Inwardly rectifying potassium conductances in AtT-20 clonal pituitary cells

We have detected two inwardly rectifying potassium conductances in AtT-20 clonal corticotrophs, a cell line derived from the mouse pituitary gland. An agonist-independent potassium conductance was activated by voltage steps negative to the reversal potential for potassium (V _K) and was completely blocked by 1 mM barium in the bathing solution. The conductance was transient and inactivated completely with a time constant of about 80 ms. Reducing the external sodium concentration from 140 mM to 14 mM attenuated inactivation. In the presence of 100 nM somatostatin an inwardly rectifying conductance, which reversed at potentials close to V _K, was also elicited. This conductance exhibited a maximal slope conductance that increased with increasing extracellular potassium. Rectification depends on both voltage and extracellular potassium concentration (V _m–V _K). The inward current induced by somatostatin during voltage steps negative to V _K was completely blocked by 1 mM extracellular barium, whereas the outward somatostatin-induced current activated at the holding current, which was about 30 mV positive to V _K, was unaffected by 1 mM extracellular barium. The muscarinic agonist carbachol (10 (M) also induces an inwardly rectifying conductance of similar magnitude to that induced by somatostatin. Since the agonist-independent potassium current exhibits sodium-dependent inactivation, whereas the hormone-induced current does not inactivate, these currents are probably carried by different populations of potassium channels.     

Somatostatin activates an inwardly rectifying K+ channel in neonatal rat atrial cells

Somatostatin, localized throughout the central and peripheral nervous systems has been found in neurons of the vagal inhibitory pathway of the heart and has been shown to have negative inotropic effects in cardiac tissue. Using patch clamp techniques we show that somatostatin activates an inwardly rectifying K⁺ channel in rat atrial cells. Loss of somatostatin-induced K⁺ channel activity in excised inside-out patches is restored by the addition of GTP to the bath. Pertussis toxin pretreatment blocked GTP-dependent somatostatin activation of the inwardly rectifying K+ channel. This K+ channel has a conductance of 34 pS and a mean open time of approximately 1 ms. It is apparently the same K+ channel activated by muscarinic and adenosine receptors in atrial and cardiac pacemaker cells. Thus, atrial cells have at least three receptors which act via pertussis toxin-sensitive G proteins to activate the same class of K+ channels.     

Characterization of whole-cell currents in mucosal and connective tissue rat mast cells using amphotericin-B-perforated patches and temperature control

 Rat mucosal type mast cells are thought to possess only a K⁺-selective inwardly rectifying (IR_K) current in the resting state. We used rat-bone-marrow-derived mast cells (BMMCs) as a model of mucosal mast cells and recorded whole-cell membrane currents from cells perforated with amphotericin B. Under these conditions, both inwardly rectifying (IR) and outwardly rectifying (OR) currents were observed. The reversal potential and conductance of the IR current depended on the extracellular K⁺ concentration, indicating that the channel was K⁺ selective. The OR current was not affected by changes in extracellular K⁺ concentration, but lowering extracellular Cl^–concentration reduced the conductance and shifted the reversal potential in a positive direction. The OR current was not affected by K⁺ channel blockers, but was reversibly blocked by the chloride channel blocker 4,4’-diisothiocyanato-2,2’-stilbenedisulphonate (DIDS), again indicating a Cl^–conductance. The IR_K current was also detected in the majority of cells using the conventional whole-cell recording configuration at room temperature. In contrast, the OR_Cl current was only observed in 7% of recordings made at room temperature with the conventional whole-cell voltage-clamp mode, but was detected in 66% of cells if the bath temperature was increased and the integrity of the cell’s cytoplasm was preserved by using the perforated-patch technique. Under similar conditions, the OR_Cl current was also present in rat peritoneal mast cells, a connective tissue phenotype previously thought to have no whole-cell currents in the resting state. The role of this current and factors affecting its activation are discussed. Received: 10 May 1996 / Received after revision: 4 July 1996 / Accepted: 8 July 1996     

Antisense oligodeoxynucleotides of sulfonylurea receptors inhibit ATP-sensitive K+ channels in cultured neonatal rat ventricular cells

