Cell swelling activates K+ and Cl− channels as well as nonselective,stretch-activated cation channels in ehrlich ascites tumor cells

Summary Cell-attached patch-clamp recordings from Ehrlich ascites tumor cells reveal nonselective cation channels which are activated by mechanical deformation of the membrane. These channels are seen when suction is applied to the patch pipette or after osmotic cell swelling. The channel activation does not occur instantaneously but within a time delay of 1/2 to 1 min. The channel is permeable to Ba²⁺ and hence presumably to Ca²⁺. It seems likely that the function of the nonselective, stretch-activated channels is correlated with their inferred Ca²⁺ permeability, as part of the volume-activated signal system. In isolated insideout patches a Ca²⁺-dependent, inwardly rectifying K⁺ channel is demonstrated. The single-channel conductance recorded with symmetrical 150 mm K⁺ solutions is for inward current estimated at 40 pS and for outward current at 15 pS. Activation of the K⁺ channel takes place after an increase in Ca²⁺ from 10^–7 to 10^–6 m which is in the physiological range. Patch-clamp studies in cellattached mode show K⁺ channels with spontaneous activity and with characteristics similar to those of the K⁺ channel seen in excised patches. The single-channel conductance for outward current at 5 mm external K⁺ is estimated at about 7 pS. A K⁺ channel with similar properties can be activated in the cellattached mode by addition of Ca²⁺ plus ionophore A23187. The channel is also activated by cell swelling, within 1 min following hypotonic exposure. No evidence was found of channel activation by membrane stretch (suction). The time-averaged number of open K⁺ channels during regulatory volume decrease (RVD) can be estimated at 40 per cell. The number of open K⁺ channels following addition of Ca²⁺ plus ionophore A23187 was estimated at 250 per cell. Concurrent activation in cell-attached patches of stretch-activated, nonselective cation channels and K⁺ channels in the presence of 3 mm Ca²⁺ in the pipette suggests a close spatial relationship between the two channels. In excised inside-out patches (with NMDG chloride on both sides) a small 5-pS chloride channel with low spontaneous activity is observed. The channel activity was not dependent on Ca²⁺ and could not be activated by membrane stretch (suction). In cell-attached mode singlechannel currents with characteristics similar to the channels seen in isolated patches are seen. In contrast to the channels seen in isolated patches, the channels in the cell-attached mode could be activated by addition of Ca²⁺ plus ionophore A23187. The channel is also activated by hypotonic exposure with a single-channel conductance at 7 pS (or less) and with a time delay at about 1 min. The number of open channels during RVD is estimated at 80 per cell. Two other types of Cl^– channels were regularly recorded in excised inside-out patches: a voltage-activated 400-pS channel and a 34-pS Cl^– channel which show properties similar to the Cl^– channel in the apical membrane in human airway epithelial cells. There is no evidence for a role in RVD for either of these two channels.     

Identification and regulation of whole-cell Cl− and Ca2+-activated K+ currents in cultured medullary thick ascending limb cells

The whole-cell patch-clamp technique has been used to study membrane currents in cultured rabbit medullary thick ascending limb (MTAL) epithelial cells. A Ca²⁺-activated K⁺ current was characterized by its voltage-dependent and Ca²⁺-dependent properties. When the extracellular K⁺ ion concentration was increased from 2 to 140 mm, the rereversal potential (E_k) was shifted from –85 to 0 mV with a slope of 46 mV per e-fold change. The Ca²⁺-activated K⁺ current is blocked by charybdotoxin (CTX) in a manner similar to the apical membrane Ca²⁺-activated K⁺ channel studied with the single channel patch-clamp technique. The results suggest that the Ca²⁺-activated K⁺ current is the predominant, large conductance and Ca²⁺-dependent K⁺ pathway in the cultured MTAL cell apical membrane. The biophysical properties and physiological regulation of a Cl^– current were also investigated. This current was activated by stimulation of intracellular cAMP using forskolin and isobutyl-1-methylxanthine (IBMX). The current-voltage (I–V) relationship of the Cl^– current showed an outward-rectifying pattern in symmetrical Cl^– solution. The Cl^– selectivity of the whole-cell current was confirmed by tail current analysis in different Cl^– concentration bath solutions. Several Cl^– channel blockers were found to be effective in blocking the outward-rectifying Cl^– current in MTAL cells. The cAMP-dependent Cl^– transport in MTAL cells was further confirmed by measuring changes in the intensity of Cl^– sensitive dye using fluorescence microscopy. These results suggest that the Cl^– channel in the apical or basolateral membrane of MTAL cells may be regulated by cAMP-dependent protein-kinase-induced phosphorylation.This study was supported by the National Institutes of Health grants GM46834 to L.L. and DK32753 to W.B.G., and by a Grant-in-Aid from the American Heart Association of Ohio to L.L.     

