Alternative Shaker transcripts express either rapidly inactivating or noninactivating K+ channels.

Two members of the Shaker K+ channel family designated ShA2 and ShD2 were characterized in the Xenopus oocyte expression system. The predicted amino acid sequences of ShA2 and ShD2 differ only in the amino terminus, which is located intracellularly according to the present topological model of K+ channels. The differing amino termini have profound effects on the electrophysiological and pharmacological properties of the K+ channel. Most markedly, the nature of the amino terminus determines whether the K+ channel mediates rapidly inactivating or noninactivating K+ currents. It also affects the 4-aminopyridine, tetraethylammonium, and charybdotoxin sensitivities of the K+ channels. These results suggest that the amino terminus of Shaker proteins affects K+ channel structures on both sides of the membrane.     

Ethanol selectively blocks a noninactivating K+ current expressed in Xenopus oocytes.

There is presently a debate regarding the relative merits of lipid-based and protein-based theories of anesthesia and the action of ethanol in the central nervous system. Voltage-sensitive K+ channels play a key role as regulators of neuronal electrical activity and are potential targets of ethanol and other anesthetic agents. We investigated the action of low concentrations of ethanol on four structurally homologous cloned K+ channels expressed in Xenopus oocytes. We report that only the Drosophila Shaw2 channel, which does not inactivate upon prolonged depolarization, is rapidly and reversibly blocked by ethanol in a concentration-dependent manner (17-170 mM). The concentration dependence of the blockade can be explained by assuming a bimolecular interaction between ethanol and the channel. We also found that Shaw2 K+ channels were selectively blocked by halothane (1 mM). Our results support the "protein hypothesis" of ethanol and anesthetic action. These findings open ways to elucidate directly the molecular mechanism of interaction between general anesthetics and a voltage-sensitive K+ channel.     

Hormone-regulated K+ channels in follicle-enclosed oocytes are activated by vasorelaxing K+ channel openers and blocked by antidiabetic sulfonylureas.

Follicular oocytes from Xenopus laevis contain K+ channels activated by members of the recently recognized class of vasorelaxants that include cromakalim and pinacidil and blocked by antidiabetic sulfonylureas, such as glibenclamide. These channels are situated on the adherent follicular cells and are not present in denuded oocytes. Cromakalim-activated K+ channels are also activated by increases in intracellular cAMP, and cAMP-activated K+ channels are blocked by glibenclamide. Although cromakalim and cAMP effects are synergistic, cromakalim activation of K+ channels is drastically reduced or abolished by treatments that stimulate protein kinase C (e.g., muscarinic effectors, phorbol esters). Gonadotropins, known to play an essential role in ovarian physiology, also activate cromakalim and sulfonylurea-sensitive K+ channels. Follicular oocytes constitute an excellent system for studying regulation of cromakalim-sensitive K+ channels that are important in relation to a variety of disease processes, such as cardiovascular dysfunction and asthma, as well as brain function.     

Phencyclidine in nanomolar concentrations binds to synaptosomes and blocks certain potassium channels.

Phencyclidine [1-(phenylcyclohexyl)piperidine; PCP], in low dose (approximately equal to 0.1-0.2 mg/kg of body weight), induces a schizophrenia-like behavioral syndrome in man; this effect has been attributed to block of neuronal K channels. We used a K-stimulated 86Rb efflux assay to demonstrate that low concentrations of PCP (10-50 nM) block a class of depolarization-activated K channels in rat brain synaptosomes--pinched-off presynaptic nerve terminals. The dose-response curve is biphasic, and much higher PCP concentrations (greater than 10 microM) are required to block the remainder of the K-stimulated 86Rb efflux. The [3H]PCP binding curve for synaptosomes is also biphasic: PCP binds to some components with high affinity (Kd approximately equal to 6.0 X 10(-8) M), and to other components with much lower affinity (Kd approximately equal to 1.15 X 10(4) M). PCP can be photoactivated with UV light to form covalent bonds: after UV irradiation, previously-bound [3H]PCP is no longer displaceable by a large excess of unlabeled PCP. Preliminary data from NaDodSO4/polyacrylamide gel electrophoresis studies after covalent binding of [3H]PCP to synaptosomes, suggest that the high-affinity binding site may be on a large protein (Mr approximately equal to 220,000). We conclude that the high-affinity PCP binding protein is associated with the K channels that are blocked by nanomolar concentrations of PCP. Block of these channels could, by prolonging action-potential duration in presynaptic nerve terminals, enhance calcium entry and neurotransmitter release, thereby altering transmission at central synapses involved in behavioral expression.     