 To identify the functional sulfonylurea receptor (SUR), a subunit of the adenosine 5′-triphosphate (ATP)-sensitive K⁺ (K_ATP) channels, in neonatal rat ventricular cells, such cells in primary culture were treated for 6 days with antisense (AS) oligodeoxynucleotides (ODNs) complementary to the mRNA for SURs. For quantification, single-channel (inside-out patches) and whole-cell currents were measured using the patch-clamp technique. The maximal K_ATP currents (at 0 mV) induced by metabolic inhibition were 48.9±2.8 pA/pF in control (n=48), 34.3±3.5 pA/pF in AS-SUR1 (n=21, P<0.05 vs control), and 23.5±3.4 pA/pF in AS-SUR2 (n=17, P<0.01 vs control). As a control, scramble oligonucleotides had no effect. The fast Na⁺ current and inward-rectifying K⁺ current were not affected by AS-SURs. Treatment with both AS-SUR1 and AS-SUR2 had no additive effects on inhibition of K_ATP currents compared with AS-SUR2 alone. The single-channel conductance, open probability, and kinetics (in ATP-free solution) were not significantly different between control, AS-SUR1, and AS-SUR2. These results suggest that treatment with AS-ODN for SUR1 or SUR2 reduced the number of functional K_ATP channels. Furthermore, in four out of seven control cells tested, outward K⁺ currents were stimulated by diazoxide, which is a potent K⁺ channel-opening drug for the constructed SUR1/Kir6.2 and SUR2B/Kir6.2 channels, but not for the SUR2A/Kir6.2 channel. Therefore, in neonatal rat ventricular cells, both SUR2 and SUR1 subtypes could be integral components of the functional K_ATP channels. The larger population of K_ATP channels may be constructed with SUR2, whereas a smaller population may be constructed with a combination of SUR1 and SUR2. Received: 29 May 1998 / Received after revision: 8 September 1998 / Accepted: 13 October 1998     

Hypertonic cell shrinkage reduces the K+ conductance of rat colonic crypts

 It has previously been shown in studies of a renal epithelial cell line that nonselective cation (NSC) channels are activated by exposure to hypertonic solution. We have also found such channels in excised patches of colonic crypt cells. They require high Ca²⁺ activities on the cytosolic side and a low ATP concentration for their activation and have not been recorded from cell-attached patches of colonic crypts. We examine here whether this type of channel is activated by hypertonic cell shrinkage. Bath osmolality was increased by addition of 25, 50 or 100 mmol/l mannitol. Cell-attached and whole-cell patch recordings were obtained from rat base and mid-crypt cells. In whole-cell recordings we found that addition of 50 or 100 mmol/l mannitol depolarized these cells significantly from –78±2.0 to –66±3.8 mV (n=22) and from –78±1.3 to –56±2.6 mV (n=61), respectively, and reduced the whole-cell conductance from 20±8.0 to 14±6.6 nS (n=7) and from 20±3.0 to 9.8±1.6 nS (n=19), respectively. In cell-attached patches K⁺ channels with a single-channel conductance of ≈16 pS were found in most recordings. The activity of these channels (N×P _o, N=number, P _o=open channel probability) was reduced from 2.08±0.37 to 0.98±0.23 (n=15) by the addition of 50 mmol/l mannitol and from 1.75±0.26 to 0.77±0.20 (n=12) by 100 mmol/l mannitol. No NSC channel activity was apparent in any of these recordings. Previously we have shown that the 16-pS K⁺ channel is controlled by cytosolic Ca²⁺ ([Ca²⁺]_i). Therefore we measured [Ca²⁺]_i by the fura-2 method and found that hypertonic solution reduced [Ca²⁺]_i significantly (n=16). These data indicate that exposure of rat colonic crypts to hypertonic solutions does not activate NSC channels; [Ca²⁺]_i falls in hypertonic solution leading to a reduction in the value of K⁺ channel N×Po, a reduced whole-cell conductance and depolarization of mid-crypt cells. These processes probably assist volume regulation inasmuch as they reduce KCl losses from the cell. Received: 21 July 1997 / Received after revision: 24 November 1997 / Accepted: 15 December 1997     

