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.     

Verapamil, a phenylalkylamine Ca2 + channel blocker, inhibits ATP-sensitive K + channels in insulin-secreting cells from rats

Summary Radioisotopic and electrophysiological techniques were used to assess the effects of verapamil, a phenylalkylamine Ca^{2 +} channel blocker, on K ⁺ permeability of insulin-secreting cells. Verapamil provoked a concentration-dependent inhibition of ⁸⁶Rb (⁴²K substitute) outflow from prelabelled and perifused rat pancreatic islets. This property appears to be inherent to the phenylalkylamine Ca^{2 +} channel blockers since gallopamil, a methoxyderivative of verapamil, but not nifedipine, a 1,4-dihydropyridine Ca^{2 +} channel blocker, inhibited ⁸⁶Rb outflow. The experimental data further revealed that verapamil interacted with a Ca^{2 +} -independent, glucose- and glibenclamide-sensitive modality of ⁸⁶Rb extrusion. Moreover, verapamil prevented the increase in ⁸⁶Rb outflow brought about by BPDZ 44; a potent activator of the ATP-sensitive K ⁺ channel. Single-channel current recordings by the patch clamp technique confirmed that verapamil elicited a dose-dependent inhibition of the ATP-dependent K ⁺ channel. Lastly, under experimental conditions in which verapamil clearly inhibited the ATP-sensitive K ⁺ channels, the drug did not affect ⁴⁵Ca outflow, the cytosolic free Ca^{2 +} concentration or insulin release. It is concluded that the Ca^{2 +} entry blocker verapamil inhibits ATP-sensitive K ⁺ channels in pancreatic beta cells. This effect was not associated with stimulation of insulin release [Diabetologia (1997) 40: 1403–1410]. Received: 21 May 1997 and in final revised form: 28 August 1997     

ATP-modulated K+ channels sensitive to antidiabetic sulfonylureas are present in adenohypophysis and are involved in growth hormone release.

The adenohypophysis contains high-affinity binding sites for antidiabetic sulfonylureas that are specific blockers of ATP-sensitive K+ channels. The binding protein has a M(r) of 145,000 +/- 5000. The presence of ATP-sensitive K+ channels (26 pS) has been demonstrated by electrophysiological techniques. Intracellular perfusion of adenohypophysis cells with an ATP-free medium to activate ATP-sensitive K+ channels induces a large hyperpolarization (approximately 30 mV) that is antagonized by antidiabetic sulfonylureas. Diazoxide opens ATP-sensitive K+ channels in adenohypophysis cells as it does in pancreatic beta cells and also induces a hyperpolarization (approximately 30 mV) that is also suppressed by antidiabetic sulfonylureas. As in pancreatic beta cells, glucose and antidiabetic sulfonylureas depolarize the adenohypophysis cells and thereby indirectly increase Ca2+ influx through L-type Ca2+ channels. The K+ channel opener diazoxide has an opposite effect. Opening ATP-sensitive K+ channels inhibits growth hormone secretion and this inhibition is eliminated by antidiabetic sulfonylureas.     

Effects of sulfonylureas on mitochondrial ATP-sensitive K+ channels in cardiac myocytes: implications for sulfonylurea controversy

