, and they were unaffected by 0.5 mM SITS, H₂O₂, propranolol, quinine or amitriptyline, or by 2 mM ATP, or by variations in pH (5.5–8.8). Ca²⁺ and Mg²⁺ did not alter single channel activity, but did modify single current amplitudes in the lower-conductance channel. This effect, together with voltage-dependent substate behavior, is described in the following paper.     

Anion Channels in Chara corallina Tonoplast Membrane: Calcium Dependence and Rectification

Tonoplast K⁺ channels of Chara corallina are well characterized but only a few reports mention anion channels, which are likely to play an important role in the tonoplast action potential and osmoregulation of this plant. For experiments internodal cells were isolated. Cytoplasmic droplets were formed in an iso-osmotic bath solution according to a modified procedure. Ion channels with conductances of 48 pS and 170 pS were detected by the patch-clamp technique. In the absence of K⁺ in the bath solution the 170 pS channel was not observed at negative pipette potential values. When Cl⁻ on either the vacuolar side or the cytoplasmic side was partly replaced with F⁻, the reversal potential of the 48 pS channel shifted conform to the Cl⁻ equilibrium potential with similar behavior in droplet-attached and excised patch mode. These results showed that the 48 pS channel was a Cl⁻ channel. In droplet-attached mode the channel rectified outward current flow, and the slope conductance was smaller. When Chara droplets were formed in a bath solution containing low (10⁻⁸ m) Ca²⁺, then no Cl⁻ channels could be detected either in droplet-attached or in inside-out patch mode. Channel activity was restored if Ca²⁺ was applied to the cytoplasmic side of inside-out patches. Rectification properties in the inside-out patch configuration could be controlled by the holding pipette potential. Holding potential values negative or positive to the calculated reversal potential for Cl⁻ ions induced opposite rectification properties. Our results show Ca²⁺-activated Cl⁻ channels in the tonoplast of Chara with holding potential dependent rectification. Received: 30 March 1999/Revised: 10 August 1999     

Involvement of Protein Tyrosine Kinase in the InsP3-Induced Activation of Ca2+-Dependent Cl− Currents in Cultured Cells of the Rat Retinal Pigment Epithelium

This combined study of patch-clamp and intracellular Ca²⁺ ([Ca²⁺] _i ) measurement was undertaken in order to identify signaling pathways that lead to activation of Ca²⁺-dependent Cl⁻ channels in cultured rat retinal pigment epithelial (RPE) cells. Intracellular application of InsP3 (10 μm) led to an increase in [Ca²⁺] _i and activation of Cl⁻ currents. In contrast, intracellular application of Ca²⁺ (10 μm) only induced transient activation of Cl⁻ currents. After full activation by InsP3, currents were insensitive to removal of extracellular Ca²⁺ and to the blocker of I _CRAC, La³⁺ (10 μm), despite the fact that both maneuvers led to a decline in [Ca²⁺] _i . The InsP3-induced rise in Cl⁻ conductance could be prevented either by thapsigargin-induced (1 μm) depletion of intracellular Ca²⁺ stores or by removal of Ca²⁺ prior to the experiment. The effect of InsP3 could be mimicked by intracellular application of the Ca²⁺-chelator BAPTA (10 mm). Block of PKC (chelerythrine, 1 μm) had no effect. Inhibition of Ca²⁺/calmodulin kinase (KN-63, KN-92; 5 μm) reduced Cl⁻-conductance in 50% of the cells investigated without affecting [Ca²⁺] _i . Inhibition of protein tyrosine kinase (50 μm tyrphostin 51, 5 μm genistein, 5 μm lavendustin) reduced an increase in [Ca²⁺] _i and Cl⁻ conductance. In summary, elevation of [Ca] _i by InsP3 leads to activation of Cl⁻ channels involving cytosolic Ca²⁺ stores and Ca²⁺ influx from extracellular space. Tyrosine kinases are essential for the Ca²⁺-independent maintenance of this conductance. Received: 15 October 1998/Revised: 3 March 1999     

A Ca2+-activated Whole-Cell Cl− Conductance In Human Placental Cytotrophoblast Cells Activated Via a G Protein

Whole-cell patch clamp experiments were performed on cultured human cytotrophoblast cells incubated for 24–48 hr after their isolation from term placentas. Cl⁻-selective currents were examined using K⁺-free solutions. Under nonstimulated conditions, most cells initially expressed only small background leak currents. However, inclusion of 0.2 mm GTPγS in the electrode solution caused activation of an outwardly rectifying conductance which showed marked time-dependent activation at depolarized potentials above +20 mV. Stimulation of this conductance by GTPγS was found to be Ca²⁺-dependent since GTPγS failed to activate currents when included in a Ca²⁺-free electrode solution. In addition, similar currents could be activated by increasing the [Ca²⁺] of the pipette solution to 500 nm. The Ca²⁺-activated conductance was judged to be Cl⁻-selective, since reversal potentials were predicted by Nernst equilibrium potentials for Cl⁻. This conductance could also be reversibly inhibited by addition of the anion channel blocker DIDS to the bath solution at a dose of 100 μm. Preliminary experiments indicated the presence of a second whole-cell anion conductance in human cytotrophoblast cells, which may be activated by cell swelling. Possible roles for the Ca²⁺-activated Cl⁻ conductance in human placental trophoblast are discussed. Received: 9 November 1995/Revised: 18 January 1996     