HL-1 cells express an inwardly rectifying K+ current activated via muscarinic receptors comparable to that in mouse atrial myocytes

An inwardly rectifying K⁺ current is present in atrial cardiac myocytes that is activated by acetylcholine (I_KACh). Physiologically, activation of the current in the SA node is important in slowing the heart rate with increased parasympathetic tone. It is a paradigm for the direct regulation of signaling effectors by the Gβγ G-protein subunit. Many questions have been addressed in heterologous expression systems with less focus on the behaviour in native myocytes partly because of the technical difficulties in undertaking comparable studies in native cells. In this study, we characterise a potassium current in the atrial-derived cell line HL-1. Using an electrophysiological approach, we compare the characteristics of the potassium current with those in native atrial cells and in a HEK cell line expressing the cloned Kir3.1/3.4 channel. The potassium current recorded in HL-1 is inwardly rectifying and activated by the muscarinic agonist carbachol. Carbachol-activated currents were inhibited by pertussis toxin and tertiapin-Q. The basal current was time-dependently increased when GTP was substituted in the patch-clamp pipette by the non-hydrolysable analogue GTPγS. We compared the kinetics of current modulation in HL-1 with those of freshly isolated atrial mouse cardiomyocytes. The current activation and deactivation kinetics in HL-1 cells are comparable to those measured in atrial cardiomyocytes. Using immunofluorescence, we found GIRK4 at the membrane in HL-1 cells. Real-time RT-PCR confirms the presence of mRNA for the main G-protein subunits, as well as for M2 muscarinic and A1 adenosine receptors. The data suggest HL-1 cells are a good model to study IKAch.     

Phasic and tonic attenuation of EPSPs by inward rectifier K+ channels in rat hippocampal pyramidal cells

We made whole-cell recordings from CA1 pyramidal cells of hippocampal slices in combination with brief dendritic glutamate pulses to study the role of constitutive inwardly rectifying K⁺ channels (IRK, Kir2.0) and G-protein-activated inwardly rectifying K⁺ channels (GIRK, Kir3.0) in the processing of excitatory inputs. Phasic activation of GIRK channels by baclofen (20 μ m ) produced a reversible reduction of glutamate-evoked postsynaptic potentials (GPSPs), our equivalent of EPSPs, by about one-third. Conversely, tertiapin (30 n m ), a selective inhibitor of GIRK channels, and Ba²⁺ (200 μ m ), a non-selective blocker of inwardly rectifying K⁺ channels, enhanced GPSPs and, in voltage-clamp experiments, reduced the underlying K⁺ conductances, indicating a functionally significant background GIRK conductance, in addition to constitutive IRK channel activity. When examined after suppression of endogenous adenosinergic inhibition, using either adenosine deaminase or the selective A1 receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine, tertiapin failed to influence either the GPSPs or the inwardly rectifying K⁺ conductance. Voltage-clamp recordings from acutely isolated CA1 pyramidal cells not exposed to ambient adenosine exhibited no response to tertiapin, whereas Ba²⁺ was still capable of reducing hyperpolarizing inward rectification. Our data indicate that in hippocampal pyramidal cells, two components of the inwardly rectifying K⁺ conductance can be identified, which together exert a tonic modulation of excitatory synaptic input: one arises from constitutive putative IRK channels, the other is mediated by the background activity of GIRK channels that results from the tonic activation of A1 receptors by ambient adenosine.     