Separate,Ca2+-activated K+ and Cl− transport pathways in Ehrlich ascites tumor cells

Summary The net loss of KCl observed in Ehrlich ascites cells during regulatory volume decrease (RVD) following hypotonic exposure involves activation of separate conductive K⁺ and Cl^– transport pathways. RVD is accelerated when a parallel K⁺ transport pathway is provided by addition of gramicidin, indicating that the K⁺ conductance is rate limiting. Addition of ionophore A23187 plus Ca²⁺ also activates separate K⁺ and Cl^– transport pathways, resulting in a hyperpolarization of the cell membrane. A calculation shows that the K⁺ and Cl^– conductance is increased 14-and 10-fold, respectively. Gramicidin fails to accelerate the A23187-induced cell shrinkage, indicating that the Cl^– conductance is rate limiting. An A23187-induced activation of⁴²K and³⁶Cl tracer fluxes is directly demonstrated. RVD and the A23187-induced cell shrinkage both are: (i) inhibited by quinine which blocks the Ca²⁺-activated K⁺ channel. (ii) unaffected by substitution of NO ₃ ^– or SCN^– for Cl^–, and (iii) inhibited by the anti-calmodulin drug pimozide. When the K⁺ channel is blocked by quinine but bypassed by addition of gramicidin, the rate of cell shrinkage can be used to monitor the Cl^– conductance. The Cl^– conductance is increased about 60-fold during RVD. The volume-induced activation of the Cl^– transport pathway is transient, with inactivation within about 10 min. The activation induced by ionophore A23187 in Ca²⁺-free media (probably by release of Ca²⁺ from internal stores) is also transient, whereas the activation is persistent in Ca²⁺-containing media. In the latter case, addition of excess EGTA is followed by inactivation of the Cl^– transport pathway. These findings suggest that a transient increase in free cytosolic Ca²⁺ may account for the transient activation of the Cl^– transport pathway. The activated anion transport pathway is unselective, carrying both Cl^–, Br^–, NO ₃ ^– , and SCN^–. The anti-calmodulin drug pimozide blocks the volume- or A23187-induced Cl^– transport pathway and also blocks the activation of the K⁺ transport pathway. This is demonstrated directly by⁴²K flux experiments and indirectly in media where the dominating anion (SCN^–) has a high ground permeability. A comparison of the A23187-induced K⁺ conductance estimated from⁴²K flux measurements at high external K⁺, and from net K^– flux measurements suggests single-file behavior of the Ca²⁺-activated K⁺ channel. The number of Ca²⁺-activated K⁺ channels is estimated at about 100 per cell.     

Identification of K+, Cl−, and Ca2+ channels in the vacuolar membrane of tobacco cell suspension cultures

