ATP maintains ATP-inhibited K+ channels in an operational state

In patch-clamp records of K⁺ _ATP channels in an insulin-secreting cell line (RINm5F) inhibition evoked by exposing the internal surface of the membrane to ATP is followed not just by the recovery of K⁺ _ATP channel activity when the ATP is removed but by a marked activation of K⁺ _ATP channels. This phenomenon is not a direct consequence of channel closure as inhibition induced by quinidine and quinine is followed upon the removal of the drug only by the recovery of K⁺ _ATP channel activity and not by post-inhibitory activation. If ATP is applied to the exposed internal surface of a membrane patch when all of its K⁺ _ATP channel have run down subsequent removal of the ATP causes their activation. The magnitude and duration of the reactivation of K⁺ _ATP channels is shown to depend upon both the concentration of ATP and the length of time for which the membrane is exposed to ATP. We therefore have a paradoxical situation in that K⁺ channels which are inhibited by intracellular ATP require intracellular ATP to retain the ability to open.     

GTP and GDP activation of K+ channels that can be inhibited by ATP

K⁺ channels that can be inhibited by intracellular ATP have been found in many different cell types. In the insulin-secreting pancreatic islet cells these channels are of crucial importance for stimulus-secretion coupling as glucose stimulation closes the ATP-sensitive channels which leads to depolarization and firing of Ca²⁺ action potentials. We now demonstrate that nucleotides other than ATP also influence the gating of these K⁺ channels. In contrast to the action of ATP, GTP (10 M – 1 mM) and GDP (100 M to 1 mM) evoke dose-dependent channel activation and this effect is immediately reversible. Phosphorylation is not directly involved as non-hydrolysable GTP-and GDP-analogues also evoke channel opening. ATP reversibly inhibits opening of the GTP- or GDP- activated K⁺ channels.     

Activation of K+ and Cl− channels by Ca2+ and cyclic AMP in dissociated kidney epithelial (MDCK) cells

In dissociated MDCK cells, activators of the cyclic AMP system cause depolarization detectable by changes in fluorescence of the membrane potential sensitive dye bisoxonol. Addition of forskolin (60 M), vasopressin (2 M), 8-bromo-cyclic AMP (0.5 mM) or l-epinephrine (10 M) depolarized the cells substantially in low Cl^– (5 mM) but had little effect in high Cl^– (140 mM) solution. These results are consistent with cyclic AMP activation of Cl^– channels. The Ca²⁺-ionophore ionomycin (1 M) produced a rapid hyperpolarization in low and high Cl^– solutions, consistent with K⁺ channel opening. Using a clonal subline, MDCK-14, the magnitude of the ionomycin hyperpolarization was roughly proportional to the concomitant rise in [Ca²⁺]_i as measured with the intracellular Ca²⁺ probe indo-I. Both l-epinephrine and isoproterenol appeared to activate the Cl^– channels. However only l-epinephrine produced a [Ca²⁺]_i rise and a transient hyperpolarization (due to K⁺ channel opening), which preceeded the depolarization due to Cl^– channel opening. The l-epinephrine-induced [Ca²⁺]_i response of the heterogeneous MDCK cell population but not of the clonal subline MDCK-14 was inhibited by removal of extracellular Ca²⁺. In the latter only the slow secondary phase of the [Ca²⁺]_i rise was affected by Ca²⁺ removal. It is concluded that l-epinephrine activates K⁺ and Cl^– channels in a sequential manner in MDCK cells by Ca²⁺ and cAMP signals, presumably via - and -adrenergic receptors located on the same cell.Abbreviations MDCK cells Madin Darby Canine Kidney cells - [Ca²⁺]_i intracellular calcium concentration - [Cl^–]_i intracellular chloride concentration - [Cl^–]_o extracellular chloride concentration - [Na⁺+K⁺]_i intracellular concentration of Na⁺ and K⁺ - [Na⁺+K⁺]_o extracellular concentration of Na⁺ and K⁺ - E_M transmembrane potential - E_Cl chloride equilibrium potential - E_K potassium equilibrium potential - bis-oxonol [bis(1,3-diethylthio-barbiturate)] trimethine oxonol - DMSO dimethylsulfoxide - EDTA ethylenediaminetetraacetic acid - EGTA ethylene glycol bis (-aminoethyl ether) N,N-tetraacetic acid - Hepes 4-(2-hydroxyethyl)-1 piperazineethanesulfonic acid - NMG⁺ N-methylglucamine - RPMI medium Rosewell Park Memorial Institute medium     

