Identification of a voltage- and calcium-dependent non-selective cation channel in cultured adult and fetal human nasal epithelial cells

The apical membranes of cultured human nasal epithelial cells from adults and fetuses were investigated with the patch-clamp technique. Amiloride-insensitive, calcium- and voltage-dependent, non-selective cation channels were found in 4% of the cell-attached, and 18% of the inside-out and outside-out patches (n=412). Multiple functional channels were present in more than 90% of these patches, with a mean of 3.9 channels per patch (n=55). The current-voltage relationship can be described by the Goldman equations and the single channel conductance was 20.1±0.3 pS (n=29) in adult and 20.7±0.4 pS (n=44) in fetal cells in symmetrical 150mM NaCl solutions. The channels were highly selective for cations: P_Na/P_Cl was 30 in adult and 45 in fetal experiments. They were equally permeable for K⁺ and Na⁺, somewhat less for Cs⁺, and impermeable for choline⁺ and tetraethylammonium⁺. The open probability was voltage dependent: it increased approximately 2-fold with 30mV depolarization in the potential range from −60mV to +60mV. The channels were activated by Ca²⁺ concentrations of about 10⁻⁴M at the cytoplasmic side, but were insensitive to extracellular Ca²⁺ and amiloride (10⁻⁴M). The non-selective cation channels found in apical membranes of cultured fetal nasal epithelial cells were not different from the adult ones.     

Histamine-activated,non-selective cation currents and Ca2+ transients in endothelial cells from human umbilical vein

Permeation properties and modulation of an ionic current gated by histamine were measured in single endothelial cells from human umbilical cord veins by use of the patch-clamp technique in the ruptured-whole-cell mode or using perforated patches. We combined these current measurements with a microfluorimetric method to measure concomitantly free intracellular calcium concentration ([Ca²⁺]_i). Application of histamine induced an intracellular calcium transient and an ionic current that reversed near 0 mV. The amplitude of the current ranged from –0.2 to –2nA at –100mV. The tonic rise in [Ca²⁺]_i and the ionic current are partly due to Ca²⁺ influx. This Ca²⁺ entry pathway is also permeable for Ba²⁺ and Mn²⁺. The amplitude of the histamine-activated current was also closely correlated with the amplitude of the concomitant Ca²⁺ transient, suggesting that the latter is at least partially due to Ca²⁺ influx through histamine-activated channels. The reversal potential of the histamine-induced current was 7.6±4.1 mV (n=14) when the calcium concentration in the bath solution ([Ca²⁺]_o) was 1.5mmol/l. With 10 mmol/l [Ca²⁺]_o it was –13.7±4.7 mV and shifted to +13.0±1.5 mV in nominally Ca²⁺-free solution (n=3 cells). The amplitude of the current in Ca²⁺-free solution was enhanced compared to that in 10 mmol/l [Ca²⁺]_o. The shift of the reversal potential and the concomitant change of the current amplitude suggest that the channel is permeable for calcium but has a smaller permeability for calcium than for monovalent cations. The latency between the application of histamine and the appearance of the current was voltage dependent and was much smaller at more negative potentials. This effect is unlikely to be due to desensitization, but may suggest a voltage-dependent step in the signal transduction chain. Similar histamine-induced Ca²⁺ signals were observed if the currents were measured in patches perforated with nystatin. The onset of the agonist-activated current was, however, much more delayed and its amplitude significantly lower than in ruptured patches. The histamine-induced currents and intracellular Ca²⁺-transients were largely reduced after incubation of endothelial cells with the phorbol ester TPA. H7, a blocker of protein kinase C, induced membrane currents and Ca²⁺ signals in the absence of an agonist. It is concluded that the agonist-activated Ca²⁺-entry in endothelial cells occurs through non-selective cation channels which can be down-regulated by protein kinase C activation.     