Turgor regulation in a brackish water charophyte,Lamprothamnium succinctum

Internodal cells of a brackish water charophyte,Lamprothamnium succinctum (A. Br. in Ash.) R.D.W. regulate the turgor pressure in response to changes in both the cellular and the external osmotic pressures. During turgor regulation upon hypotonic treatment, net effluxes of K⁺ and Cl⁻ from the vacuole, membrane depolarization, a transient increase in the electrical membrane conductance and a transient increase in concentration of cytoplasmic Ca²⁺ are induced. Activation of the plasmalemma Ca²⁺ channels and the Ca²⁺-controlled passive effluxes of K⁺ and Cl⁻ through the plasmalemma ion channels are postulated.     

Chloride-induced Ca2+ Release from the Sarcoplasmic Reticulum of Chemically Skinned Rabbit Psoas Fibers and Isolated Vesicles of Terminal Cisternae

There is increasing evidence that Ca²⁺ release from sarcoplasmic reticulum (SR) of mammalian skeletal muscle is regulated or modified by several factors including ionic composition of the myoplasm. We have studied the effect of Cl⁻ on the release of Ca²⁺ from the SR of rabbit skeletal muscle in both skinned psoas fibers and in isolated terminal cisternae vesicles. Ca²⁺ release from the SR in skinned fibers was inferred from increases in isometric tension and the amount of release was assessed by integrating the area under each tension transient. Ca²⁺ release from isolated SR was measured by rapid filtration of vesicles passively loaded with ⁴⁵Ca²⁺. Ca²⁺ release from SR was stimulated in both preparations by exposure to a solution containing 191 mm choline-Cl, following pre-equilibration in Ca²⁺-loading solution that had propionate as the major anion. Controls using saponin (50 μg/ml), indicated that the release of Ca²⁺ was due to direct action of Cl⁻ on the SR rather than via depolarization of T-tubules. Procaine (10 mm) totally blocked Cl⁻- and caffeine-elicited tension transients recorded using loading and release solutions having ([Na⁺] + [K⁺]) × [Cl⁻] product of 6487.69 mm ² and 12361.52 mm ², respectively, and blocked 60% of Ca²⁺ release in isolated SR vesicles. Surprisingly, procaine had only a minor effect on tension transients elicited by Cl⁻ and caffeine together. The data from both preparations suggests that Cl⁻ induces a relatively small amount of Ca²⁺ release from the SR by activating receptors other than RYR-1. In addition, Cl⁻ may increase the Ca²⁺ sensitivity of RYR-1, which would then allow the small initial release of Ca²⁺ to facilitate further release of Ca²⁺ from the SR by Ca²⁺-induced Ca²⁺ release. Received: 6 February 1996/Revised: 17 July 1996     

ATP-sensitive voltage- and calcium-dependent chloride channels in sarcoplasmic reticulum vesicles from rabbit skeletal muscle

Chloride channels in the sarcoplasmic reticulum (SR) are thought to play an essential role in excitation-contraction (E-C) coupling by balancing charge movement during calcium release and uptake. In this study the nucleotide-sensitivity of Cl⁻ channels in the SR from rabbit skeletal muscle was investigated using the lipid bilayer technique. Two distinct ATP-sensitive Cl⁻ channels that differ in their conductance and kinetic properties and in the mechanism of ATP-induced channel inhibition were observed. The first, a nonfrequent 150 pS channel was inhibited by trans (luminal) ATP, and the second, a common 75 pS small chloride (SCl) channel was inhibited by cis (cytoplasmic) ATP. In the case of the SCl channel the ATP-induced reversible decline in the values of current (maximal current amplitude, I _max and integral current, I′) and kinetic parameters (frequency of opening F _O , probability of the channel being open P _O , mean open T _O and closed T _c times) show a nonspecific block of the voltage- and Ca²⁺-dependent SCl channel. ATP was a more potent blocker from the cytoplasmic side than from the luminal side of the channel. The SCl channel block was not due to Ca²⁺ chelation by ATP, nor to phosphorylation of the channel protein. The inhibitory action of ATP was mimicked by the nonhydrolyzable analogue adenylylimidodiphosphate (AMP-PNP) in the absence of Mg²⁺. The inhibitory potency of the adenine nucleotides was charge dependent in the following order ATP⁴⁻ > ADP³⁻ > > > AMP²⁻. The data suggest that ATP-induced effects are mediated via an open channel block mechanism. Modulation of the SCl channel by [ATP] _cis and [Ca²⁺] _cis indicates that (i) this channel senses the bioenergetic state of the muscle fiber and (ii) it is linked to the ATP-dependent cycling of the Ca²⁺ between the SR and the sarcoplasm. Received: 4 September 1996/Revised: 6 December 1996     