Summary K⁺, Cl^–, and Ca²⁺ channels in the vacuolar membrane of tobacco cell suspension cultures have been investigated using the patch-clamp technique. In symmetrical 100mM K⁺, K⁺ channels opened at positive vacuolar membrane potentials (cytoplasmic side as reference) had different conductances of 57 pS and 24 pS. K⁺ channel opened at negative vacuolar membrane potentials had a conductance of 43 pS. The K⁺ channels showed a significant discrimination against Na⁺ and Cl^–. The Cl^– channel opened at positive vacuolar membrane potentials for cytoplasmic Cl^– influx had a high conductance of 110pS in symmetrical 100mM Cl^–. When K⁺ and Cl^– channels were excluded from opening, no traces were found of Ca²⁺ channel activity for vacuolar Ca²⁺ release induced by inositol 1,4,5-trisphosphate or other events. However, we found a 19pS Ca²⁺ channel which allowed influx of cytoplasmic Ca²⁺ into the vacuole when the Ca²⁺ concentration on the cytoplasmic side was high. When Ca²⁺ was substituted by Ba²⁺, the conductance of the 19 pS channel became 30 pS and the channel showed a selectivity sequence of Ba²⁺Sr²⁺Ca²⁺Mg²⁺=10.60.60.21. The reversal potentials of the channel shifted with the change in Ca²⁺ concentration on the vacuolar side. The channel could be efficiently blocked from the cytoplasmic side by Cd²⁺, but was insensitive to La³⁺, Gd³⁺, Ni²⁺, verapamil, and nifedipine. The related ion channels in freshly isolated vacuoles from red beet root cells were also recorded. The coexistence of the K⁺, Cl^–, and Ca²⁺ channels in the vacuolar membrane of tobacco cells might imply a precise classification and cooperation of the channels in the physiological process of plant cells.     

Ionic Selectivity and Permeation Properties of Human PIEZO1 Channels

Members of the eukaryotic PIEZO family (the human orthologs are noted hPIEZO1 and hPIEZO2) form cation-selective mechanically-gated channels. We characterized the selectivity of human PIEZO1 (hPIEZO1) for alkali ions: K⁺, Na⁺, Cs⁺ and Li⁺; organic cations: TMA and TEA, and divalents: Ba²⁺, Ca²⁺, Mg²⁺ and Mn²⁺. All monovalent ions permeated the channel. At a membrane potential of -100 mV, Cs⁺, Na⁺ and K⁺ had chord conductances in the range of 35–55 pS with the exception of Li⁺, which had a significantly lower conductance of ~ 23 pS. The divalents decreased the single-channel permeability of K⁺, presumably because the divalents permeated slowly and occupied the open channel for a significant fraction of the time. In cell-attached mode, 90 mM extracellular divalents had a conductance for inward currents carried by the divalents of: 25 pS for Ba²⁺ and 15 pS for Ca²⁺ at -80 mV and 10 pS for Mg²⁺ at -50 mV. The organic cations, TMA and TEA, permeated slowly and attenuated K⁺ currents much like the divalents. As expected, the channel K⁺ conductance increased with K⁺ concentration saturating at ~ 45 pS and the K_D of K⁺ for the channel was 32 mM. Pure divalent ion currents were of lower amplitude than those with alkali ions and the channel opening rate was lower in the presence of divalents than in the presence of monovalents. Exposing cells to the actin disrupting reagent cytochalasin D increased the frequency of openings in cell-attached patches probably by reducing mechanoprotection.     

Ion Channel Permeable for Divalent and Monovalent Cations in Native Spinach Thylakoid Membranes

A cation-selective channel was characterized in isolated patches from osmotically swollen thylakoids of spinach (Spinacea oleracea). This channel was permeable for K⁺ as well as for Mg²⁺ and Ca²⁺ but not for Cl⁻. When K⁺ was the main permeant ion (symmetrical 105 mm KCl) the conductance of the channel was about 60 pS. The single channel conductance for different cations followed a sequence K⁺ > Mg²⁺≥ Ca²⁺. The permeabilities determined by reversal potential measurements were comparable for K⁺, Ca²⁺, and Mg²⁺. The cation channel displayed bursting behavior. The total open probability of the channel increased at more positive membrane potentials. Kinetic analysis demonstrated that voltage dependence of the total open probability was determined by the probability of bursts formation while the probability to find the channel in open state within a burst of activity was hardly voltage-dependent. The cation permeability of intact spinach thylakoids can be explained on the single channel level by the data presented here. Received: 26 December 1995/Revised: 17 April 1996     