Changes of membrane currents in cardiac cells induced by long whole-cell recordings and tolbutamide

Single isolated myocytes were obtained from the ventricles of adult guinea pig hearts. The whole-cell recording configuration of the patch-clamp technique was used to measure membrane currents. A decrease (run-down) of the Ca²⁺ inward current and an increase of a time-independent K⁺ outward current were observed during long lasting (1–3 h) recordings. The time at which the outward current developed depended on the intracellular ATP concentration in the pipette, suggesting that this current is identical to the ATP-dependent K⁺ current described by Noma and Shibasaki (1985). However, the maximum outward current reached in the experiments was independent of the ATP concentration indicating a limited diffusion of ATP in the cell interior. In single-channel experiments on isolated patches of cell membrane and in whole-cell recordings the ATP-dependent K⁺ current could be blocked by the hypoglycaemic sulphonylurea tolbutamide. The IC₅₀ of 0.38 mM was about 50 times higher than that reported for pancreatic -cells (Trube et al. 1986). The Ca²⁺ inward current and the inwardly retifying K⁺ current were not affected by tolbutamide (3 mM).     

ATP reduces voltage-activated K+ current in cultured rat hippocampal neurons

Effects of extracellular ATP were investigated in cultured rat hippocampal neurons using whole-cell voltage-clamp techniques. When a depolarizing step to +10 mV was applied from a holding potential of -60 mV, an outward K⁺ current was activated. ATP (3 to 300 μM) reduced the K⁺ current. Among adenosine derivatives, ADP (100 μM) slightly inhibited the K⁺ current, and AMP or adenosine (100 μM) was ineffective. UTP was as potent as ATP and α,β-methylene ATP was less effective than ATP. The inhibition by ATP of the K⁺ current was abolished by inclusion of 2 mM GDPβS in the intracellular solution. The results indicate that ATP inhibits K⁺ channels in rat hippocampal neurons through UTP-responsive P₂-purinoceptors coupled with GTP-binding proteins.     

Regulation of ionic currents by protein kinase A and intracellular calcium in outer hair cells isolated from the guinea-pig cochlea

 Two prominent potassium currents, termed I _K and I _K,n, and a cation current are found in outer hair cells (OHCs) of the guinea-pig cochlea. We report here whole-cell recordings which indicate that the currents are regulated by intracellular factors. 8-bromo-cAMP (500 μM), a membrane-permeable cAMP analogue, activated potassium currents in OHCs in both apical and basal turns of the cochlea. In OHCs from the cochlear apex, the drug effect was largest at potentials positive to –40 mV, indicating I _K as the target. In short cells from the cochlear base, both I _K and I _K,n were affected. The effects of 8-bromo-cAMP could be blocked by the presence of 1 μM H-89 (a protein kinase A inhibitor) in the patch pipette solution. Extracellular application of 10 nM okadaic acid, a protein phosphatase inhibitor, also activated both potassium currents. Currents were also modulated by intracellular calcium. I _K was activated in long cells by photorelease of calcium from the caged compound nitr5. Cation current activation required calcium release by photolysis of DM-nitrophen, a compound releasing more calcium. The results show that OHC potassium channels are regulated by background phosphorylation through protein kinase A and dephosphorylation by protein phosphatase. Cellular calcium also activates I _K and the cation channel, but with different sensitivities. Received: 1 September 1998 / Received after revision: 21 October 1998 / Accepted: 22 October 1998     

m4 muscarinic receptor subtype activates an inwardly rectifying potassium conductance in AtT20 cells.

The modulation of an inwardly rectifying potassium conductance by muscarinic receptor stimulation was studied in the AtT-20 pituitary cell line, using the whole-cell patch-clamp technique. Only m4 mRNA was detected in these cells, thus, it is assumed that the actions of muscarinic receptor stimulation are mediated by the m4 receptor. AtT-20 cells express a slowly activating inwardly rectifying potassium conductance. Application of acetylcholine (ACh), resulted in an atropine sensitive, reversible increase in inwardly rectifying current. The ACh-induced current differed from the current recorded in control, in that it was fast activating, while the control current was slowly activating. Inclusion of GTP gamma S in the patch pipette activated an inward current with characteristics similar to the ACh-induced current, and the ACh-induced current response could be inhibited by pre-incubation with pertussis toxin (PTX). It is concluded that the m4 muscarinic receptor is coupled to an inwardly rectifying potassium conductance via a PTX sensitive G-protein.     