Small-conductance Ca2+-activated K+ channels in bovine chromaffin cells

Simultaneous whole-cell patch-clamp and fura-2 fluorescence [Ca²⁺]_i measurements were used to characterize Ca²⁺-activated K⁺ currents in cultured bovine chromaffin cells. Extracellular application of histamine (10 M) induced a rise of [Ca²⁺]_i concomitantly with an outward current at holding potentials positive to –80 mV. The activation of the current reflected an increase in conductance, which did not depend on membrane potential in the range –80 mV to –40 mV. Increasing the extracellular K⁺ concentration to 20 mM at the holding potential of –78 mV was associated with inwardly directed currents during the [Ca²⁺]_i elevations induced either by histamine (10 M) or short voltage-clamp depolarizations. The current reversal potential was close to the K⁺ equilibrium potential, being a function of external K⁺ concentration. Current fluctuation analysis suggested a unit conductance of 3–5 pS for the channel that underlies this K⁺ current. The current could be blocked by apamin (1 M). Whole-cell current-clamp recordings snowed that histamine (10 M) application caused a transient hyperpolarization, which evolved in parallel with the [Ca²⁺]_i changes. It is proposed that a small-conductance Ca²⁺-activated K⁺ channel is present in the membrane of bovine chromaffin cells and may be involved in regulating catecholamine secretion by the adrenal glands of various species.     

Aldosterone actions on basolateral Na+/H+ exchange in Madin-Darby canine kidney cells

In recent studies, there has been a re-evaluation of the polarity of Na⁺/H⁺ exchange in Madin-Darby canine kidney (MDCK) cells. This study was designed to examine aldosterone actions on basolaterally located Na⁺/H⁺ exchange of MDCK cell monolayers grown on permeant filter supports; pH_i was analysed in the absence of bicarbonate by using the pH-sensitive fluorescent probe 2,7-bis(carboxyethyl)-5,6-carboxyfluorescein. Pre-exposure of MDCK cells to aldosterone led within 10–20 min to an alkalization of pH_i ( 0.3 pH unit); this effect is prevented by an addition of dimethylamiloride to the basolateral superfusate. Addition of aldosterone led to stimulation of the basolaterally located Na⁺/H⁺ exchange activity (Na⁺-dependent recovery from an acid load); this effect required preincubation (more then 3 min) and was observed at 0.1 nM aldosterone. Preexposure (15 min) of MDCK monolayers to phorbol 12-myristate 13-acetate also led to an activation of Na⁺/H⁺ exchange; pre-exposure to 8-bromo-cAMP led to inhibition of Na⁺/H⁺ exchange activity. An inhibitory effect of aldosterone was observed if Na⁺/H⁺ exchange activity was analysed in the presence of aldosterone; the highest inhibitory effects (20%–30%) occurred at concentrations of 5 nM and higher. Aldosterone-dependent inhibition does not require preincubation and is fully reversible; it was only observed at low (20 mM) but not at high Na⁺ concentrations (130 mM). The data suggest that aldosterone has an instantaneous inhibitory effect on basolaterally located Na⁺/H⁺ exchange activity under conditions of low Na⁺, but stimulates the rate of transport activity upon preincubation under conditions of physiological Na⁺ concentrations.     

L-alanine evokes opening of single Ca2+-activated K+ channels in rat liver cells

Cellular uptake of neutral amino acids via Na⁺ cotransporters is known to be associated with an increased membrane K⁺ conductance mediated by an unknown mechanism that is essential for avoiding excessive cell swelling. We now demonstrate by patch-clamp single-channel current recording that exposure of rat liver cells to L-alanine, but not the poorly transported D-stereoisomer, evokes opening of single K⁺ channels and that this effect is reversible upon removal of the amino acid. The nature of the conductance pathways opened in the intact cell by L-alanine has been investigated in cell-free excised membrane patches where it can be shown that the K⁺-selective channels are opened by Ca²⁺ acting from the inside of the membrane at a concentration as low as 0.1 M.     