Electrophysiological properties of membrane currents in single myometrial cells isolated from pregnant rats

The whole-cell voltage-clamp method was applied to single smooth muscle cells prepared from the longitudinal layer of the pregnant rat myometrium (17–20 days of gestation). It was found that the transient inward current mainly consists of Ca²⁺ current, because the removal of Ca²⁺ ions from the external medium and 10 M nifedipine eliminated this inward current. Its steady-state inactivation curve was obtained by the standard method, in which the membrane potential of half inactivation and the slope factor were estimated to be –58.0±4.9 mV (n=11) and 8.9±1.4 mV (n=11), respectively. In a small number of preparations (in 2 out of 30 preparations), there remained a very fast inward current in Ca²⁺-free medium containing Mg²⁺. Tetrodotoxin (TTX, 10 M) can abolish this current, suggesting that the channel for this current is equivalent to the Na⁺ channel in nerve cells. Two major phases of outward currents were identified by voltage jumps from negative holding levels to more positive levels. The first phase was a fast transient outward current. This current remained intact after external tetraethylammonium (TEA, 20 mM) was added. Following the transient current, a large delayed rectified outward current reached its peak over a period of 50 ms and then decayed. The reversal potential for this outward current was determined by observing the change of polarity of the tail currents with the change in extracellular K⁺ concentration ([K⁺]₀). The slope for the change of reversal potential per ten-fold change in [K⁺]₀ is 57.7 mV at more than 23.2 mM [K⁺]_o, indicating that this current is mostly carried by K⁺ ions. Voltage-dependent inactivation of the delayed rectified outward current was determined by the standard method. The membrane potential for half inactivation and the slope factor were estimated to be –42.8±3.9 mV (n=3) and 10.1±1.5 mV (n=3), respectively. External TEA (20 mM) effectively eliminated the delayed rectified outward currents. Nifedipine (10 M) suppressed not only Ca²⁺ current but also outward K⁺ currents.     

Thapsigargin discharges intracellular calcium stores and induces transmembrane currents in human endothelial cells

We have measured the effects of thapsigargin, a specific inhibitor of endoplasmic Ca²⁺-adenosine 5-triphosphatase (Ca²⁺-ATPase), on membrane currents and on the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in single endothelial cells from the human umbilical cord vein. Currents were recorded by means of the patchclamp technique in the whole-cell mode and [Ca²⁺]_i was measured using Fura II. Application of thapsigargin at concentrations between 0.2 and 2 mol/l induced a slow increase in [Ca²⁺]_i to a peak value of 400±110 nmol/l above a resting level of 120±35 nmol/l, and then slowly declined to a new steady-state level of 315±90 nmol/l (n=33). The thapsigargin-induced increase in [Ca²⁺]_i depended on the extracellular Ca²⁺ concentration ([Ca²⁺]_o: it declined after removal of extracellular Ca²⁺, but increased again when [Ca²⁺]_o was augmented, indicating that the response depends on a transmembrane influx of Ca²⁺ ions. The peak amplitude of the histamine-induced Ca²⁺ transient was reduced in the presence of thapsigargin. This reduction was more pronounced when histamine was applied at the peak of the increase in [Ca²⁺]_i induced by thapsigargin than during the rising phase of the changes in [Ca²⁺]_i. The decline of the Ca²⁺ transient induced by histamine after washing out the agonist was also affected by thapsigargin. Before application of thapsigargin, this decline could be described by a single exponential with a time constant equal to 24.5±5 s (n=7). In the presence of thapsigargin, the decline was much slower (n =8 cells), although in four cells a fraction of about 23% still exchanged with a similar fast value of 29.4±4 s. Thapsigargin also induced a slowly developing inward current in endothelial cells at a holding potential of –40 mV. Voltage ramps applied before and during the development of this current indicated that a non-selective cation channel with a reversal potential near 0 mV was activated. In contrast with the Ca²⁺ transients, these currents did not show a declining phase. These results indicate that inhibition of the endoplasmic Ca²⁺ pump in endothelial cells increases [Ca²⁺]_i. The tonic component of this increase might be partly due to opening of non-selective Ca²⁺-permeable cation channels activated by depletion of intracellular stores.     

