Ca2+-dependent K+ Channels in Bovine Adrenal Chromaffin Cells are Modulated by Lipoxygenase Metabolites of Arachidonic Acid

Fatty acids play an important role in a variety of physiological processes including ion channel modulation and catecholamine release. Using patch-clamp techniques we show that arachidonic acid (AA) is converted to lipoxygenase metabolites (LOMs) to potentiate activity of the Ca²⁺ and voltage-dependent, large-conductance K⁺ channel (BK) in bovine adrenal medullary chromaffin cells (BAMCCs). AA and LOM potentiation of BK current and recovery from potentiation were unaffected by the nonhydrolyzable ATP analogue AMP-PNP, or by exclusion of nucleotides in excised patch recordings. Also, AA and LOM potentiation of BK channel activity in outside-out patches exposed to strong Ca²⁺ buffering ruled out cytoplasmic messengers or changes in intracellular Ca²⁺ levels as causative factors. Lipoxygenase inhibitor attenuated AA, but not LOM potentiation of BK activity in outside-out patches, indicating that lipoxygenase processing of AA is possible in excised membrane patches, possibly via a membrane associated lipoxygenase. AA and LOM release have been implicated in the mechanics of catecholamine secretion from BAMCCs. By limiting action potential duration and thus voltage-gated Ca²⁺ influx, fatty acid potentiation of BK current may serve an inhibitory feedback function in regulating secretion from BAMCCs. Received: 15 July 1997/Revised: 27 January 1997     

Single K+ Channels in Embryonic Leech Ganglion Cells

We investigated the properties of single K⁺ channels in the soma membrane of embryonic leech ganglion cells using the patch-clamp technique. We compared these K⁺ channels with the K⁺ channels found previously in Retzius neurons of the adult leech. In ganglion cells of 9- to 15-day-old embryos we characterized eight different types of K⁺ channels with mean conductances of 21, 55, 84, 111, 122, 132, 149 and 223 pS. The 55 pS and 84 pS channels showed flickering and were active for less than 2 min after excising the patch. The 111 pS channel was an outward rectifier, and the open state probability (p _o ) decreased in the inside-out configuration when the Ca²⁺ concentration was raised from pCa 7 to pCa 3. The 122 pS channel also showed outward rectification. This type of channel was activated after changing from the cell-attached to the inside-out configuration and it did not inactivate during more than 30 min. The p _o was Ca²⁺- and voltage-insensitive. One hundred μm glibenclamide reversibly reduced p _o . The 132 pS channel was an outward rectifier and was Ca²⁺-insensitive. The 149 pS channel inactivated in the inside-out configuration. The 149- and the 223 pS channel showed inward rectification. The 111 pS channel had similar properties to the Ca²⁺-dependent K⁺ channel and the 122 pS channel resembled the ATP-inhibited K⁺ channel found previously in Retzius neurons of the adult leech. Received: 20 April 1995/Revised: 18 January 1996     

Single calcium-dependent cation channels in mouse pancreatic acinar cells

Summary The Ca²⁺-activated nonselective cation channel in mouse pancreatic acini has been studied with the help of patch-clamp single-channel current recording in both the cell-attached conformation and in excised inside-out membrane patches. In intact resting mouse pancreatic acinar cells no unitary activity was observed. Adding saponin to the bath solution to disrupt the plasma membrane (apart from the isolated patch membrane from which current recording was made) evoked unitary inward current steps when the free ionized Ca²⁺ concentration in the bath ([Ca²⁺] _i ) was 5×10^–8 m or above. When an electrically isolated patch membrane was excised and the internal aspects of the plasma membrane were exposed to the bath solution, channel activation could be obtained when [Ca²⁺] _i was 10^–7 m or above. However, with the passage of time the total inward current declined and about 1 min after excision no unitary current steps could be observed. At this stage Ca²⁺ in micromolar concentration was needed to open the channels and several hundred micromoles of Ca²⁺ per liter were required for maximal channel activation. Our results indicate that the Ca²⁺-activated nonselective cation channel is more sensitive to internal Ca²⁺ than hitherto understood and that it may therefore play a role under physiological conditions in intact cells.     