Changes in inward rectifier K+ channels in hepatic stellate cells during primary culture

PURPOSE: This study examined the expression and function of inward rectifier K(+) channels in cultured rat hepatic stellate cells (HSC). MATERIALS AND METHODS: The expression of inward rectifier K(+) channels was measured using real-time RT-PCR, and electrophysiological properties were determined using the gramicidin-perforated patch-clamp technique. RESULTS: The dominant inward rectifier K(+) channel subtypes were K(ir)2.1 and K(ir)6.1. These dominant K(+) channel subtypes decreased significantly during the primary culture throughout activation process. HSC can be classified into two subgroups: one with an inward-rectifying K(+) current (type 1) and the other without (type 2). The inward current was blocked by Ba(2+) (100 microM) and enhanced by high K(+) (140 mM), more prominently in type 1 HSC. There was a correlation between the amplitude of the Ba(2+)-sensitive current and the membrane potential. In addition, Ba(2+) (300 microM) depolarized the membrane potential. After the culture period, the amplitude of the inward current decreased and the membrane potential became depolarized. CONCLUSION: HSC express inward rectifier K(+) channels, which physiologically regulate membrane potential and decrease during the activation process. These results will potentially help determine properties of the inward rectifier K(+) channels in HSC as well as their roles in the activation process.     

Roles of K+ channels in regulating tumour cell proliferation and apoptosis

K⁺ channels are a most diverse class of ion channels in the cytoplasmic membrane and are distributed widely in a variety of cells including cancer cells. Cell proliferation and apoptosis (programmed cell death or cell suicide) are two counterparts that share the responsibility for maintaining normal tissue homeostasis. Evidence has been accumulating from fundamental studies indicating that tumour cells possess various types of K⁺ channels, and that these K⁺ channels play important roles in regulating tumour cell proliferation and apoptosis, i.e. facilitating unlimited growth and promoting apoptotic death of tumour cells. The potential implications of K⁺ channels as a pharmacological target for cancer therapy and a biomarker for diagnosis of carcinogenesis are attracting increasing interest. This review aims to provide a comprehensive overview of current status of research on K⁺ channels/currents in tumour cells. Focus is placed on the roles of K⁺ channels/currents in regulating tumour cell proliferation and apoptosis. The possible mechanisms by which K⁺ channels affect tumour cell growth and death are discussed. Speculations are also made on the potential implications of regulation of tumour cell proliferation and apoptosis by K⁺ channels.     

Acetylcholine and ATP hyperpolarize endothelium via activation of different types of Ca(2+)-activated K+ channels

Experiments on rat thoracic aorta showed that ATP and acetylcholine hyperpolarize endothelial cell via selective activation of low (SK) and intermediate (IR) conductance Ca²⁺-activated K⁺ channels, respectively. It was hypothesized that ATP- and acetylcholine-activated Ca²⁺-signals are spatially separated and generated in plasma membrane regions with predominant localization of SK- and IR-channels, respectively.     

K+ channels in PC12 cells are affected by propofol

The effect on K⁺ currents (I _K) of the general anaesthetic propofol (PR) (2,6-diisopropylphenol) was tested in undifferentiated clonal pheochromocytoma (PC12) cells using the patch-clamp technique in whole-cell and single-channel configurations. PR decreased macroscopic I _K amplitudes in a concentration-dependent way from 50 M to 1 mM. The blocking effect was unchanged by repetitive depolarizing pulses and it was independent of the holding potential. Whereas activation of I _K in control conditions was fitted by sigmoidal plus exponential time courses, only the sigmoidal time course gave an adequate fit with PR in the bath. The above effects were reversible. PR concentrations below 140 M decreased single-channel activity for K⁺ channels with unitary conductance of 22 pS, in the voltage range between –40 and 60 mV from a holding potential of –50 mV. In contrast, the anaesthetic had nearly no effect on the opening probability of a channel with conductance of 10 pS. The unitary current amplitudes were unaffected in both channel types. These results suggest that PR action on I _K may depend on the different blocking mechanisms of the K⁺ channels.     