Effect of hydro-osmotic pressure on polycystin-2 channel function in the human syncytiotrophoblast

Polycystin-2 (PC2), one of the gene products whose mutations cause autosomal dominant polycystic kidney disease is a transient receptor potential (TRP)-type (TRPP2) Ca²⁺-permeable, non-selective cation channel. PC2 is localized in the plasma membrane, the primary cilium, and other cellular organelles of renal epithelial and other cells. Recent studies indicate that PC2 is involved in signal transduction events associated with the transient increase in cytosolic Ca²⁺. Proof of evidence now hinges on involvement of the PC2 channel in the transduction of environmental signals. PC2 is abundantly expressed in the apical membrane of human syncytiotrophoblast (hST), a highly intricate epithelial tissue, which is essential for the maternal–fetal transfer of solutes, including ions. Physical forces such as hydrostatic (H) and osmotic () pressure play important roles in placenta homeostasis. In this study, we provide new information on PC2 channel regulation in the hST by these environmental factors, and propose a model as to how they may trigger the activation of PC2. Using apical hST vesicles reconstituted in a lipid bilayer system, we found that a change in either H or modified PC2 channel activity. This stimulatory effect was no longer observed in hST vesicles pre-treated with the actin cytoskeleton disrupter cytochalasin D. As shown by immunofluorescence analysis PC2 co-localized with actin filaments in the vicinity of the plasma membrane. This co-localization was disrupted by cytochalasin D. Taken together, our findings indicate that physical forces exerted on cells regulate PC2 channel activity by a sensory mechanism involving the actin cytoskeleton.     

Na+ entry and modulation of Na+/Ca2+ exchange as a key mechanism of TRPC signaling

Ion channels formed by canonical transient receptor potential (TRPC) proteins are considered to be key players in cellular Ca²⁺ homeostasis. As permeation of Ca²⁺ through TRPC homo- and/or heteromeric channels has been repeatedly demonstrated, analysis of the physiological role of TRPC proteins was so far based on the concept that these proteins form regulated Ca²⁺ entry channels. The well-recognized lack of cation selectivity of TRPC channels and the ability to generate substantial monovalent conductances that govern membrane potential and cation gradients were barely appreciated as a physiologically relevant issue. Nonetheless, recent studies suggest monovalent, specifically Na⁺ permeation through TRPC cation channels as an important event in TRPC signaling. TRPC-mediated Na⁺ entry may be converted into a distinct pattern of cellular Ca²⁺ signals by interaction with Na⁺/Ca²⁺ exchanger proteins. This review discusses current concepts regarding the link between Na⁺ entry through TRPC channels and cellular Ca²⁺ signaling.     

Comparison of Ca2+-dependent K+ channels in the membrane of smooth muscle cells isolated from adult and foetal human aorta

Ca²⁺-activated K⁺ ionic currents in the membrane of cultured smooth muscle cells isolated from foetal and adult human aorta were studied using whole cell and single-channel patch-clamp techniques. Whole cell currents in adult smooth muscle cells were 3–8 times larger than in foetal cells of similar sizes. The elementary conductance and ionic selectivity of single Ca²⁺-activated K⁺ were identical for both types of cells. Channel openings occurred in burst, the duration of which was 3–5-fold longer in adult than in foetal cells. The voltage dependency of the channel activating mechanism and the dependency of the mean open time on the Ca²⁺ concentration on the inner side of the membrane were similar for both types of cells. These results suggest that the main reason for the increase in potassium conductance during development is an alteration in the open time of the Ca²⁺-activated K⁺ channels.     