Effects of Thiol-Modifying Agents on a K(Ca2+) Channel of Intermediate Conductance in Bovine Aortic Endothelial Cells

Ca²⁺-activated K⁺ channels (K(Ca²⁺)) constitute key regulators of the endothelial cell electrophysiological response to InsP₃-mobilizing agonists. Inside-out and outside-out patch clamp experiments were thus undertaken to determine if the gating properties of a voltage-insensitive K(Ca²⁺) channel of intermediate conductance present in bovine aortic endothelial (BAE) cells could be modified by specific sulfhydryl (SH) oxidative and/or reducing reagents. The results obtained first indicate that cytosolic application of hydrophilic oxidative reagents such as 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB) (0.2 to 5 mm) or [(O-carboxyphenyl)thio]ethyl mercury sodium salt (thimerosal) (0.5 to 5 mm) reduces gradually the K(Ca²⁺) channel activity with no modification of the channel unitary conductance. The inhibitory action of DTNB (1 to 5 mm) or thimerosal (1 to 5 mm) was not reserved following withdrawal of the oxidative agents, but channel activity could partly be restored by the addition of the SH group reducing agents dithiothreitol (DTT) (5 mm) or reduced glutathione (GSH) (5 mm) in 53% and 50% of the inside-out experiments performed with DTNB and thimerosal respectively. Similar results were obtained using H₂O₂ at concentrations ranging from 500 μm to 10 mm as oxidative reagent. In contrast, the lipid soluble oxidative agent 4,4′-dithiodipyridine (4-PDS) (1 mm) appeared in inside-out experiments less potent than DTNB and thimerosal at inhibiting the K(Ca²⁺) channel activity, suggesting that the critical SH groups involved in channel gating are localized at the inner face of the cell membrane. This conclusion was further substantiated by a series of outside-out patch clamp experiments which showed that DTNB (5 mm) and thimerosal (5 mm) were unable to inhibit the K(Ca²⁺) channel activity when applied to the external surface of the excised membrane. Finally, no significant changes of the gating properties of the K(Ca²⁺) channel were observed in inside-out experiments where the SH group reducing agents DTT and GSH were applied immediately following membrane excision. However, the application of either GSH or DTT was found to partly restore channel activity in experiments where the K(Ca²⁺) channels showed significant rundown. Received: 7 August 1996/Revised: 20 February 1997     

Single Mechanosensitive and Ca2+-Sensitive Channel Currents Recorded from Mouse and Human Embryonic Stem Cells

Cell-attached and inside-out patch clamp recording was used to compare the functional expression of membrane ion channels in mouse and human embryonic stem cells (ESCs). Both ESCs express mechanosensitive Ca²⁺ permeant cation channels (MscCa) and large conductance (200 pS) Ca²⁺-sensitive K⁺ (BK_Ca2+) channels but with markedly different patch densities. MscCa is expressed at higher density in mESCs compared with hESCs (70 % vs. 3 % of patches), whereas the BK_Ca2+ channel is more highly expressed in hESCs compared with mESCs (~50 % vs. 1 % of patches). ESCs of both species express a smaller conductance (25 pS) nonselective cation channel that is activated upon inside-out patch formation but is neither mechanosensitive nor strictly Ca²⁺-dependent. The finding that mouse and human ESCs express different channels that sense membrane tension and intracellular [Ca²⁺] may contribute to their different patterns of growth and differentiation in response to mechanical and chemical cues.     

Proteolytic activation of a hyperpolarization- and calcium-dependent potassium channel inParamecium

Summary The effects of proteolysis on a hyperpolarization- and Ca²⁺-dependent K channel from the surface membrane ofParamecium tetraurelia were examined in the inside-out excised patch mode. Treatment with trypsin, pronase or thermolysin removed the Ca²⁺-dependence of the channel activation, yielding an increase in channel activity greater than 2.5-fold at all Ca²⁺ concentrations between 10^–4 and 10^–8 m. Thermolysin addition-ally removed the voltage dependence of channel opening and gave the most activation among the three proteases tested. Proteolysis did not affect the single-channel conductance. In an analogy to the mechanism of activation of many Ca²⁺-dependent enzymes it is suggested that thisParamecium channel has a cytoplasmic inhibitory domain which can be removed by proteolysis, and that the physiological activation by Ca²⁺ is due to a temporary removal of this inhibition. Moreover, these findings indicate structural differences between depolarization-, Ca²⁺-dependent K channels (BK channels) and the hyperpolarization-, Ca²⁺-dependent K channels inParamecium.     