Aldosterone-regulated ion transporters in the kidney

Summary Madin-Darby canine kidney (MDCK) cells resemble intercalated cells of the renal collecting duct. In these cultured epithelial cells aldosterone activates apical Na⁺/H⁺ exchange, initiating a cascade of intracellular events such as cell growth, epithelial cell polarity, and stimulation of transepithelial ion transport. Transepithelial K⁺ secretion is triggered by the insertion of new ion channels and the activation of previously quiescent channels with increasing cytoplasmic pH. Aldosterone supplies the cell with ion transporters necessary for adequate function of the renal collecting duct when the organism is metabolically challenged.     

Reciprocal effects of Ca2+ and Mg-ATP on the ‘run-down’ of the K+ channels in opossum kidney cells

Using the patch clamp technique, we identified an inwardly rectifying K⁺ channel in the membrane of opossum kidney cells. The single channel conductance was about 90 pS for inward currents and 30 pS for outward currents under a symmetrical high-K⁺ condition. The activity of the channel was found to decrease with time during recording from inside-out patches. In the solution with submicromolar Ca²⁺, the activity disappeared within 4–20 min. Intracellular Ca²⁺ promoted the run-down of the channel activity at 0.1–1 mM, whereas millimolar Mg-ATP restored the activity after run-down. The run-down channels could never be reactivated by ATP in the absence of Mg²⁺, or by a nonhydrolyzable ATP analog, AMPPNP, even in the presence of Mg²⁺.     

Regulation of inwardly rectifying K+ channels by intracellular pH in opossum kidney cells

The effects of intracellular pH on an inwardly rectifying K⁺ channel (K_in channel) in opossum kidney (OK) cells were examined using the patch-clamp technique. Experiments with inside-out patches were first carried out in Mg²⁺-and adenosine triphosphate (ATP)-free conditions, where Mg²⁺-induced inactivation and ATP-induced reactivation of K_in channels were suppressed. When the bath (cytoplasmic side) pH was decreased from 7.3 to either 6.8 or 6.3, K_in channels were markedly inhibited. The effect of acid pH was not fully reversible. When the bath pH was increased from 7.3 to 7.8, 8.3 or 8.8, the channels were activated reversibly. The channel activity exhibited a sigmoidal pH dependence with a maximum sensitivity at pH 7.5. Inside-out experiments were also carried out with a solution containing 3 mM Mg-ATP and a similar pH sensitivity was observed. However, in contrast with the results obtained in the absence of Mg²⁺ and ATP, the effect of acid pH was fully reversible. Experiments with cell-attached patches demonstrated that changes in intracellular pH, which were induced by changing extracellular pH in the presence of an H⁺ ionophore, could influence the channel activity reversibly. It is concluded that the activity of K_in channels can be controlled by the intracellular pH under physiological conditions.     

Determination of intracellular K+ activity in rat kidney proximal tubular cells

The intracellular K⁺ activity of rat kidney proximal tubular cells was determined in vivo, using intracellular microelectrodes. In order to minimize damage from the impaling electrodes, separate measurements on separate cells, were performed with single-barrelled KCl-filled non-selective electrodes and single-barrelled, K⁺-sensitive microelectrodes, which were filled with a liquid K⁺-exchanger resin that has also a small sensitivity to Na⁺. Both electrodes had tip diameters of 0.2 m or below. The proper intracellular localization of the electrodes was ascertained by recording the cell potential response to intermittent luminal perfusions with glucose. The membrane potential measured with the non-selective microelectrodes was –76.3±8.1 mV (n=81) and the potential difference measured with the K⁺-sensitive microelectrode was –7.2±5.8 mV (n=32). Based on the activity of K⁺ in the extracellular fluid of 3 mmol/l the intracellular K⁺ activity was estimated to be 82 mmol/l. Assuming equal K⁺-activity coefficients to prevail inside and outside the cell, this figure suggests that the intracellular K⁺ concentration is 113 mmol/l which must be considered as a lower estimate, however. The data indicate that the K⁺-ion distribution between cytoplasm and extracellular fluid is not in equilibrium with the membrane potential, but that K⁺ is actively accumulated inside the cell. This result provides direct evidence for the presence of an active K⁺ pump in the tubular cell membranes, which in view of other observations, must be envisaged as a (not necessarily electroneutral) Na⁺/K⁺-exchange pump which operates in the peritubular cell membrane and is eventually responsible for the major part of the tubular solute and water absorption.     