Potassium transport in epithelial cells isolated from small intestine of the chicken

The transport of potassium has been studied in epithelial cells isolated from chicken small intestine using⁸⁶Rb as a tracer for K⁺. (i) The uptake studies revealed that about 60% of the total K⁺ net flux is inhibited by ouabain and therefore mediated by the Na⁺–K⁺-ATPase. About 20% of the ouabain-insensitive K⁺ net influx was inhibited by furosemide, bumetanide and by either Na⁺ or Cl^– removal from the incubation solution, suggesting that a Na⁺/Cl^–/K⁺ cotransport system might be present in chicken enterocytes. (ii) The efflux of K⁺ was measured from cells preloaded with⁸⁶Rb. K⁺ efflux was inhibited by Ba²⁺, quinine and verapamil; it was stimulated by A23187, and it was unaffected by 3,4-diaminopyridine. Apamin, that has no effect on basal rates of K⁺ efflux, abolished the effect of A23187. These findings suggest that K⁺ efflux appears to occur through Ca²⁺-activated K⁺ channels.     

Capacitative Ca2+ influx and a Ca2u+-dependent nonselective cation pathway are discriminated by genistein in mouse pancreatic acinar cells

We have investigated the effect of genistein on the hormone-stimulated Ca²⁺ influx and on a 28 pS nonselective cation channel in mouse pancreatic acinar cells using the Ca²⁺ indicator fluo-3 and the patch-clamp technique. The identity of the Ca^2u+ influx pathway has not been established in this cell type so far. Therefore we have investigated the Ca²⁺-dependent nonselective cation channel as a potential pathway for Ca²⁺ influx. Capacitative Ca²⁺ entry was induced by depletion of intracellular Ca²⁺ stores with 500 nM acetylcholine or with the Ca²⁺ ATPase inhibitor 2,5-di-tert- butylhydroquinone. In the presence of 100 M genistein, Ca²⁺ release was unimpaired, whereas Ca²⁺ influx was reversibly suppressed. Patch-clamp experiments demonstrated that genistein had no effect on Ca²⁺-activated nonselective cation channels, the activity of which was measured in excised membrane patches (inside/out) or in the whole-cell configuration. Therefore we conclude that this 28 pS nonselective cation channel does not contribute to Ca²⁺ influx into mouse exocrine pancreatic cells. With the exception of genistein and tyrphostin 25, other tyrosine kinase inhibitors such as methyl-2,5-dihydroxycinnamate, lavendustin A, herbimycin A, and tyrphostin B56 were without effect on Ca²⁺ signalling. Thus, the involvement of tyrosine phosphorylation in the activation of the Ca²⁺ entry mechanism in mouse pancreatic acinar cells is unclear.     

Activation of non-selective cation conductance by carbachol in freshly isolated bovine ciliary muscle cells

 In smooth muscle cells freshly isolated from the bovine ciliary body, effects of carbachol (CCh) on the membrane potential and current were examined by the whole-cell clamp method. The resting membrane potential of the muscle cells used was –60 ± 1 mV (n=111). Extracellular application of CCh (2 μM) depolarized the cells to –15 ± 5 mV (n=50) with an apparent increase in membrane conductance. Under voltage-clamp conditions, CCh (2 μM) evoked an inward current which exhibited inward-going rectification and reversed the polarity at about 0 mV. Removal of Na⁺ from the external solution caused a reduction of the amplitude of the current and a shift of the reversal potential to the negative direction. CCh was able to elicit an inward current even under a condition where Ca²⁺ was the only cation producing an inwardly directed electrochemical gradient. The current was not affected by verapamil or by tetrodotoxin. The CCh-induced current was inhibited by antimuscarinic agents with the affinity sequence: atropine ≈4–DAMP >> pirenzepine > AF-DX116, indicating that the response is mediated by a muscarinic cholinoceptor that belongs to the M₃-subtype. Unlike the non-selective cation channel current in intestinal smooth muscles, which is activated by elevation of the intracellular Ca²⁺ concentration ([Ca²⁺]_i), the current of the ciliary muscle was inactivated when the [Ca²⁺]_i was increased. The conductance, which admits Ca²⁺, may serve as a pathway for Ca²⁺ entry required for contraction. Received: 2 December 1996 / Received after revision: 7 January 1997 / Accepted: 8 January 1997     