Ca2+ dependence and pharmacology of large-conductance K+ channels in nonlabor and labor human uterine myocytes

Two populations,Ca²⁺-dependent(BK_Ca) andCa²⁺-independentK⁺ (BK) channels of largeconductance were identified in inside-out patches of nonlabor and laborfreshly dispersed human pregnant myometrial cells, respectively.Cell-attached recordings from nonlabor myometrial cells frequentlydisplayed BK_Ca channel openings characterized by a relatively low open-state probability, whereas similar recordings from labor tissue displayed either no channel openings or consistently high levels of channel activity that oftenexhibited clear, oscillatory activity. In inside-out patch recordings,Ba²⁺ (2-10 mM),4-aminopyridine (0.1-1 mM), andShaker B inactivating peptide("ball peptide") blocked theBK_Ca channel but were much lesseffective on BK channels. Application of tetraethylammonium toinside-out membrane patches reduced unitary current amplitude ofBK_Ca and BK channels, withdissociation constants of 46 mM and 53 µM, respectively.Tetraethylammonium applied to outside-out patches decreased the unitaryconductance of BK_Ca and BKchannels, with dissociation constants of 423 and 395 µM,respectively. These results demonstrate that the properties of humanmyometrial large-conductance K⁺channels in myocytes isolated from laboring patients are significantly different from those isolated from nonlaboring patients.

     

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     

The N-terminal domain of Orai3 determines selectivity for activation of the store-independent ARC channel by arachidonic acid

Although highly selective Ca²⁺ entry pathways play a critical role in agonist-activated Ca²⁺ signals in non-excitable cells, only with the recent discovery of the Orai proteins have the first insights into the molecular nature of these pathways been possible. To date, just two such highly Ca²⁺-selective “Orai channels” have been identified in native cells—the storeoperated CRAC channels and the store-independent, arachidonic acid-activated ARC channels. Studies have shown that the functional CRAC channel pore is formed by a tetrameric arrangement of Orai1 subunits, whilst a heteropentamer of three Orai1 subunits and two Orai3 subunits forms the functional ARC channel pore. Importantly, this inclusion of Orai3 subunits in the ARC channel structure has been shown to play a specific role in determining the selectivity of these channels for activation by arachidonic acid. Using an approach based on the expression of various concatenated constructs, we examined the basis for this Orai3-dependent effect on selectivity for arachidonic acid. We show that, whilst heteropentamers containing only one Orai3 subunit are sensitive to arachidonic acid, specific selectivity for activation by this fatty acid is only achieved on inclusion of the second Orai3 subunit in the pentamer. Further studies identified the cytosolic N-terminal domain of Orai3 as the region specifically responsible for this switch in selectivity. Substitution of just this domain into an otherwise complete single Orai1 subunit within a concatenated 31111 pentamer is sufficient to change the resulting channel from one that is predominantly store-operated, to one that is exclusively activated by arachidonic acid.Key words: STIM1, Orai1, Orai3, calcium channel, calcium entry, arachidonic acid     

Different contributions of calcium channel subtypes to electrical excitability of chromaffin cells in rat adrenal slices