Protein kinase C activation causes inhibition of Na/K-ATPase activity in Madin-Darby canine kidney epithelial (MDCK) cells

To evaluate the influence of protein kinase C (PKC) activation on Na/K-ATPase activity in MDCK cells, we studied the effect of phorbol myristate acetate (PMA) and two diacylglycerol analogues, oleoylacetylglycerol and dioctanoylglycerol, on the enzyme activity. Na/K-ATPase activity was determined by cytochemistry. PMA induced a time- and dose-dependent inhibition of Na/K-ATPase activity and at 100 ng/ml decreased the enzyme activity by 55% of the initial value. These effects were mimicked by oleoylacetylglycerol and dioctanoylglycerol, and were abolished by two inhibitors of PKC, 1-(5-isoquinolinylsulphonyl)-2-methylpiperazine (H7) and sphingosine. A phorbol ester that does not activate PKC, 4-phorbol 12, 13-didecanoate, did not inhibit Na/ K-ATPase activity. PMA inhibition persisted in the presence of cycloheximide and actinomycin D but not in the presence of amiloride. Dopamine (10 M) inhibition of Na/K-ATPase activity was abolished in a dose-dependent manner by sphingosine. Results suggest that in MDCK cells Na/K-ATPase is an effector protein for PKC and that dopamine inhibition of its activity may be mediated by PKC.     

K+-dependent stability and ion conduction of Shab K+ channels: a comparison with Shaker channels

K⁺ depletion exerts dramatically variable effects on different potassium channels. Here we report that Shab channels are rather stable in the absence of either internal or external K⁺ alone; however, its stability is greater with K⁺ outside the cell. In contrast, with 0 K⁺ (non-added) solutions on both sides of the membrane, the conductance (G_K) is rapidly and irreversibly lost. G_K is lost with the channels closed and regardless of the composition of the 0 K⁺ solutions. In comparison, it is known that the Shaker B G_K collapses only if the channels are gated in 0 K⁺, Na⁺-containing solutions. In order to compare the behavior of Shab to that of Shaker, we show that after extensively gating the channels in 0 K⁺ N-methyl-D-glucamine solutions, most Shaker channels remain stable, and in a conformation where G_K collapses as soon as there is Na⁺ in the solutions. Regarding ion conduction, in contrast to Kv2.1 and Shaker A463C that have a sizable G_Na in 0 K⁺, Shab, which shares a 463-cysteine and an identical signature sequence with these channels, does not appreciably conduct Na⁺, although it presents a significant Cs⁺ conductance. The observations suggest that there are at least two sites where K⁺ binds and thus maintains Shab G_K stable, one internal and the other(s) most likely located outside the selectivity filter.     

Ca2+-activated K+ channels in airway smooth muscle are inhibited by cytoplasmic adenosine triphosphate

Large-conductance Ca²⁺-activated K⁺ channels were studied in membranes of cultured rabbit airway smooth muscle cells, using the patch-clamp technique. In cell-attached recordings, channel openings were rare and occurred only at very positive potentials. Bradykinin (10 M), an agonist which releases Ca²⁺ from the sarcoplasmic reticulum, transiently increased channel activity. The metabolic blocker 2,4-dinitrophenol (20 M), which lowers cellular adenosine triphosphate (ATP) levels, induced a sustained increase of channel activity in cell-attached patches. In excised patches, these channels had a slope conductance of 155 pS at 0 mV, were activated by depolarization and by increasing the Ca²⁺ concentration at the cytoplasmic side above 10^–7 mol/l. ATP, applied to the cytoplasmic side of the patches, dose-dependently decreased the channel's open-state probability. An inhibition constant (K _i) of 0.2 mmol/l was found for the ATP-induced inhibition. ATP reduced the Ca²⁺ sensitivity of the channel, shifting the Ca²⁺ activation curve to the right and additionally reducing its steepness. Our results demonstrate that cytoplasmic ATP inhibits a large-conductance Ca²⁺-activated K⁺ channel in airway smooth muscle. This ATP modulation of Ca²⁺-activated K⁺ channels might serve as an important mechanism linking energy status and the contractile state of the cells.     