Permselectivity for cations over anions in the upper portion of descending limbs of Henle's loop of long-loop nephron isolated from hamsters

The effects of extracellular and intracellular tetraethylammonium (TEA) ions on single Ca²⁺-activated K⁺ channels were studied in excised membrane patches from the anterior pituitary clone AtT-20/D16-16 with the patch-clamp technique. The presence of TEA on either surface of the membrane resulted in a decrease in the single-channel current. Dissociation constants at zero voltage for the TEA-receptor complex were calculated to be 52.2 mM and 0.08 mM for external and internal TEA, respectively. The high sensitivity of AtT-20/D16-16 celsl to internal TEA is of considerable interest, since in other preparations, the greater TEA sensitivity for Ca²⁺-activated K⁺ channels has thus far been found to occur on the external membrane surface. Hill plot analysis of the dose-response data yielded a slope of 0.92, indicating a one-to-one stoichiometry for TEA-receptor binding. The blockade by TEA showed little voltage or current sensitivity over the membrane potential range studied, and could be fully reversed by washout in drug-free solution. The results suggest the presence of TEA receptors on both the external and internal membrane surfaces but with different binding affinities. Occupancy of either site by TEA leads to an apparent decrease in the single-channel conductance of Ca²⁺-activated K⁺ channels.     

Effects of caffeine on intracellular calcium,calcium current and calcium-dependent potassium current in anterior pituitary GH3 cells

Caffeine elicits physiological responses in a variety of cell types by triggering the mobilization of Ca²⁺ from intracellular organelles. Here we investigate the effects of caffeine on intracellular Ca²⁺ concentration ([Ca²⁺]_i) and ionic currents in anterior pituitary cells (GH₃) cells. Caffeine has a biphasic effect on Ca²⁺-activated K⁺ current [I _K(Ca)]: it induces a transient increase superimposed upon a sustained inhibition. While the transient increase coincides with a rise in [Ca²⁺]_i, the sustained inhibition of I _K(Ca) is correlated with a sustained inhibition of the L-type Ca²⁺ current. The L-type Ca²⁺ current is also inhibited by other agents that mobilize intracellular Ca²⁺, including thyrotropin releasing hormone (TRH) and ryanodine, but in a matter distinct from caffeine. Unlike the caffeine effect, the TRH-induced inhibition washes-out under whole-cell patch-clamp conditions and is eliminated by intracellular Ca²⁺ chelators. Likewise, the ryanodine-induced inhibition desensitizes while the caffeine-induced inhibition does not. Simultaneous [Ca²⁺]_i and Ca²⁺ current measurements show that caffeine can inhibit Ca²⁺ current without changing [Ca²⁺]_i. Single-channel recordings show that caffeine reduces mean open time without affecting single-channel conductance of L-type channels. Hence the effects of caffeine on ion channels in GH₃ cells are attributable both to mobilization of intracellular Ca²⁺ and to a direct effect on the gating of L-type Ca²⁺ channels.     

Regulation of a non-selective cation channel of cultured porcine coronary artery smooth muscle cells by tyrosine kinase

A non-selective cation channel was found in primary cultured porcine coronary artery smooth muscle cells. In patch-clamp studies in the cell-attached mode, this channel was activated by bath application of genistein, a specific inhibitor of tyrosine kinase, but not by daidzein, which is similar in structure to genistein but has no inhibitory effect on tyrosine kinase. This channel discriminated poorly between Na⁺ and K⁺ (permeability ratio P _Na/P _K=1.03), and also transported Ca²⁺. The single-channel conductance measured with a pipette solution containing 150mM Na⁺ was 139±24 pS (mean ± SD, n=5), and that for the inward current measured with 100 mM Ca²⁺ solution was 25±9 pS (n=3). This non-selective cation channel was also activated by staurosporine, a non-specific protein kinase inhibitor, but not by H-7, an inhibitor of protein serine/ threonine kinase. These results suggest that the activity of the non-selective cation channel is negatively regulated by tyrosine kinase activity, and thus a decrease of the enzyme activity in porcine coronary artery smooth muscle cells may result in membrane depolarization and Ca²⁺ entry.     