We characterized the ionic currents underlying the cellular excitability and the Ca²⁺‐channel subtypes involved in action potential (AP) firing of rat adrenal chromaffin cells (RCCs) preserved in their natural environment, the adrenal gland slices, through the perforated patch‐clamp recording technique. RCCs prepared from adrenal slices exhibit a resting potential of ?54 mV, firing spontaneous APs (2–3 spikes/s) generated by the opening of Na⁺ and Ca²⁺‐channels, and terminated by the activation of voltage and Ca²⁺‐activated K⁺‐channels (BK). Ca²⁺ influx via L‐type Ca²⁺‐channels is involved in reaching threshold potential for AP firing, and is responsible for activation of BK‐channels contributing to AP‐repolarization and afterhyperpolarization, whereas P/Q‐type Ca²⁺‐channels are involved only in the repolarization phase. BK‐channels carry total outward current during AP‐repolarization. Blockade of L‐type Ca²⁺‐channels reduces BK‐current ~60%, whereas blockade of N‐ or P/Q‐type produces little effect. This study demonstrates that Ca²⁺ influx through L‐type Ca²⁺‐channels plays a key role in modulating the threshold potential from RCCs in situ.

     

Membrane Potassium Channels and Human Bladder Tumor Cells. I. Electrical Properties

These experiments were conducted to determine the membrane K⁺ currents and channels in human urinary bladder (HTB-9) carcinoma cells in vitro. K⁺ currents and channel activity were assessed by the whole-cell voltage clamp and by either inside-out or outside-out patch clamp recordings. Cell depolarization resulted in activation of a Ca²⁺-dependent outward K⁺ current, 0.57 ± 0.13 nS/pF at −70 mV holding potential and 3.10 ± 0.15 nS/pF at 30 mV holding potential. Corresponding patch clamp measurements demonstrated a Ca²⁺-activated, voltage-dependent K⁺ channel (K_Ca) of 214 ± 3.0 pS. Scorpion venom peptides, charybdotoxin (ChTx) and iberiotoxin (IbTx), inhibited both the activated current and the K_Ca activity. In addition, on-cell patch recordings demonstrated an inwardly rectifying K⁺ channel, 21 ± 1 pS at positive transmembrane potential (V _m ) and 145 ± 13 pS at negative V _m . Glibenclamide (50 μm), Ba²⁺ (1 mm) and quinine (100 μm) each inhibited the corresponding nonactivated, basal whole-cell current. Moreover, glibenclamide inhibited K⁺ channels in inside/out patches in a dose-dependent manner, and the IC₅₀= 46 μm. The identity of this K⁺ channel with an ATP-sensitive K⁺ channel (K_ATP) was confirmed by its inhibition with ATP (2 mm) and by its activation with diazoxide (100 μm). We conclude that plasma membranes of HTB-9 cells contain the K_Ca and a lower conductance K⁺ channel with properties consistent with a sulfonylurea receptor-linked K_ATP. Received: 12 June 1997/Revised: 21 October 1997     

L-type Ca<Superscript>2+</Superscript> channel and Ca<Superscript>2+</Superscript>-permeable nonselective cation channel as a Ca<Superscript>2+</Superscript> conducting pathway in myocytes isolated from the cricket lateral oviduct

The Ca²⁺-conducting pathway of myocytes isolated from the cricket lateral oviduct was investigated by means of the whole-cell patch clamp technique. In voltage-clamp configuration, two types of whole cell inward currents were identified. One was voltage-dependent, initially activated at –40 mV and reaching a maximum at 10 mV with the use of 140 mM Cs²⁺-aspartate in the patch pipette and normal saline in the bath solution. Replacement of the external Ca²⁺ with Ba²⁺ slowed the current decay. Increasing the external Ca²⁺ or Ba²⁺ concentration increased the amplitude of the inward current and the current–voltage (I–V) relationship was shifted as expected from a screening effect on negative surface charges. The inward current could be carried by Na⁺ in the absence of extracellular Ca²⁺. Current carried by Na⁺ (I _Na) was almost completely blocked by the dihydropyridine Ca²⁺ channel antagonist, nifedipine, suggesting that the I _Na is through voltage-dependent L-type Ca²⁺ channels. The other inward current is voltage-independent and its I–V relationship was linear between –100 mV to 0 mV with a slight inward rectification at more hyperpolarizing membrane potentials when 140 mM Cs⁺-aspartate and 140 mM Na⁺-gluconate were used in the patch pipette and in the bath solution, respectively. A similar current was observed even when the external Na⁺ was replaced with an equimolar amount of K⁺ or Cs⁺, or 50 mM Ca²⁺ or Ba²⁺. When the osmolarity of the bath solution was reduced by removing mannitol from the bath solution, the inward current became larger at negative potentials. The I–V relationship for the current evoked by the hypotonic solution also showed a linear relationship between –100 mV to 0 mV. Bath application of Gd³⁺ (10 M) decreased the inward current activated by membrane hyperpolarization. These results clearly indicate that the majority of current activated by a membrane hyperpolarization is through a stretch-activated Ca²⁺-permeable nonselective cation channel (NSCC). Here, for the first time, we have identified voltage-dependent L-type Ca²⁺ channel and stretch-activated Ca²⁺-permeable NSCCs from enzymatically isolated muscle cells of the cricket using the whole-cell patch clamp recording technique.Abbreviations I _Ca Ca²⁺ current - I _Na Na⁺ current - I–V current–voltage - NSCC nonselective cation channel Communicated by G. Heldmaier     