The GPR55 agonist lysophosphatidylinositol directly activates intermediate-conductance Ca<Superscript>2+</Superscript>-activated K<Superscript>+</Superscript> channels

Lysophosphatidylinositol (LPI) was recently shown to act both as an extracellular mediator binding to G protein-coupled receptor 55 (GPR55) and as an intracellular messenger directly affecting a number of ion channels including large-conductance Ca²⁺ and voltage-gated potassium (BK_Ca) channels. Here, we explored the effect of LPI on intermediate-conductance Ca²⁺-activated K⁺ (IK_Ca) channels using excised inside-out patches from endothelial cells. The functional expression of IK_Ca was confirmed by the charybdotoxin- and TRAM-34-sensitive hyperpolarization to histamine and ATP. Moreover, the presence of single IK_Ca channels with a slope conductance of 39 pS in symmetric K⁺ gradient was directly confirmed in inside-out patches. When cytosolically applied in the range of concentrations of 0.3–10 μM, which are well below the herein determined critical micelle concentration of approximately 30 μM, LPI potentiated the IK_Ca single-channel activity in a concentration-dependent manner, while single-channel current amplitude was not affected. In the whole-cell configuration, LPI in the pipette was found to facilitate membrane hyperpolarization in response to low (0.5 μM) histamine concentrations in a TRAM-34-sensitive manner. These results demonstrate a so far not-described receptor-independent effect of LPI on the IK_Ca single-channel activity of endothelial cells, thus, highlighting LPI as a potent intracellular messenger capable of modulating electrical responses in the vasculature.     

Acetylcholine-mediated K+ channel activity in guinea-pig atrial cells is supported by nucleoside diphosphate kinase

We studied the role of nucleoside diphosphate kinase (NDPK) in acetylcholine-mediated muscarinic K⁺ channel activation in inside-out patches of guinea-pig atrial cells. NDPK-catalysed activation of the muscarinic K⁺ channels by adenosine triphosphate-Mg²⁺ (ATP-Mg²⁺) is not prevented by occupation of the muscarinic receptor [by acetylcholine (ACh) or atropine], nor by uncoupling of the receptor from the G protein by pertussis-toxin-catalysed adenosine diphosphate (ADP)-ribosylation of G_K. In the presence of ACh, addition of 0.1 mM guanosine triphosphate (GTP) after activation of the channels by 4 mM ATP alone resulted in a moderate increase of channel activity (in contrast to block in the absence of ACh): NDPK-mediated direct transphosphorylation is uncoupled by the G nucleotide but agonist-induced guanosine diphosphate (GDP)-to-GTP exchange takes over activation of the channels. Moreover, ACh-dependent channel stimulation was possible in inside-out patches while ATP and GDP were present in the bathing solution (in contrast to the complete absence of channel activation in the absence of ACh). This indicates that NDPK synthesises sufficient GTP to support channel activation by exchange. Hence, it is postulated that the main functional role of NDPK under physiological conditions is to provide a local supply of GTP (using GDP and ATP) in the immediate vicinity of the G protein, thereby maintaining a high local GTP/GDP ratio and ensuring adequate receptor-mediated regulation of muscarinic K⁺ channel activity.     

Galanin activates an inwardly rectifying potassium conductance in mudpuppy atrial myocytes

Galanin- and bethanechol-activated K⁺ currents have been studied in mudpuppy atrial myocytes. The galanin and bethanechol K⁺ currents were time-dependent and inwardly rectifying. In GTPS, the galanin and bethanechol currents were reduced progressively as G-protein gated K⁺ channels became activated. GDPS inhibited agonist-induced outward currents. We conclude that galanin and bethanechol activate the same or a very similar inwardly rectifying K⁺ conductance and that activation of a G protein is required.     