Endothelin blocks ATP-sensitive K+ channels and depolarizes smooth muscle cells of porcine coronary artery.

ATP-sensitive K+ channels with a conductance of 30 pS in smooth muscle cells of porcine coronary artery were found to be highly active in the intact cell-attached patch configuration when the pipette contained a physiological concentration of Ca2+ (greater than 10(-4) M). In the inside-out configuration, these channels were activated by extracellular Ca2+ and blocked by cytosolic ATP and glibenclamide. Endothelin applied to the pipette specifically blocked these channels in a concentration-dependent manner in the cell-attached configuration (half-maximal inhibition, 1.3 x 10(-9) M). A K+ channel opener, nicorandil, activated these channels even in the presence of 10(-8) M endothelin. In the whole-cell current-clamp method, the cell membrane was depolarized by endothelin and then repolarized by nicorandil. The membrane depolarization is closely related to contraction of smooth muscle cells. These results suggest that the ATP-sensitive K+ channels are important in controlling the vascular tone of the coronary artery and that endothelin can increase vascular tone by blocking these channels.     

Primary structure of a beta subunit of alpha-dendrotoxin-sensitive K+ channels from bovine brain.

Voltage-dependent cation channels are largeheterooligomeric proteins. Heterologous expression of cDNAs encoding the alphasubunits alone of K+, Na+, or Ca2+ channels produces functional multimericproteins; however, coexpression of those for the latter two with their auxiliaryproteins causes dramatic changes in the resultant membrane currents.Fast-activating, voltage-sensitive K+ channels from brain contain four alpha andbeta subunits, tightly associated in a 400-kDa complex; although moleculardetails of the alpha-subunit proteins have been determined, little is knownabout the beta-subunit constituent. Proteolytic fragments of a beta subunit frombovine alpha-dendrotoxin-sensitive neuronal K+ channels yielded nine differentsequences. In the polymerase chain reaction, primers corresponding to two ofthese peptides amplified a 329-base-pair fragment in a lambda gt10 cDNA libraryfrom bovine brain; a full-length clone subsequently isolated encodes a proteinof 367 amino acids (M(r) approximately 40,983). It shows no significant homologywith any known protein. Unlike the channels' alpha subunits, the hydropathyprofile of this sequence failed to reveal transmembrane domains. Severalconsensus phosphorylation motifs are apparent and, accordingly, the beta subunitcould be phosphorylated in the intact K+ channels. These results, including theabsence of a leader sequence and N-glycosylation, are consistent with the betasubunit being firmly associated on the inside of the membrane with alphasubunits, as speculated in a simplified model of these authentic K+ channels.Importantly, this first primary structure of a K(+)-channel beta subunitindicates that none of the cloned auxiliary proteins of voltage-dependent cationchannels, unlike their alpha subunits, belong to a super-family ofgenes.     

K+ channel openers activate brain sulfonylurea-sensitive K+ channels and block neurosecretion.

Vascular K+ channel openers such as cromakalim, nicorandil, and pinacidil potently stimulate 86Rb+ efflux from slices of substantia nigra. This 86Rb+ efflux is blocked by antidiabetic sulfonylureas, which are known to be potent and specific blockers of ATP-regulated K+ channels in pancreatic beta cells, cardiac cells, and smooth muscle cells. K0.5, the half-maximal effect of the enantiomer (-)-cromakalim, is as low as 10 nM, whereas K0.5 for nicorandil is 100 nM. These two compounds appear to have a much higher affinity for nerve cells than for smooth muscle cells. Openers of sulfonylurea-sensitive K+ channels lead to inhibition of gamma-aminobutyric acid release. There is an excellent relationship between potency to activate 86Rb+ efflux and potency to inhibit neurotransmitter release.     

ATP-sensitive K+ channels that are blocked by hypoglycemia-inducing sulfonylureas in insulin-secreting cells are activated by galanin, a hyperglycemia-inducing hormone.