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.     

Pituitary adenylate cyclase-activating peptide (PACAP) recruits low voltage-activated T-type calcium influx under acute sympathetic stimulation in mouse adrenal chromaffin cells

Low voltage-activated T-type Ca_v3.2 calcium channels are expressed in neurosecretory chromaffin cells of the adrenal medulla. Previous studies have shown that naïve adrenal chromaffin cells express a nominal Ca_v3.2-dependent conductance. However, Ca_v3.2 conductance is up-regulated following chronic hypoxia or long term exposure to cAMP analogs. Thus, although a link between chronic stressors and up-regulation of Ca_v3.2 exists, there are no reports testing the specific role of Ca_v3.2 channels in the acute sympathoadrenal stress response. In this study, we examined the effects of acute sympathetic stress on T-type Ca_v3.2 calcium influx in mouse chromaffin cells in situ. Pituitary adenylate cyclase-activating peptide (PACAP) is an excitatory neuroactive peptide transmitter released by the splanchnic nerve under elevated sympathetic activity to stimulate the adrenal medulla. PACAP stimulation did not evoke action potential firing in chromaffin cells but did cause a persistent subthreshold membrane depolarization that resulted in an immediate and robust Ca²⁺-dependent catecholamine secretion. Moreover, PACAP-evoked secretion was sensitive to block by nickel chloride and was acutely inhibited by protein kinase C blockers. We utilized perforated patch electrophysiological recordings conducted in adrenal tissue slices to investigate the mechanism of PACAP-evoked calcium entry. We provide evidence that stimulation with exogenous PACAP and native neuronal stress stimulation both lead to a protein kinase C-mediated phosphodependent recruitment of a T-type Ca_v3.2 Ca²⁺ influx. This in turn evokes catecholamine release during the acute sympathetic stress response.     

Modulation of small conductance calcium-activated potassium channels in C6 glioma cells

Using the patch clamp technique, we have characterized a small conductance, calcium-activated potassium (SK) channel in the C6 glioma cell line. Elevation of cytosolic Ca²⁺ concentration ([Ca²⁺] _i ) by applications of serotonin or ionomycin induced bursts of channel openings recorded in the cell-attached configuration. These channels underlie the serotonin-induced, [Ca²⁺] _i -activated whole-cell K⁺ conductance described previously. [Ca²⁺] _i directly activated SK channels in inside-out patches with a biphasic concentration dependence. Submicromolar [Ca²⁺] _i induced bursts of channel openings with a unitary conductance of about 25 pS, similar to that of the serotonin-induced channels. Supramicromolar [Ca²⁺] _i caused prolonged openings with a unitary conductance of about 35 pS, resulting in a pronounced increase of the average current in patches exposed to [Ca²⁺] _i above 100 m. The two modes of opening reflect the activity of the same SK channel. The channel conductance depended on external K⁺ concentration with K _Dof 5 m. The channel was slightly permeable to cations other than K⁺, with a permeability ratio for K⁺Ca²⁺Na⁺ of 10.0400.030, respectively. ATP was required to maintain channel activity in outside-out patches but was not essential in inside-out patches. The modulation of SK channels in C6 cells by components in their microenvironment may be related to the role of glial cells in controlling the extracellular milieu in the CNS.The authors are grateful to Dr. M. Segal for continuous support, stimulating discussions and criticism throughout the course of this work, to Dr. I. Steinberg for helpful suggestions and to Dr. H. Jarosch, for helping with the Fortran application. N.M.'s research was supported in part by BARD, the U.S.-Israel Binational Agricultural Research and Development Fund, grant no. IS-1670-89RC.     