ADH Action on Whole-Cell Currents by Cytosolic Ca2+-dependent Pathways in Aldosterone-treated A6 Cells

We studied the characteristics of the basal and antidiuretic hormone (arginine vasotocin, AVT)-activated whole cell currents of an aldosterone-treated distal nephron cell line (A6) at two different cytosolic Ca²⁺ concentrations ([Ca²⁺] _c , 2 and 30 nm). A6 cells were cultured on a permeable support filter for 10 ∼ 14 days in media with supplemental aldosterone (1 μm). At 30 nm [Ca²⁺] _c , basal conductances mainly consisted of Cl⁻ conductances, which were sensitive to 5-nitro-2-(3-phenylpropylamino)-benzoate. Reduction of [Ca²⁺] _c to 2 nm abolished the basal Cl⁻ conductance. AVT evoked Cl⁻ conductances at 2 as well as 30 nm [Ca²⁺] _c . In addition to Cl⁻ conductances, AVT induced benzamil-insensitive nonselective cation (NSC) conductances. This action on NSC conductances was observed at 30 nm [Ca²⁺] _c but not at 2 nm [Ca²⁺] _c . Thus, cytosolic Ca²⁺ regulates NSC and Cl⁻ conductances in a distal nephron cell line (A6) in response to AVT. Keeping [Ca²⁺] _c at an adequate level seems likely to be an important requirement for AVT regulation of ion conductances in aldosterone-treated A6 cells. Received: 6 May 1996/Revised: 28 June 1996     

Putative ClC-2 Chloride Channel Mediates Inward Rectification in <Emphasis Type="Italic">Drosophila </Emphasis>Retinal Photoreceptors

We report that Drosophila retinal photoreceptors express inwardly rectifying chloride channels that seem to be orthologous to mammalian ClC-2 inward rectifier channels. We measured inwardly rectifying Cl⁻ currents in photoreceptor plasma membranes: Hyperpolarization under whole-cell tight-seal voltage clamp induced inward Cl⁻ currents; and hyperpolarization of voltage-clamped inside-out patches excised from plasma membrane induced Cl⁻ currents that have a unitary channel conductance of ∼3.7 pS. The channel was inhibited by 1 mM Zn²⁺ and by 1 mM 9-anthracene, but was insensitive to DIDS. Its anion permeability sequence is Cl⁻ = SCN⁻> Br⁻>> I⁻, characteristic of ClC-2 channels. Exogenous polyunsaturated fatty acid, linolenic acid, enhanced or activated the inward rectifier Cl⁻ currents in both whole-cell and excised patch-clamp recordings. Using RT-PCR, we found expression in Drosophila retina of a ClC-2 gene orthologous to mammalian ClC-2 channels. Antibodies to rat ClC-2 channels labeled Drosophila photoreceptor plasma membranes and synaptic regions. Our results provide evidence that the inward rectification in Drosophila retinal photoreceptors is mediated by ClC-2-like channels in the non-transducing (extra-rhabdomeral) plasma membrane, and that this inward rectification can be modulated by polyunsaturated fatty acid. G. Ugarte and R. Delgado contributed equally to this work.     

Transient Activity of Excitatory Cl− Channels in Chara: Evidence for Quantal Release of a Gating Factor

In attached patches on the plasma membrane of nonexcited Chara corallina cells, randomly activating, transient Cl⁻ currents with variable amplitudes were recorded. The peak amplitudes of these currents could be grouped into distinct populations with approximately equidistant mean peak currents. Generally, the mean current of the smallest population measured about half of the distance between the means of subsequent populations. Currents of the smallest population occurred most frequently at all voltages; the frequency of observations decreased with increasing amplitudes of the currents. At all voltages transient currents from different populations were similar in duration with the exception of the smallest currents, which lasted only 0.6 times as long as larger currents. Furthermore, transient currents were most frequent at positive voltages, but once initiated at a positive conditioning pulse they were also observed during subsequent pulses to negative voltages. The results are consistent with the idea that Chara contains Ca²⁺ stores in the vicinity of the plasma membrane, which are indirectly filled from the external medium. Upon quantal Ca²⁺ discharge from adjacent stores, a process independent of membrane voltage, the concentration of Ca²⁺ in the cytoplasm increases transiently. Depending on the number of discharging stores, distinct numbers of Ca²⁺-stimulated Cl⁻ channels activate, giving rise to the macroscopic excitatory Cl⁻ current in these cells. Received: 27 October 1997/Revised: 26 February 1998