BACKGROUND: Mitochondrial ATP-sensitive K(+) (mitoK(ATP)) channel plays a key role in cardioprotection. Hence, a sulfonylurea that does not block mitoK(ATP) channels would be desirable to avoid damage to the heart. Accordingly, we examined the effects of sulfonylureas on the mitoK(ATP) channel and mitochondrial Ca(2+) overload. METHODS: Flavoprotein fluorescence in rabbit ventricular myocytes was measured to assay mitoK(ATP) channel activity. The mitochondrial Ca(2+) concentration was measured by loading cells with rhod-2. RESULTS: The mitoK(ATP) channel opener diazoxide (100 microM) reversibly increased flavoprotein oxidation to 31.8 +/- 4.3% (n = 5) of the maximum value induced by 2,4-dinitrophenol. Glimepiride (10 microM) alone did not oxidize the flavoprotein, and the oxidative effect of diazoxide was unaffected by glimepiride (35.4 +/- 3.2%, n = 5). Similarly, the diazoxide-induced flavoprotein oxidation was unaffected both by gliclazide (10 microM) and by tolbutamide (100 microM). Exposure to ouabain (1 mM) for 30 min produced mitochondrial Ca(2+) overload, and the intensity of rhod-2 fluorescence increased to 197.4 +/- 7.2% of baseline (n = 11). Treatment with diazoxide significantly reduced the ouabain-induced mitochondrial Ca(2+) overload (149.6 +/- 5.1%, n = 11, p < 0.05 versus ouabain alone), and the effect was antagonized by the mitoK(ATP) channel blocker 5-hydroxydecanoate (189.8 +/- 27.8%, n = 5) and glibenclamide (193.1 +/- 7.7%, n = 8). On the contrary, cardioprotective effect of diazoxide was not abolished by glimepiride (141.8 +/- 7.8%, n = 6), gliclazide (139.0 +/- 9.4%, n = 5), and tolbutamide (141.1 +/- 4.5%, n = 7). CONCLUSIONS: Our results indicate that glimepiride, gliclazide, and tolbutamide have no effect on mitoK(ATP) channel, and do not abolish the cardioprotective effects of diazoxide. Therefore, these sulfonylureas, unlike glibenclamide, do not interfere with the cellular pathways that confer cardioprotection.     

Hypoxia does not activate ATP-sensitive K+ channels in arteriolar muscle cells

OBJECTIVE: To test the hypothesis that hypoxia activates ATP-sensitive K+ (KATP) channels in cremasteric arteriolar muscle cells, resulting in membrane hyperpolarization and inhibition of norepinephrine-induced contraction. METHODS: Arteriolar muscle cells were isolated enzymatically from second- and third-order arterioles that were surgically removed from hamster cremaster muscles. The effects of hypoxia (PO2 = 12-15 mm Hg) were then examined on norepinephrine-induced contraction, membrane currents, and membrane potential in these cells at room temperature. Whole-cell currents and membrane potential were recorded using the perforated patch technique. RESULTS: Hypoxia (12-15 mm Hg PO2) reversibly inhibited norepinephrine-induced contraction to 52 +/- 6% of the response in normoxic solutions (156 mm Hg, n = 12 digests, p < 0.05). These effects of hypoxia could be prevented by superfusion of the cells with either solutions containing the KATP channel antagonist glibenclamide (1 microM) or solutions containing 35 mM K+ to reduce the electrochemical gradient for K+ diffusion. Cromakalim, an activator of KATP channels, also inhibited norepinephrine-induced contraction to a similar extent as hypoxia, and in a glibenclamide and 35 mM K(+)-sensitive manner. These results are consistent with the KATP channel hypothesis. In contrast, hypoxia had no effect on estimated whole-cell membrane conductance between -40 and -90 mV in voltage-clamp experiments; on holding current measured at -60 mV in cells superfused with 143 mM K+ under voltage-clamp conditions; or on membrane potential in current-clamp experiments, despite positive effects of cromakalim in all three protocols. These electrophysiological data lead to rejection of the hypothesis that hypoxia activates KATP channels. CONCLUSIONS: Hypoxia inhibits norepinephrine-induced contraction of cremasteric arteriolar muscle cells by a mechanism that does not involve KATP channels. It is speculated that the inhibitory effects of glibenclamide and 35 mM K+ on the effects of hypoxia on contraction resulted from depolarization induced by these treatments rather than specific inhibition of KATP channels.     

Evidence that neuronal G-protein-gated inwardly rectifying K+ channels are activated by G beta gamma subunits and function as heteromultimers.

Guanine nucleotide-binding proteins (G proteins) activate K+ conductances in cardiac atrial cells to slow heart rate and in neurons to decrease excitability. cDNAs encoding three isoforms of a G-protein-coupled, inwardly rectifying K+ channel (GIRK) have recently been cloned from cardiac (GIRK1/Kir 3.1) and brain cDNA libraries (GIRK2/Kir 3.2 and GIRK3/Kir 3.3). Here we report that GIRK2 but not GIRK3 can be activated by G protein subunits G beta 1 and G gamma 2 in Xenopus oocytes. Furthermore, when either GIRK3 or GIRK2 was coexpressed with GIRK1 and activated either by muscarinic receptors or by G beta gamma subunits, G-protein-mediated inward currents were increased by 5- to 40-fold. The single-channel conductance for GIRK1 plus GIRK2 coexpression was intermediate between those for GIRK1 alone and for GIRK2 alone, and voltage-jump kinetics for the coexpressed channels displayed new kinetic properties. On the other hand, coexpression of GIRK3 with GIRK2 suppressed the GIRK2 alone response. These studies suggest that formation of heteromultimers involving the several GIRKs is an important mechanism for generating diversity in expression level and function of neurotransmitter-coupled, inward rectifier K+ channels.     