I K.ACh activation by arachidonic acid occurs via a G-protein-independent pathway mediated by the GIRK1 subunit

The molecular target of arachidonic-acid-derived metabolites, serving as second messengers that activate atrial acetylcholine-activated potassium current (IK.ACh) in addition to G-protein beta/gamma subunits (Gbeta/gamma), is unknown. Co-expression of two isoforms of G-protein-activated, inwardly rectifying potassium channels (GIRKs) in oocytes of Xenopus laevis revealed that these heterologous co-expressed GIRKs, which are responsible for the formation of IK.ACh in the atrium, are activated by arachidonic acid metabolites, like their counterparts in atrial cells. The expression of homooligomeric GIRK1(F137S) and GIRK4wt channels revealed that this activatory mechanism is specific to the GIRKI subunit. Sequestrating available Gbeta/gamma by overexpression of C-betaARK (a Gbeta/gamma binding protein) failed to abolish the activation of GIRK currents by arachidonic acid. From our experiments we conclude that the GIRKI subunit itself is the molecular target for regulation of GIRK channels by arachidonic acid metabolites.     

KATP channel current increases in postinfarction remodeled cardiomyocytes

Adenosintriphosphate-sensitive potassium channels (K_ATP channels) are an important linkage between the metabolic state of a cell and electrophysiological membrane properties. In this study, K_ATP channels were studied in myocytes of normal and remodeled myocardium of the rat. Myocardial infarction was induced by ligature of the left anterior descending artery. Remodeled myocytes were obtained from the hypertrophied posterior left ventricular wall and interventricular septum 3 months after infarction. The current through K_ATP channels was measured in whole-cell and inside-out patches by using the patch-clamp technique. After myocardial infarction, the heart weight/body weight ratio was doubled and the myocytes were hypertrophied yielding a cell capacitance of 266±16 pF compared to 122±12 pF in control cells. The amount of Kir6.2 protein was indistinguishable in corresponding regions of control and remodeled hearts. The ATP sensitivity of K_ATP channels in remodeled cells was significantly lower than in control cells (half maximum block at 115 μmol/l ATP in remodeled and at 71 μmol/l ATP in control cells). The maximum I _KATP density induced by metabolic inhibition was higher in small remodeled (176±15 pA/pF) than in control cells (127±11 pA/pF), but was unchanged in large remodeled cells. Both, the higher I _KATP density and the lower sensitivity of the K_ATP channels to ATP suggest that remodeled cardiomyocytes develop an improved tolerance to ischemia by stabilizing the resting potential and decreasing excitability.     

Single-channel properties and regulation of pinacidil/glibenclamide-sensitive K+ channels in follicular cells from Xenopus oocyte

Follicular oocytes from Xenopus laevis contain K⁺ channels that are activated by members of the recently recognized class of vasorelaxants that includes the pinacidil derivative P1060. These channels are blocked by antidiabetic sulphonylureas such as glibenclamide. Opening of the glibenclamide-sensitive K⁺ channels with P1060 promotes follicular oocyte maturation. Whole-cell and single-channel patch-clamp configurations were used to monitor K⁺ channel activity in isolated follicular cells. In the presence of micromolar concentrations of intracellular Mg²⁺ ATP, P1060 activated a whole-cell K⁺ current that was blocked by glibenclamide. The P1060 response was depressed by millimolar concentrations of intracellular ATP and ATP[S]. Single-channel recordings identified two different types of K⁺ channel. These channels differed in their unitary conductances (19 pS and 150 pS), in their sensitivities to internal Ca²⁺, to charybdotoxin and to pinacidil and glibenclamide. Only the Ca²⁺-independent K⁺ channel (19 pS) was activated by the pinacidil derivative and blocked by glibenclamide. Opening of the 19-pS glibenclamide-sensitive K⁺ channel by P1060 critically required the presence of a low concentration of Mg²⁺ATP in the intracellular medium. The 19-pS K⁺ channel was opened by increasing intracellular cAMP. Similar effects were obtained by intracellular application of the catalytic subunit of protein kinase A in the presence of micromolar concentrations of Mg²⁺ATP. Both acetylcholine and the phorbol ester phorbol 12-myristate 13-acetate blocked the 19-pS K⁺ channel after it was activated by P1060.This work was supported by the Centre National de la Recherche Scientifique (CNRS) and the Fondation pour la Recherche Médicale (FRM)     

Demonstration of an inwardly rectifying K+ current component modulated by thyrotropin-releasing hormone and caffeine in GH3 rat anterior pituitary cells