Single nonselective cation channels and Ca2+-activated K+ channels in aortic endothelial cells

Summary In cultured bovine aortic endothelial cells, elementary K⁺ currents were studied in cell-attached and inside-out patches using the standard patch-clamp technique. Two different cationic channels were found, a large channel with a mean unitary conductance of 150±10 pS and a small channel with a mean unitary conductance of 12.5±1.1 pS. The 150-pS channel proved to be voltag- and Ca²⁺-activatable and seems to be a K⁺ channel. Its open probability increased on membrane depolarization and, at a given membrane potential, was greatly enhanced by elevating the Ca²⁺ concentration at the cytoplasmic side of the membrane from 10^–7 to 10^–4 m. 150-pS channels were not influenced by the patch configuration in that patch excision neither induced rundown nor evoked channel activity in silent cell-attached patches. However, they were only seen in two out of 55 patches. The 12-pS channel was predominant, a nonselective cationic channel with almost the same permeability for K⁺ and Na⁺ whose open probability was minimal near –60 mV but increased on membrane hyperpolarization. An increase in internal Ca²⁺ from 10^–7 to 10^–4 m left the open probability unchanged. Although the K⁺ selectivity of the 150-pS channels remains to be elucidated, it is concluded that they may be involved in controlling Ca²⁺-dependent cellular functions. Under physiological conditions, 12-pS nonselective channels may provide an inward cationic pathway for Na⁺.     

Chloride channels in human platelets: Evidence for activation by internal calcium

Summary Whole-cell patch-clamp recordings were made from freshly isolated human platelets. The pipette contained a high concentration of divalent cations, which permitted easy disruption of cell-attached membrane patches by suction. Single-channel currents were measured when the pipette contained isotonic BaCl₂ or MgCl₂ saline; over 30 sec –5 min an increasing number of channels appeared until conductance steps through individual channels could no longer be distinguished. The current-voltage relationship was curvilinear; chord conductance at –35 mV was 25 pS increasing to 45 to 52 pS at +45 mV. Ion substitution experiments showed the current to be primarily carried by Cl^–.E _rev was shifted 30 mV/10-fold change in external Cl^– (replaced by gluconate), was similar with BaCl₂ or MgCl₂ in the pipette and was not significantly shifted by replacing external Na⁺ with K⁺. Addition of 1mm BAPTA to the MgCl₂ pipette saline prevented activation of Cl^– currents; with isotonic CaCl₂ internal saline, current appeared immediately upon patch rupture, suggesting that the Cl^– channels are dependent on internal Ca²⁺, 5-nitro-2-(3-phenylpropylamino)-benzoate, reported to block a Cl^– conductance in studies of rat epithelial cells, caused a potent flickery block and may be a useful tool with which to investigate the physiological role of Cl^– currents in human platelets.     

Regulation of K+ channels in the basolateral membrane ofNecturus oxyntic cells

Summary Patch-clamp methods were used to study single-channel events in isolated oxyntic cells and gastric glands fromNecturus maculosa. Cell-attached, excised inside-out and outside-out patches from the basolateral membrane frequently contained channels which had conductances of 67±21 pS in 24% of the patches and channels of smaller conductance, 33±6 pS in 56% of the patches. Channels in both classes were highly selective for K⁺ over Na⁺ and Cl^–, and shared linear current-voltage relations. The 67-pS channel was activated by membrane depolarization, whereas the activity of the 33-pS channel was relatively voltage independent. The larger conductance channels were activated by intracellular Ca²⁺ in the range between 5 and 500nm, but unaffected by cAMP. The smaller conductance channels were activated by cAMP, but not Ca²⁺. The presence of K⁺ channels in the basolateral membrane which are regulated by these known second messengers can account for the increase in conductance and the hyperpolarization of the membrane observed upon secretagogue stimulation.     