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.     

The structure and function of the ATP-sensitive K+ channel in insulin-secreting pancreatic beta-cells

ATP-sensitive K+ channels (KATP channels) play important roles in many cellular functions by coupling cell metabolism to electrical activity. The KATP channels in pancreatic beta-cells are thought to be critical in the regulation of glucose-induced and sulfonylurea-induced insulin secretion. Until recently, however, the molecular structure of the KATP channel was not known. Cloning members of the novel inwardly rectifying K+ channel subfamily Kir6.0 (Kir6.1 and Kir6.2) and the sulfonylurea receptors (SUR1 and SUR2) has clarified the molecular structure of KATP channels. The pancreatic beta-cell KATP channel comprises two subunits: a Kir6.2 subunit and an SUR1 subunit. Molecular biological and molecular genetic studies have provided insights into the physiological and pathophysiological roles of the pancreatic beta-cell KATP channel in insulin secretion.     

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.     

ATP-sensitive K+ channels in a plasma membrane H+-ATPase mutant of the yeast Saccharomyces cerevisiae.

A mutant in the plasma membrane H+-ATPase gene of the yeast Saccharomyces cerevisiae with a reduced H+-ATPase activity, when examined at the single-channel level with the patch-clamp technique, was found to exhibit K+ channels activated by intracellular application of ATP. In the parent strain, the same channel, identified by its conductance and selectivity, is not activated by ATP. This activity in the mutant is blocked by the ATPase inhibitor N,N'-dicyclohexylcarbodiimide. ADP and the ATP analog adenosine 5'-[gamma-[35S]thio]triphosphate do not activate the channel. These findings suggest a tight physical coupling between the plasma membrane ATPase and the K+ channel.     

Intracellular ATP-sensitive K+ channels in mouse pancreatic beta cells: against a role in organelle cation homeostasis

Aims/hypothesis ATP-sensitive K⁺ (K_ATP) channels located on the beta cell plasma membrane play a critical role in regulating insulin secretion and are targets for the sulfonylurea class of antihyperglycaemic drugs. Recent reports suggest that these channels may also reside on insulin-containing dense-core vesicles and mitochondria. The aim of this study was to explore these possibilities and to test the hypothesis that vesicle-resident channels play a role in the control of organellar Ca²⁺ concentration or pH.Methods To quantify the subcellular distribution of the pore-forming subunit Kir6.2 and the sulfonylurea binding subunit SUR1 in isolated mouse islets and clonal pancreatic MIN6 beta cells, we used four complementary techniques: immunoelectron microscopy, density gradient fractionation, vesicle immunopurification and fluorescence-activated vesicle isolation. Intravesicular and mitochondrial concentrations of free Ca²⁺ were measured in intact or digitonin-permeabilised MIN6 cells using recombinant, targeted aequorins, and intravesicular pH was measured with the recombinant fluorescent probe pHluorin.Results SUR1 and Kir6.2 immunoreactivity were concentrated on dense-core vesicles and on vesicles plus the endoplasmic reticulum/Golgi network, respectively, in both islets and MIN6 cells. Reactivity to neither subunit was detected on mitochondria. Glibenclamide, tolbutamide and diazoxide all failed to affect Ca²⁺ uptake into mitochondria, and K_ATP channel regulators had no significant effect on intravesicular free Ca²⁺ concentrations or vesicular pH.Conclusions/Interpretation A significant proportion of Kir6.2 and SUR1 subunits reside on insulin-secretory vesicles and the distal secretory pathway in mouse beta cells but do not influence intravesicular ion homeostasis. We propose that dense-core vesicles may serve instead as sorting stations for the delivery of channels to the plasma membrane.Electronic Supplementary Material Supplementary material is available for this article at     

Mitochondrial ATP-sensitive K+ channels play a role in cardioprotection by Na+-H+ exchange inhibition against ischemia/reperfusion injury.