The action of the hyperglycemia-inducing hormone galanin, a 29-amino acid peptide named from its N-terminal glycine and C-terminal amidated alanine, was studied in rat insulinoma (RINm5F) cells using electrophysiological and 86Rb+ flux techniques. Galanin hyperpolarizes and reduces spontaneous electrical activity by activating a population of ATP-sensitive K+ channels with a single-channel conductance of 30 pS (at -60 mV). Galanin-induced hyperpolarization and reduction of spike activity are reversed by the hypoglycemia-inducing sulfonylurea glibenclamide. Glibenclamide blocks the galanin-activated ATP-sensitive K+ channel. 86Rb+ efflux from insulinoma cells is stimulated by galanin in a dose-dependent manner. The half-maximum value of activation is found at 1.6 nM. Galanin-induced 86Rb+ efflux is abolished by glibenclamide. The half-maximum value of inhibition is found at 0.3 nM, which is close to the half-maximum value of inhibition of the ATP-dependent K+ channel reported earlier. 86Rb+ efflux studies confirm the electrophysiological demonstration that galanin activates an ATP-dependent K+ channel.     

Inward-rectifying K+ channels in guard cells provide a mechanism for low-affinity K+ uptake.

The molecular mechanisms by which higher plant cells take up K+ across the plasma membrane (plasmalemma) remain unknown. Physiological transport studies in a large number of higher plant cell types, including guard cells, have suggested that at least two distinct types of K(+)-uptake mechanisms exist, permitting low-affinity and high-affinity K+ accumulation, respectively. Recent patch clamp studies have revealed the presence of inward-conducting (inward-rectifying) K+ channels in the plasma membrane of higher plant cells. Research on guard cells has suggested that these K+ channels provide a major pathway for proton pump-driven K+ uptake during stomatal opening. In the present study the contribution of inward-rectifying K+ channels to higher plant cell K+ uptake was investigated by examining kinetic properties of guard cell K+ channels in Vicia faba in response to changes in the extracellular K+ concentration. Increasing the extracellular K+ concentration in the range from 0.3 mM to 11.25 mM led to enhancement of inward K+ currents and changes in current-voltage characteristics of K+ channels. The increase in K+ conductance as a function of the extracellular K+ concentration revealed a K(+)-equilibrium dissociation constant (Km) of approximately 3.5 mM, which suggests that inward-rectifying K+ channels can function as a molecular mechanism for low-affinity K+ uptake. Lowering the extracellular K+ concentration in the range from 11 mM to 1 mM induced negative shifts in the activation potential of K+ channels, such that these channels function as a K+ sensor, permitting only K+ uptake. At low extracellular K+ concentrations of 0.3 mM K+, inward-rectifying K+ channels induce hyperpolarization. Results from the present study suggest that inward-rectifying K+ channels constitute an essential molecular mechanism for plant nutrition and growth control by providing a K(+)-sensing and voltage-dependent pathway for low-affinity K+ uptake into higher plant cells and additionally by contributing to plasma membrane potential regulation.     

Ca2+ influx through stretch-activated cation channels activates maxi K+ channels in porcine endocardial endothelium.

The endocardial endothelium is an important modulator of myocardial function. The present study demonstrates the existence of a stretch-activated Ca(2+)-permeable cation channel and of a Ca(2+)-activated K+ channel in the endocardial endothelium of the porcine right atrium. The stretch-activated channel is permeable for K+, Na+, Ca2+, and Ba2+, with mean conductances of approximately 32 pS for the monovalent cations and approximately 13 pS for divalent cations. The Ca(2+)-activated K+ channel has a mean conductance of 192 pS in symmetrical KCl. solution. Channel activity is strongly dependent on membrane potential and the cytosolic Ca2+ concentration. Half-maximal activation occurs at a cytosolic Ca2+ concentration of approximately 5 microM. The influx of Ca2+ through the stretch-activated channel is sufficient to activate the Ca(2+)-activated K+ channel in cell-attached patches. Upon activation of the stretch-activated channel, the cytosolic Ca2+ concentration increases, at least locally, to values of approximately 0.5 microM, as deduced from the open probability of the Ca(2+)-dependent K+ channel that was activated simultaneously. The stretch-activated channels are capable of inducing an intracellular Ca2+ signal and may have a role as mechanosensors in the atrial endothelium, possibly activated by atrial overload.     