Potassium Channels in Motor Cells of Samanea saman: A Patch-Clamp Study

Leaflet movements in Samanea saman are driven by the shrinking and swelling of cells in opposing (extensor and flexor) regions of the motor organ (pulvinus). Changes in cell volume, in turn, depend upon large changes in motor cell content of K⁺, Cl⁻ and other ions. We performed patch-clamp experiments on extensor and flexor protoplasts, to determine whether their plasma membranes contain channels capable of carrying the large K⁺ currents that flow during leaflet movement. Recordings in the “whole-cell” mode reveal depolarization-activated K⁺ currents in extensor and flexor cells that increase slowly (t_½ = ca. 2 seconds) and remain active for minutes. Recordings from excised patches reveal a single channel conductance of ca. 20 picosiemens in both cell types. The magnitude of the K⁺ currents is adequate to account quantitatively for K⁺ loss, previously measured in vivo during cell shrinkage. The K⁺ channel blockers tetraethylammonium (5 millimolar) or quinine (1 millimolar) blocked channel opening and decreased light- and dark-promoted movements of excised leaflets. These results provide evidence for the role of potassium channels in leaflet movement.     

Ion channels are linked to differentiation in keratinocytes

Summary In vivo and in vitro, keratinocyte differentiation is linked with increased extracellular Ca²⁺. In order to correlate ion channels with cell differentiation and investigate keratinocyte membrane responses to Ca²⁺, keratinocyte single channel currents were studied using the patch-clamp technique. The most frequently observed channel was a 14 pS nonspecific cation channel. This channel was permeable to Ca²⁺ and activated by physiological concentrations of Ca²⁺. We also found a 35 pS Cl^– channel whose open probability increased with depolarization. Finally, a 70 pS K⁺ channel was seen only in cell-attached or nystatin-permeabilized patches. We correlated channel types with staining for involucrin, an early marker of keratinocyte differentiation. While the nonspecific cation channel and Cl^– channel were seen in both involucrin positive and involucrin negative cells, all channels in which the K⁺ channel activity was present were involucrin positive. Membrane currents through these channels may be one pathway by which signals for keratinocyte proliferation or differentiation are sent.This work was supported in part by a National Institutes of Health grant K08 AR01853-03 and a National Science Foundation grant DCB-9009915 (to T.M.M.); National Institutes of Health Research Career Development Award K04 ARO 1803 and AR 39031 (to R.R.I.) and a National Institutes of Health grant GM-44840 (to P.A.P.).     

Properties of Single K and Cl Channels in Asclepias tuberosa Protoplasts

Potassium and chloride channels were characterized in Asclepias tuberosa suspension cell derived protoplasts by patch voltage-clamp. Whole-cell currents and single channels in excised patches had linear instantaneous current-voltage relations, reversing at the Nernst potentials for K⁺ and Cl⁻, respectively. Whole cell K⁺ currents activated exponentially during step depolarizations, while voltage-dependent Cl⁻ channels were activated by hyperpolarizations. Single K⁺ channel conductance was 40 ± 5 pS with a mean open time of 4.5 milliseconds at 100 millivolts. Potassium channels were blocked by Cs⁺ and tetraethylammonium, but were insensitive to 4-aminopyridine. Chloride channels had a single-channel conductance of 100 ± 17 picosiemens, mean open time of 8.8 milliseconds, and were blocked by Zn²⁺ and ethacrynic acid. Whole-cell Cl⁻ currents were inhibited by abscisic acid, and were unaffected by indole-3-acetic acid and 2,4-dichlorophenoxyacetic acid. Since internal and external composition can be controlled, patch-clamped protoplasts are ideal systems for studying the role of ion channels in plant physiology and development.     