ZD 7288 inhibits T-type calcium current in rat hippocampal pyramidal cells

Many studies have used the channel blocker ZD 7288 to assess possible physiological and pathophysiological roles of hyperpolarization-activated cation currents (I_h). In view of the known interplay between I_h and other membrane conductances, the effects in Wistar rats of ZD 7288 on low-voltage-activated (LVA (− or T-type)) Ca²⁺ channels were examined in whole-cell patch-clamp recordings from CA1 pyramidal cells in the presence of TTX, TEA, 4-AP, CsCl, BaCl₂ and nifedipine. ZD 7288 reduced T-type calcium channel currents and this effect was concentration dependant. ZD 7288 blocked T-type currents when applied extracellularly, but not when included in the recording pipette. Furthermore, ZD 7288 altered the steady-state voltage-dependent inactivation of T-currents. These results indicate that the blocker ZD 7288 has effects on voltage sensitive channels additional to those reported for the I_h current.     

Characterization of calcium-activated potassium channels in single smooth muscle cells using the patch-clamp technique

Single-channel currents were recorded with the patch-clamp technique from freshly dissociated vertebrate smooth muscle cells from the stomach ofBufo marinus. Of the variety of channels observed, one displayed a large linear conductance of 250 pS (in symmetric 130 mM KCl) which in excised patches was shown to be highly K⁺ selective. The probability of the channel being open (P _o) increased when [Ca²⁺]_i was elevated and/or when the membrane potential was made more positive. Thus, the features of this channel resemble the large-conductance Ca²⁺-activated K⁺ channel found in a wide variety of cell types. The voltage sensitivity of the channel was studied in detail. For patches containing a single large-conductance channel a plot ofP _o versus membrane potential followed the Boltzman relationship. Increasing [Ca²⁺]_i shifted this plot to the left along the voltage axis to more negative potentials. Both the mean closed time and mean open time varied with potential as a single exponential with almost all of the voltage sensitivity ofP _o residing in the mean closed time. These results were verified with a series of experiments carried out at lowP _o (<0.1) in patches containing multiple (N) large-conductance channels. Here the ln (NP _o) was a linear function of potential with an inverse slope of 9 mV. Almost all of the potential sensitivity lay in the mean closed time the natural log of which was also a linear function of potential with an inverse slope 11 mV in magnitude. The characteristics of this channel as well as the appearance of several of them in almost every patch suggest that they underlie the large peak outward macroscopic current found with whole-cell voltage-clamp studies.     

Ca2+-activated K+-channels in the nuclear envelope isolated from single pancreatic acinar cells

The patch-clamp techniques are applied to the outer membrane of the nuclear envelope isolated from rat pancreatic acinar cells. The nucleus identified under an inverted microscope was removed by cell surgery from enzymatically dispersed single cells. All the patch-clamp techniques, in situ, excised, and whole-material recordings were applied to the envelope. We have found voltage- and Ca²⁺-activated K⁺-channels with an unitary conductance of 200 pS in the outer membrane. The channels are activated by lumen positive potentials and by an increase in luminal Ca²⁺ concentration. They may play a role for controlling Ca²⁺-release from the lumen of the nuclear envelope (endoplasmic reticulum) to the nucleoplasm and the perinuclear cytoplasm.     