Sarcolemmal versus mitochondrial ATP-sensitive K+ channels and myocardial preconditioning.

Ischemic preconditioning (IPC) is a phenomenon in which single or multiple brief periods of ischemia have been shown to protect the heart against a more prolonged ischemic insult, the result of which is a marked reduction in myocardial infarct size, severity of stunning, or incidence of cardiac arrhythmias. Although a number of substances and signaling pathways have been proposed to be involved in mediating the cardioprotective effect of IPC, the overwhelming majority of evidence suggests that the ATP-sensitive potassium channel (KATP channel) is an important component of this phenomenon and may serve as the end effector in this process. Initially, it was hypothesized that the surface or sarcolemmal KATP (sarc KATP) channel mediated protection observed after IPC; however, subsequent evidence suggested that the recently identified mitochondrial KATP channel (mito KATP) may be the potassium channel mediating IPC-induced cardioprotection. In this review, evidence will be presented supporting a role for either the sarc KATP or the mito KATP in IPC and potential mechanisms by which opening these channels may produce cardioprotection; additionally, we will address important questions that still need to be investigated to define the role of the sarc or mito KATP channel, or both, in cardiac pathophysiology.     

Local Ca2+ entry through L-type Ca2+ channels activates Ca2+-dependent K+ channels in rabbit coronary myocytes.

Large-conductance Ca2+-dependent K+ channels (KCa), which are abundant on the sarcolemma of vascular myocytes, provide negative feedback via membrane hyperpolarization that limits Ca2+ entry through L-type Ca2+ channels (ICaL). We hypothesize that local accumulation of subsarcolemmal Ca2+ during ICaL openings amplifies this feedback. Our goal was to demonstrate that Ca2+ entry through voltage-gated ICaL channels can stimulate adjacent KCa channels by a localized interaction in enzymatically isolated rabbit coronary arterial myocytes voltage clamped in whole-cell or in cell-attached patch clamp mode. During slow-voltage-ramp protocols, we identified an outward KCa current that is activated by a subsarcolemmal Ca2+ pool dissociated from bulk cytosolic Ca2+ pool (measured with indo 1) and is dependent on L-type Ca2+ channel activity. Transient activation of unitary KCa channels in cell-attached patches could be detected during long step depolarizations to +40 mV (holding potential, -40 mV; 219 pS in near-symmetrical K+). This local interaction between the channels required the presence of Ca2+ in the pipette solution, was enhanced by the ICaL agonist Bay K 8644, and persisted after impairment of the sarcoplasmic reticulum by incubation with 10 micromol/L ryanodine and 30 micromol/L cyclopiazonic acid for at least 60 minutes. Furthermore, we provide the first direct evidence of simultaneous openings of single KCa (67 pS) and ICaL (3.9 pS) channels in near-physiological conditions, near resting membrane potential. Our data imply a novel sensitive mechanism for regulating resting membrane potential and tone in vascular smooth muscle.     

Tetraethylammonium blockade distinguishes two inactivation mechanisms in voltage-activated K+ channels.

Voltage-activated K+ channels are a family of closely related membrane proteins that differ in their gating behavior, conductance, and pharmacology. A prominent and physiologically important difference among K+ channels is their rate of inactivation. Inactivation rates range from milliseconds to seconds, and K+ channels with different inactivation properties have very different effects on signal integration and repetitive firing properties of neurons. The cloned Shaker B (H4) potassium channel is an example of a K+ channel that inactivates in a few milliseconds. Recent experiments have shown that removal of an N-terminal region of the Shaker protein by site-directed deletion practically abolishes this fast inactivation, but the modified channel does still inactivate during a prolonged depolarization lasting many seconds. Here we report that this remnant inactivation must occur by a distinct mechanism from the rapid inactivation of the wild-type Shaker channel. Like the inactivation of another K+ channel [Grissmer, S. & Calahan, M. (1989) Biophys. J. 55, 203-206], this slow inactivation is retarded by the application of a channel blocker, tetraethylammonium, to the extracellular side of the channel. By contrast, the fast inactivation of the wild-type Shaker channel is sensitive only to intracellular application of tetraethylammonium. Intracellular tetraethylammonium slows down the fast inactivation process, as though it competes with the binding of the inactivation particle.     

The presence in pig brain of an endogenous equivalent of apamin, the bee venom peptide that specifically blocks Ca2+-dependent K+ channels.