Cl− currents of unstimulated T84 intestinal epithelial cells studied by intracellular recording

The ionic currents spontaneously present in T84 intestinal epithelial cells, a line of colonie carcinoma origin, have been studied using the whole-cell recording mode of the patch-clamp technique and the single-electrode voltage-clamp method. Patch-clamp experiments showed that nonstimulated T84 cells already possess large currents but that these tend to disappear during the course of the experiments, presumably through the dialysis of some essential cytoplasmic component against the micropipette solution. The main charge carrier in these experiments appears to be Cl^– as judged from ion replacement. Microelectrode impalement of T84 cells gave a membrane potential of around –30 mV, similar to the equilibrium potential for Cl^– estimated from previously published values for intracellular Cl^– concentration. Voltage-clamp experiments with a single microelectrode revealed three kinetically distinguishable current patterns; currents decaying during hyperpolarizing voltage pulses, currents slowly activating during hyperpolarizing pulses and time-independent currents. The appearance of these distinct kinetic patterns was not predictable from cell to cell, and was not dependent on extracellular Ca²⁺. Ionic replacement experiments suggest that the charge carrier was always Cl^–, regardless of the kinetic pattern observed. No K⁺ currents appear to be present in the nonstimulated T84 cells. Exposure of T84 cells to the muscarinic agonist carbachol induced a shift in the membrane potential towards more negative values, consistent with an activation of a K⁺ conductance. Thus, we suggest that the resting membrane potential in T84 cells is determined by the distribution of Cl^–. This might imply that activation of K⁺ conductance could by itself support secretion by T84 monolayers through tonically active Cl^– channels.G.M.V. and M.A.V. were supported by AFRC (UK) LRG 111 and DGICYT (Spain), respectively. We are grateful to John O'Brien for culturing the cells, to John Dempster (University of Strathclyde, Glasgow, UK) for providing the analysis software, and to Geoff Warhurst (Hope Hospital, Salford, UK) for generously providing the initial batch of T84 cells.     

Satellite glial cells In Situ within mammalian prevertebral ganglia express K+ channels active at rest potential

Patch-clamp experiments were performed on satellite glial cells wrapped around sympathetic neurons in the rabbit coeliac ganglion. With the cleaning method used, the glial cells could be kept in place and were directly accessible to the patch-clamp pipettes. Whole-cell recordings showed that glial cells had almost ohmic properties. Their resting potential (–79.1±1.2 mV) was found to be very nearly the same as the K⁺ reversal potential and 20 mV more negative than that of the neurons they encapsulated. Unitary currents from ionic channels present in the glial membrane were recorded in the cell-attached configuration with pipettes filled with various amounts of K⁺, Na⁺ and gluconate. Only K⁺-selective channels with slight inwardly rectifying properties (in the presence of 150 mM [K⁺]₀) were detected. These channels were active (P ₀=0.7–0.8) at the cell resting potential. The channel conductance, but not its opening probability, was dependent on the [K⁺] in the pipette. Cl^–-selective channels (outwardly rectifying and large conductance channels) were detected in excised patches.The properties of the K⁺ channels (increased inward current with [K⁺] and detectable outward current at low [K⁺]) are well suited for siphoning the K⁺ released by active neurons.     

Effects of antidiuretic hormone on cellular conductive pathways in mouse medullary thick ascending limbs of Henle: I. ADH increases transcellular conductance pathways

Summary This paper reports experiments designed to assess the relations between net salt absorption and transcellular routes for ion conductance in single mouse medullary thick ascending limbs of Henle microperfusedin vitro. The experimental data indicate that ADH significantly increased the transepithelial electrical conductance, and that this conductance increase could be rationalized in terms of transcellular conductance changes. A minimal estimate (G _c ^min ) of the transcellular conductance, estimated from Ba⁺⁺ blockade of apical membrane K⁺ channels, indicated thatG _c ^min was approximately 30–40% of the measured transepithelial conductance. In apical membranes, K⁺ was the major conductive species; and ADH increased the magnitude of a Ba⁺⁺-sensitive K⁺ conductance under conditions where net Cl^– absorption was nearly abolished. In basolateral membranes, ADH increased the magnitude of a Cl^– conductance; this ADH-dependent increase in basal Cl^– conductance depended on a simultaneous hormone-dependent increase in the rate of net Cl^– absorption. Cl^– removal from luminal solutions had no detectable effect onG _e , and net Cl^– absorption was reduced at luminal K⁺ concentrations less than 5mm; thus apical Cl^– entry may have been a Na⁺,K⁺,2Cl^– cotransport process having a negligible conductance. The net rate of K⁺ secretion was approximately 10% of the net rate of Cl^– absorption, while the chemical rate of net Cl^– absorption was virtually equal to the equivalent short-circuit current. Thus net Cl^– absorption was rheogenic; and approximately half of net Na⁺ absorption could be rationalized in terms of dissipative flux through the paracellular pathway. These findings, coupled with the observation that K⁺ was the principal conductive species in apical plasma membranes, support the view that the majority of K⁺ efflux from cell to lumen through the Ba⁺⁺-sensitive apical K⁺ conductance pathway was recycled into cells by Na⁺,K⁺,2Cl^– cotransport.     