Regulation of potassium conductance by prostaglandins in cultured renal epitheloid (Madin-Darby canine kidney) cells

Madin-Darby canine kidney (MDCK) cells form arachidonic acid metabolites following stimulation of several hormones known to modify the ion conductances at the plasma membrane. The present study has been performed to elucidate the influence of arachidonic acid on the electrical properties of subconfluent MDCK cells. As a result, arachidonic acid (1 or 10 mol/l) leads to a transient hyperpolarization of the cell membrane, followed by a transient depolarization and a second, sustained hyperpolarization. The effects are inhibited by cycloxygenase inhibitor indomethacin (1 mol/l). The initial transient hyperpolarization is mimicked by prostaglandin E₂ (PGE₂, 0.1 mol/l), the sustained hyperpolarization by both PGE₂ (0.1 mol/l) and PGF₂ (0.1 mol/l). The transient hyperpolarization is paralleled by an increase of potassium selectivity and a decrease of cell membrane resistance and is thus the result of increased potassium conductance. The transient depolarization is paralleled by an increase of chloride selectivity, reflecting an increase of chloride conductance. The sustained hyperpolarization is paralleled by an increase of cell membrane resistance, and increase of potassium selectivity and a decrease of chloride selectivity, and is thus the result of decreasing chloride conductance. The observations reveal a role of prostaglandins in the regulation of ion conductances in MDCK cells, which could well participate in the transport regulation by hormones.     

Hypertonicity in fused Madin-Darby canine kidney cells: transient rise in NaHCO3 followed by sustained KCl accumulation

We investigated mechanisms of regulatory volume increase in fused Madin-Darby canine kidney (MDCK) cells, a cell line originally derived from renal collecting duct. The intracellular ion concentrations as well as the concentration of the volume marker tetramethylammonium⁺ were measured by means of ion-selective microelectrodes. Application of hypertonic Ringer bicarbonate solution (+150 mmol/l mannitol) resulted in cell shrinkage to 84±2% of the initial cell volume (shrinkage expected for an ideal osmometer = 66%), indicating a significant regulatory volume increase. During the first 90 s of the hypertonic stress, a transient increase in intracellular Na⁺ and HCO ₃ ^– concentrations was observed. It was followed by a sustained increase in intracellular K⁺ and Cl^– concentrations. Ouabain (0.1 mmol/l) as well as amiloride (1 mmol/l) reduced K⁺ accumulation significantly, whereas the H⁺ /K⁺-ATPase inhibitor SCH 28080 had no effect. Hypertonic stress hyperpolarized the cell membrane potential by 19±2 mV, owing to the decrease of the ratio of Cl^– conductance to K⁺ conductance of the cell membrane. We conclude: (a) acute hypertonic stress activates Na⁺/H⁺ exchange in MDCK cells; (b) transient alteration of intracellular Na⁺ and pH stimulates Na⁺/K⁺-ATPase and Cl^–/HCO ₃ ^– exchange, both leading to the sustained intracellular accumulation of KCl; (c) a high intracellular KCl concentration is maintained by the partial reversion of the Cl^–/K⁺ conductance ratio of the plasma membrane.     

Further characterization of volume regulatory decrease in cultured renal epitheloid (MDCK) cells

In Madin Darby canine kidney (MDCK) cells volume regulatory decrease (VRD) is paralleled by a variable, transient hyperpolarization followed by a sustained depolarization of the cell membrane. In the depolarized cells, the cell membrane selectivity is decreased for potassium and increased for chloride. Without knowledge of the cell membrane resistance (R _m), these changes of cell membrane selectivity cannot be translated into conductances, i.e. the observed alterations of ion selectivity could have been due to inhibition of potassium conductance or activation of anion conductance. In the present study R _m has been determined by cellular cable analysis. To this end, three microelectrodes were impaled into three different cells of a cell cluster, current (up to 3 nA) was injected into one cell and the corresponding voltage deflections determined in the other two cells. As a result, exposure of the cells to hypotonic perfusates leads to a marked, sustained reduction of R _m. In the absence of chloride and in the absence of bicarbonate and chloride, the decrease of R _m is only transient. The data indicate that cell swelling leads to a transient increase of potassium conductance followed by a sustained increase of anion conductance. As evident from BCECF fluorescence, exposure of MDCK cells to hypotonic perfusates leads to a significant decrease of intracellular pH, which may in part be due to loss of bicarbonate through the anion conductive pathway.