An apamin-like factor has been isolated from pig brain after extraction of the tissue and purification on sulfopropyl-Sephadex C-25 and on reversed-phase high pressure liquid chromatography. The apamin-like factor has the following properties: (i) it prevents 125I-labeled apamin binding to its specific receptor site present on rat brain synaptosomes, (ii) it is active in the radioimmunoassay for apamin (i.e., it prevents 125I-labeled apamin precipitation by anti-apamin antibodies), (iii) it induces contraction of guinea pig intestinal smooth muscle previously relaxed with epinephrine, and (iv) it blocks Ca2+-dependent K+ channels responsible for the long-lasting afterpotential hyperpolarization following the action potential in rat skeletal muscle cells in culture. All these properties are those of apamin itself. The apamin-like factor is a peptide that, like apamin, is destroyed by trypsin and unaffected by chymotrypsin. These results suggest the presence in mammalian brain of a potent Ca2+-dependent K+-channel modulator.     

Voltage-dependent K+ channels are positive regulators of alpha cell action potential generation and glucagon secretion in mice and humans

Aims/hypothesis  

The regulation of glucagon secretion from alpha cells is poorly understood. Since action potential firing at low glucose is required for glucagon secretion, we hypothesised that voltage-dependent K⁺ (Kv) currents limit glucagon secretion under these conditions, similarly to their role in insulin secretion.     

K+-induced dilation of hamster cremasteric arterioles involves both the Na+/K+-ATPase and inward-rectifier K+ channels

OBJECTIVE: The mechanism by which elevated extracellular potassium ion concentration ([K+]o) causes dilation of skeletal muscle arterioles was evaluated. METHODS: Arterioles (n = 111) were hand-dissected from hamster cremaster muscles, cannulated with glass micropipettes and pressurized to 80 cm H2O for in vitro study. The vessels were superfused with physiological salt solution containing 5 mM KCl, which could be rapidly switched to test solutions containing elevated [K+]o and/or inhibitors. The authors measured arteriolar diameter with a computer-based diameter tracking system, vascular smooth muscle cell membrane potential with sharp micropipettes filled with 200 mM KCl, and changes in intracellular Ca2+ concentration ([Ca2+]i) with Fura 2. Membrane currents and potentials also were measured in enzymatically isolated arteriolar muscle cells using patch clamp techniques. The role played by inward rectifier K+ (KIR) channels was tested using Ba2+ as an inhibitor. Ouabain and substitution of extracellular Na+ with Li+ were used to examine the function of the Na+/K+ ATPase. RESULTS: Elevation of [K+]o from 5 mM up to 20 mM caused transient dilation of isolated arterioles (27 +/- 1 microm peak dilation when [K+]o was elevated from 5 to 20 mM, n = 105, p <.05). This dilation was preceded by transient membrane hyperpolarization (10 +/-1 mV, n = 23, p <.05) and by a fall in [Ca2+]i as indexed by a decrease in the Fura 2 fluorescence ratio of 22 +/- 5% (n = 4, p <.05). Ba(2+) (50 or 100 microM) attenuated the peak dilation (40 +/- 8% inhibition, n = 22) and hyperpolarization (31 +/- 12% inhibition, n = 7, p <.05) and decreased the duration of responses by 37 +/-11% (n = 20, p < 0.05). Both ouabain (1 mM or 100 microM) and replacement of Na+ with Li+ essentially abolished both the hyperpolarization and vasodilation. CONCLUSIONS: Elevated [K+]o causes transient vasodilation of skeletal muscle arterioles that appears to be an intrinsic property of the arterioles. The results suggest that K+-induced dilation involves activation of both the Na+/K+ ATPase and KIR channels, leading to membrane hyperpolarization, a fall in [Ca2+]i, and culminating in vasodilation. The Na+/K+ ATPase appears to play the major role and is largely responsible for the transient nature of the response to elevated [K+]o, whereas KIR channels primarily affect the duration and kinetics of the response.     

Activation of ATP-sensitive K+ channels by cromakalim. Effects on cellular K+ loss and cardiac function in ischemic and reperfused mammalian ventricle.