Ensemble noise and current relaxation analysis of K+ current in single isolated salivary acinar cells from rat

The K⁺ channel in rat parotid gland acinar cells were investigated by ensemble current noise analysis in single isolated cells employing the giga-seal whole cell current recording mode. Sets of 20–40 identical de- and hyperpolarization voltage steps were applied and the resultant current records were processed by computer to obtain the mean and the variance of the current. The time-course of the mean current could be fitted by the sum of two exponentials, suggesting a 3-state model. The simplest plausible hypothesis is a model with one open and two closed states. Assuming this model, the relationship between the variance (₂) and the mean current (I) could be fitted by the function ₂/I=i–I/N. The estimated single channeli/V-relations were similar to those taken from single channel current recordings, and the size of the population of channels per cell (N) was 76±26 (n=12). The validity of the model was tested by a successful simulation of the time-course of the variance.     

Mechanism of the ATP-induced rise in cytosolic Ca2+ in freshly isolated smooth muscle cells from human saphenous vein

Effects of exogenous adenosine 5-triphosphate (ATP) were studied by measurements of intracellular Ca²⁺ concentration ([Ca²⁺]_i) and membrane currents in myocytes freshly isolated from the human saphenous vein. At a holding potential of –60 mV, ATP (10 M) elicited a transient inward current and increased [Ca²⁺]_i. These effects of ATP were inhibited by ,-methylene adenosine 5-triphosphate (AMPCPP, 10 M). The ATP-gated current corresponded to a non-selective cation conductance allowing Ca²⁺ entry. The ATP-induced [Ca²⁺]_i rise was abolished in Ca²⁺-free solution and was reduced to 30.1±5.5% (n=14) of the control response when ATP was applied immediately after caffeine, and to 23.7±3.8% (n=11) in the presence of thapsigargin. The Ca²⁺-induced Ca²⁺ release blocker tetracaine inhibited the rise in [Ca²⁺]_i induced by both caffeine and ATP, with apparent inhibitory constants of 70 M and 100 M, respectively. Of the ATP-induced increase in [Ca²⁺]_i 29.3±3.9% (n=8) was tetracaine resistant. It is concluded that the effects of ATP in human saphenous vein myocytes are only mediated by activation of P_2x receptor channels. The ATP-induced [Ca²⁺]_i rise is due to both Ca²⁺ entry and Ca²⁺ release activated by Ca²⁺ ions that enter the cell through P_2x receptor channels.     

External ATP triggers a biphasic activation process of a calcium-dependent K+ channel in cultured bovine aortic endothelial cells

We have used the patch-clamp method in order to investigate the single-channel events underlying the effect of external ATP on the potassium permeability of bovine aortic endothelial cells (BAE). The results obtained from cell-attached and inside-out experiments led first to conclude that BAE cells possess an inward rectifying potassium channel activated by internal calcium at micromolar concentrations. The channel conductance for inward currents was estimated at 40 pS in symmetrical 200 mM KCl and the open-channel probability was found to be voltage insensitive within the membrane voltage range –50 to –100 mV. Based on results obtained in the cell-attached configuration, it could next be established that external ATP and ADP at micromolar concentrations could trigger, via the stimulation of P₂ purinergic receptors, a time variable activation process of the observed calcium-dependent potassium channel. This activation process was found to occur in a biphasic manner with an initial phase independent of the presence of calcium in the cell bathing medium. The second phase which could be blocked by calcium channel blockers such as Co²⁺ or La³⁺ required, however, the presence of external calcium and could be abolished by depolarizing the cells using high K⁺ external solutions. Another important aspect related to this phenomenon was the observation that removing ATP from the external medium during the second phase led to a complete abolition of the associated calcium-dependent potassium channel activation process. It is suggested from these results that the action of ATP on the potassium permeability of BAE cells is related to a second messenger mediated release of calcium from internal calcium stores coupled to an ATP-dependent calcium influx abolished at depolarizing voltages.