Hypotonically activated chloride current in HSG cells

Hypotonically induced changes in whole-cell currents and in cell volume were studied in the HSG cloned cell line using the whole-cell, patch clamp and Coulter counter techniques, respectively. Exposures to 10 to 50% hypotonic solutions induced dose-dependent increases in whole-cell conductances when measured using K⁺ and Cl^– containing solutions. An outward current detected at 0 mV, corresponded to a K⁺ current which was transiently activated, (usually preceding activation of an inward current and had several characteristics in common with a Ca²⁺-activated K⁺ current we previously described in these cells. The hypotonically induced inward current had characteristics of a Cl^– current. This current was inhibited by NPPB (5-nitro-2-(3-phenyl-propylamino)-benzoate) and SITS (4-acetamido-4-isothiocyanostilbene), and its reversal potentials corresponded to the Cl^– equilibrium potentials at high and low external Cl^– concentrations. The induced current inactivated at voltages greater than +80 mV, and the I-V curve was outwardly rectifying. The current was unaffected by addition of BAPTA or removal of GTP from the patch pipette, but was inhibited by removal of ATP or by the presence of extracellular arachidonic acid, quinacrine, nordihydroguairetic acid, and cytochalasin D. Moreover, exposure of HSG cells to hypotonic media caused them to swell and then to undergo a regulatory volume decrease (RVD) response. Neither NPPB, SITS or quinine acting alone could inhibit RVD, but NPPB and quinine together totally inhibited RVD. These properties, plus the magnitudes of the induced currents, indicate that the hypotonically induced K⁺ and Cl^– currents may underlie the RVD response. Cytochalasin D also blocked the RVD response, indicating that intact cytoskeletal F-actin may be required for activation of the present currents. Hence, our results indicate that hypotonic stress activates K⁺ and Cl^– conductances in these cells, and that the activation pathway for the K⁺ conductance apparently involves [Ca²⁺], while the activation pathway for the Cl^– conductance does not involve [Ca²⁺] nor lipoxygenase metabolism, but does require intact cytoskeletal F-actin.We thank Mr. Louis Stamps for excellent technical support. Thanks also to Dr. Mitsunobu Sato from the Second Department of Oral and Maxillofacial Surgery, Tokushima University, Japan for sending us the HSG-PA cells, and to Dr. Englert from Hoechst company for providing us with NPPB. This work was supported by National Institute of Dental Research grants R01 DE09812 and R03 DE10535.     

ATP-sensitive inward rectifier and voltage- and calcium-activated K+ channels in cultured pancreatic islet cells

Summary K⁺ channels in cultured rat pancreatic islet cells have been studied using patch-clamp single-channel recording techniques in cell-attached and excised inside-out and outside-out membrane patches. Three different K⁺-selective channels have been found. Two inward rectifier K⁺ channels with slope conductances of about 4 and 17 pS recorded under quasi-physiological cation gradients (Na⁺ outside, K⁺ inside) and maximal conductances recorded in symmetrical K⁺-rich solutions of about 30 and 75 pS, respectively. A voltage- and calcium-activated K^– channel was recorded with a slope conductance of about 90 pS under the same conditions and a maximal conductance recorded in symmetrical K⁺-rich solutions of about 250 pS. Single-channel current recording in the cell-attached conformation revealed a continuous low level of activity in an apparently small number of both the inward rectifier K⁺ channels. But when membrane patches were excised from the intact cell a much larger number of inward rectifier K⁺ channels became transiently activated before showing an irreversible decline. In excised patches opening and closing of both the inward rectifier K⁺ channels were unaffected by voltage, internal Ca²⁺ or externally applied tetraethyl-ammonium (TEA) but the probability of opening of both inward rectifier K⁺ channels was reduced by internally applied 1–5mm adenosine-5-triphosphate (ATP). The large K⁺ channel was not operational in cell-attached membrane patches, but in excised patches it could be activated at negative membrane potentials by 10^–7 to 10^–6 m internal Ca²⁺ and blocked by 5–10mm external TEA.     