 Reduction of an inwardly rectifying K⁺ current by thyrotropin-releasing hormone (TRH) and caffeine has been considered to be an important determinant of electrical activity increases in GH₃ rat anterior pituitary cells. However, the existence of an inwardly rectifying K⁺ current component was recently regarded as a misidentification of an M-like outward current, proposed to be the TRH target in pituitary cells, including GH₃ cells. In this report, an inwardly rectifying component of K⁺ current is indeed demonstrated in perforated-patch voltage-clamped GH₃ cells. The degree of rectification varied from cell to cell, but both TRH and caffeine specifically blocked a fraction of current with strong rectification in the hyperpolarizing direction. Use of ramp pulses to continuously modify the membrane potential demonstrated a prominent blockade even in cells with no current reduction at voltages at which M-currents are active. Depolarization steps to positive voltages at the maximum of the inward current induced a caffeine-sensitive instantaneous outward current followed by a single exponential decay. The magnitude of this current was modified in a biphasic way according to the duration of the previous hyperpolarization step. The kinetic characteristics of the current are compatible with the possibility that removal from inactivation of a fast-inactivating delayed rectifier causes the hyperpolarization-induced current. Furthermore, the inwardly rectifying current was blocked by astemizole, a potent and selective inhibitor of human ether-á-go-go -related gene (HERG) K⁺ channels. Along with other pharmacological and kinetic evidence, this indicates that the secretagogue-regulated current is probably mediated by a HERG-like K⁺ channel. Addition of astemizole to current-clamped cells induced clear increases in the frequency of action potential production. Thus, an inwardly-rectifying K⁺ current and not an M-like outward current seems to be involved in TRH and caffeine modulation of electrical activity in GH₃ cells. Received: 15 May 1997 / Received after revision and accepted: 24 July 1997     

Adenosine triphosphate-dependent K currents activated by metabolic inhibition in rat ventricular myocytes differ from those elicited by the channel opener rilmakalim

Adenosine triphosphate (ATP) dependent potassium channels (K_ATP channels) in heart ventricular muscle cells can be activated by depletion of intracellular ATP stores as well as by channel openers. In the present study we examined whether properties of K_ATP channels are dependent on the mode of activation. Whole-cell and single-channel currents were investigated by use of the patch-clamp technique in isolated ventricular rat myocytes. The channel opener rilmakalim dose dependency activated whole-cell currents [concentration for half-maximal activation (EC₅₀) = 1.1 M, Hill coefficient = 3.1, saturation concentration 10 M]. Metabolic inhibition with 2-deoxy-d-glucose (10 mmol/l) also activated K_ATP currents after a time lag of several minutes. These currents were about two-fold higher than the rilmakalim-activated currents (rilmakalim-activated current 3.9 ±0.2nA, 2-deoxy-d-glucose-activated current 8.1±0.9 nA; both recorded at 0 mV clamp potential). While the rilmakalim-activated current could be blocked completely and with high affinity by the sulphonylurea glibenclamide [concentration for half-maximal inhibition (IC₅₀) = 8 nM, Hill coefficient = 0.7] the 2-deoxy-d-glucose-activated current could only be blocked partially (by maximally 46%) and higher glibenclamide concentrations were needed (IC₅₀ = 480 nM, Hill coefficient = 0.8). The partial loss of blocking efficiency after metabolic inhibition was not restricted to glibenclamide but was also observed with the sulfonylureas glimepiride and HB 985, as well as with the non-sulfonylureas HOE 511 and 5-hydroxydecanoate. Single-channel studies were in accordance with these whole-cell experiments. Both rilmakalim and metabolic inhibition with the uncoupler carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) activated single channels in the attached mode, where the number of current levels was significantly higher in the case of FCCP. Rilmakalim-activated channels were completely blocked by 10 M glibenclamide, whereas several single-channel levels appeared in the presence of 100 M glibenclamide after metabolic inhibition. In conclusion, after metabolic inhibition the amplitude of the activated K_ATP current is about twice as high as under saturating concentrations of the opener rilmakalim. Moreover, channels activated by metabolic inhibition lost part of their sensitivity to known channel blockers.