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 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⁺.     

Characterization of a Triacylglycerol Lipase That Liberates Arachidonic Acid from Bovine Chromaffin Cells During Secretion

Abstract: Primary cultures of chromaffin cells from bovine adrenal medullae were used as a model to study lipolytic events during stimulus-secretion coupling. It has been shown that chromaffin cells liberate arachidonic acid in addition to their main secretion product, the catecholamines. To understand more about the mechanism of arachidonic acid liberation, chromaffin cells were labeled with radioactive arachidonic acid, stimulated, and then analyzed for changes in lipid composition. After stimulation with 10^?4M acetylcholine, the radioactivity of triacylglycerols decreased to the same extent that the free arachidonic acid level rose. This finding suggests that in bovine chromaffin cells a stimulation-dependent triacylglycerol lipase (triacylglycerol hydrolase; EC 3.1.1.3) is involved in arachidonic acid liberation. Further work was performed on detection, characterization, and isolation of this enzyme. Triacylglycerol lipase activity was found in whole cell homogenates and in plasma membrane fractions isolated from adrenal medullary tissue. The plasma membrane lipase showed a pH optimum of 4.3. The apparent Michaelis constant was determined as 3.3 × 10^?4 mol/L. Ca²⁺ did not influence the enzymatic activity. To differentiate the plasma membrane triacylglycerol lipase from the previously described plasma membrane diacylglycerol lipase of chromaffin cells, the influence of RG 80267, a specific diacylglycerol lipase inhibitor, was examined. RG 80267 (50 μM) inhibited the triacylglycerol lipase by only 24%, although diacylglycerol lipase was totally inhibited with only 20 μM RG 80267. The pH optimum of homogenate lipase was broad, lying between 4 and 7. Starting from the soluble fraction of whole cell homogenates, the triacylglycerol lipase was partially purified by ultracentrifugation and size-exclusion chromatography. The molecular mass of the enzyme as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was found to be between 47 and 57 kDa.     

Voltage-dependent and Src-mediated regulation of NMDA receptor single channel outward currents in cortical neurons

A voltage-dependent but Ca²⁺-independent regulation of N-methyl-D-aspartate (NMDA) receptor outward activity was studied at the single channel level using outside-out patches of cultured mouse cortical neurons. Unlike the inward activity associated with Ca²⁺ and Na⁺ influx, the NMDA receptor outward K⁺ conductance was unaffected by changes in Ca²⁺ concentration. Following a depolarizing pre-pulse, the single channel open probability (NP _o), amplitude, and open duration of the NMDA inward current decreased, whereas the same pre-depolarization increased those parameters of the NMDA outward current (pre-pulse facilitation). The outward NP _o was increased by the pre-pulse facilitation, disregarding Ca²⁺ changes. The voltage–current relationships of the inward and outward currents were shifted by the pre-depolarization toward opposite directions. The Src family kinase inhibitor, PP1, and the Src kinase antibody, but not the anti-Fyn antibody, blocked the pre-pulse facilitation of the NMDA outward activity. On the other hand, a hyperpolarizing pre-pulse showed no effect on NMDA inward currents but inhibited outward currents (pre-pulse depression). Application of Src kinase, but not Fyn kinase, prevented the pre-pulse depression. We additionally showed that a depolarization pre-pulse potentiated miniature excitatory synaptic currents (mEPSCs). The effect was blocked by application of the NMDA receptor antagonist AP-5 during depolarization. These data suggest a voltage-sensitive regulation of NMDA receptor channels mediated by Src kinase. The selective changes in the NMDA receptor-mediated K⁺ efflux may represent a physiological and pathophysiological plasticity at the receptor level in response to dynamic changes in the membrane potential of central neurons.     

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.