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     

Chloride and potassium channels in U937 human monocytes

Summary Ionic channels in a human monocyte cell line (U937) were studied with the inside-out patch-clamp technique. A Ca²⁺-activated K⁺ channel and three Cl^–-selective channels were observed. The Ca²⁺-activated K⁺ channel had an inward-rectifying current-voltage relationship with slope conductance of 28 pS, and was not dependent on membrane potential. Among the three Cl^– channels, and outward-rectifying 28-pS channel was most frequently observed. The permeability ratio (Cl^–/Na⁺) was 4–5 and CH₃SO ₄ ^– was also permeant. The channel became less active with increasing polarizations in either direction, and was inactive beyond ±120 mV. The channel, observed as bursts, occasionally had rapid events within the bursts, suggesting the presence of another mode of kinetics. Diisothiocyanatostilbene-disulfonic acid (DIDS) blocked the channel reversibly in a dose-dependent manner. The second 328-pS Cl^– channel had a linear currentvoltage relationship and permeability ratio (Cl^–/Na⁺) of 5–6. This channel became less active with increasing polarizations and inactive beyond ±50 mV. DIDS blocked the channel irreversibly. The channel had multiple subconductance states. The third 15-pS Cl^– channel was least frequently observed and least voltage sensitive among the Cl^– channels. Intracellular Ca²⁺ or pH affected none of the three Cl^– channels. All three Cl^– channels had a latent period before being observed, suggesting inhibitory factor(s) presentin situ. Activation of the cells with interferon-, interferon-A or 12-O-tetradecanoylphorbol-13-acetate (TPA) caused no change in the properties on any of the channels.     

Ion conduction and selectivity in acid-sensing ion channel 1

The ability of acid-sensing ion channels (ASICs) to discriminate among cations was assessed based on changes in conductance and reversal potential with ion substitution. Human ASIC1a was expressed in Xenopus laevis oocytes, and acid-induced currents were measured using two-electrode voltage clamp. Replacement of extracellular Na⁺ with Li⁺, K⁺, Rb⁺, or Cs⁺ altered inward conductance and shifted the reversal potentials consistent with a selectivity sequence of Li ∼ Na > K > Rb > Cs. Permeability decreased more rapidly than conductance as a function of atomic size, with P_K/P_Na = 0.1 and G_K/G_Na = 0.7 and P_Rb/P_Na = 0.03 and G_Rb/G_Na = 0.3. Stimulation of Cl⁻ currents when Na⁺ was replaced with Ca²⁺, Sr²⁺, or Ba²⁺ indicated a finite permeability to divalent cations. Inward conductance increased with extracellular Na⁺ in a hyperbolic manner, consistent with an apparent affinity (K_m) for Na⁺ conduction of 25 mM. Nitrogen-containing cations, including NH₄⁺, NH₃OH⁺, and guanidinium, were also permeant. In addition to passing through the channels, guanidinium blocked Na⁺ currents, implying competition for a site within the pore. The role of negative charges in an external vestibule of the pore was evaluated using the point mutation D434N. The mutant channel had a decreased single-channel conductance, measured in excised outside-out patches, and a macroscopic slope conductance that increased with hyperpolarization. It had a weakened interaction with Na⁺ (K_m = 72 mM) and a selectivity that was shifted toward larger atomic sizes. We conclude that the selectivity of ASIC1 is based at least in part on interactions with binding sites both within and internal to the outer vestibule.