Pharmacological modulation of [K+]o accumulation and action potential changes during acute myocardial ischemia is under evaluation as a promising new antiarrhythmic and cardioprotective strategy during myocardial ischemia and reperfusion. We studied the effects of cromakalim, a K+ channel opener that activates ATP-sensitive K+ channels, in isolated arterially perfused rabbit interventricular septa subjected to ischemia and reperfusion and, through use of the patch clamp technique, in inside-out membrane patches excised from guinea pig ventricular myocytes. During aerobic perfusion, 5 microM cromakalim shortened action potential duration (APD) from 217 +/- 7 to 201 +/- 10 msec, had no effect on [K+]o, and reduced tension by 17 +/- 3% (n = 11). During ischemia, pretreatment with 5 microM cromakalim resulted in 1) more rapid APD shortening (71 +/- 9 versus 166 +/- 7 msec at 10 minutes and 63 +/- 12 versus 122 +/- 8 msec at 30 minutes), 2) similar [K+]o accumulation after 10 minutes (8.9 +/- 0.3 versus 9.6 +/- 0.5 mM) but a trend toward increased [K+]o accumulation after 30 minutes (11.0 +/- 1.7 versus 9.6 +/- 1.0 mM), and 3) similar times for tension to decline to 50% of control (2.14 +/- 0.16 versus 2.14 +/- 0.19 minutes) but shorter time to fall to 20% of control (4.34 +/- 0.33 versus 4.90 +/- 0.22 minutes; p = 0.003). After 60 minutes of reperfusion following 30 minutes of ischemia, recovery of function was similar, with a trend toward better recovery of developed tension (to 58 +/- 9% versus 39 +/- 10% of control; p = 0.18) and tissue ATP levels in cromakalim-treated hearts but no differences in APD or rest tension. Thus, 5 microM cromakalim had mild effects in normal heart but greatly accelerated APD shortening during ischemia without markedly increasing [K+]o accumulation, possibly because the more rapid APD shortening reduced the time-averaged driving force for K+ efflux through ATP-sensitive K+ channels. A significant cardioprotective effect during 30 minutes of ischemia plus 60 minutes of reperfusion could not be demonstrated in this model. In excised membrane patches studied at room temperature, the ability of cromakalim to activate ATP-sensitive K+ channels was significantly potentiated by 100 microM but not 15 microM cytosolic ADP, suggesting that in addition to the modest fall in cytosolic ATP during early ischemia, the rapid increases in cytosolic ADP may further sensitize cardiac ATP-sensitive K+ channels to activation by cromakalim.(ABSTRACT TRUNCATED AT 400 WORDS)     

Immunosuppressants implicate protein phosphatase regulation of K+ channels in guard cells.

The elevation of Ca2+ levels in the cytoplasm inactivates inward-rectifying K+ channels that play a central role in regulating the apertures of stomatal pores in higher plants. However, the mechanism for the Ca(2+)-mediated inhibition of K(+)-channel function is unknown. Using patch-clamp techniques, we show that cyclophilin-cyclosporin A and FK506-binding protein-FK506 complexes, which are highly specific inhibitors of protein phosphatase 2B (calcineurin), block Ca(2+)-induced inactivation of K+ channels in Vicia faba guard cells. A constitutively active calcineurin fragment that is Ca(2+)-independent inhibits K(+)-channel activity in the absence of Ca2+. We have also identified an endogenous Ca(2+)-dependent phosphatase activity from V. faba that is inhibited by the cyclophilin-cyclosporin A and FK506-binding protein-FK506 complexes. Our findings implicate a Ca(2+)-dependent, calcineurin-like protein phosphatase in a Ca2+ signal-transduction pathway of higher plants.     

Evidence for mitochondrial K+ channels and their role in cardioprotection

Twenty years after the discovery of sarcolemmal ATP-sensitive K+ channels and 12 years after the discovery of mitochondrial K(ATP) (mitoK(ATP)) channels, progress has been remarkable, but many questions remain. In the case of the former, detailed structural information is available, and it is well accepted that the channel couples bioenergetics to cellular electrical excitability; however, in the heart, a clear physiological or pathophysiological role has yet to be defined. For mitoK(ATP), structural information is lacking, but there is abundant evidence linking the opening of the channel to protection against ischemia-reperfusion injury or apoptosis. This review updates recent progress in understanding the physiological role of mitoK(ATP) and highlights outstanding questions and controversies, with the intent of stimulating additional investigation on this topic.