OBJECTIVES: The possible role of the ATP-sensitive potassium (KATP) channel in cardioprotection by Na+-H+ exchange (NHE) inhibition was examined. BACKGROUND: The KATP channel is suggested to be involved not only in ischemic preconditioning but also in some pharmacological cardioprotection. METHODS: Infarction was induced by 30-min coronary occlusion in rabbit hearts in situ or by 30-min global ischemia in isolated hearts. Myocardial stunning was induced by five episodes of 5-min ischemia/5-min reperfusion in situ. In these models, the effects of NHE inhibitors (cariporide and ethylisopropyl-amiloride [EIPA]) and the changes caused by KATP channel blockers were assessed. In another series of experiments, the effects of EIPA on mitochondrial KATP (mito-KATP) and sarcolemmal KATP (sarc-KATP) channels were examined in isolated cardiomyocvtes. RESULTS: Cariporide (0.6 mg/kg) reduced infarct size in situ by 40%, and this effect was abolished by glibenclamide (0.3 mg/kg), a nonselective KATP channel blocker. In vitro, 1 microM cariporide limited infarct size by 90%, and this effect was blocked by 5-hydroxydecanoate (5-HD), a mito-KATP channel blocker but not by HMR1098, a sarc-KATP channel blocker. Infarct size limitation by 1 microM EIPA was also prevented by 5-HD. Cariporide attenuated regional contractile dysfunction by stunning, and this protection was abolished by glibenclamide and 5-HD. Ethylisopropyl amiloride neither activated the mito-KATP channel nor enhanced activation of this channel by diazoxide, a KATP channel opener. CONCLUSIONS: Opening of the mito-KATP channel contributes to cardioprotection by NHE inhibition, though the interaction between NHE and this KATP channel remains unclear.     

Paramecium calcium channels are blocked by a family of calmodulin antagonists.

Although the voltage-sensitive Ca channel present in Paramecium has been subjected to detailed physiological and genetic analysis, no organic ligands have been described that block this channel with high affinity and that ultimately can be used to identify channel components. Based on a previous observation that the naphthalene sulfonamide calmodulin antagonist W-7 can block Paramecium Ca channels at high concentration, we have synthesized analogs of W-7 that block these channels at concentrations of less than 1 microM. The effectiveness of these compounds was tested both by a sensitive behavioral assay and on Ca channels that had been incorporated into planar lipid bilayers. Despite the fact that these compounds are effective Paramecium calmodulin antagonists, two independent lines of evidence suggest that W-7 and its analogs block the Ca channel by a mechanism that is independent of their action on calmodulin. In addition, the sensitivity to W-7 or dihydropyridines of Ca channels present in a number of eukaryotic phyla has been used to identify similarities in Ca channels from widely diverse organisms. It appears that the pharmacological specificity provides a means to group Ca channels.     

Tityustoxin K alpha blocks voltage-gated noninactivating K+ channels and unblocks inactivating K+ channels blocked by alpha-dendrotoxin in synaptosomes.

Two nonhomologous polypeptide toxins, tityustoxinK alpha (TsTX-K alpha) and tityustoxin K beta (TsTX-K beta), purified from thevenom of the Brazilian scorpion Tityus serrulatus, selectively blockvoltage-gated noninactivating K+ channels in synaptosomes (IC50 values of 8 nMand 30 nM, respectively). In contrast, alpha-dendrotoxin (alpha-DTX) andcharybdotoxin (ChTX) block voltage-gated inactivating K+ channels insynaptosomes (IC50 values of 90 nM and 40 nM, respectively). We studiedinteractions among these toxins in 125I-alpha-DTX binding and 86Rb effluxexperiments. Both TsTX-K alpha and ChTX completely displaced specifically bound125I-alpha-DTX from synaptic membranes, but TsTX-K beta had no effect on boundalpha-DTX. TsTX-K alpha and TsTX-K beta blocked the same noninactivatingcomponent of 100 mM K(+)-stimulated 86Rb efflux in synaptosomes. Both alpha-DTXand ChTX blocked the same inactivating component of the K(+)-stimulated 86Rbefflux in synaptosomes. Both the inactivating and the noninactivating componentsof the 100 mM K(+)-stimulated 86Rb efflux were completely blocked when 200 nMTsTX-K beta and either 600 nM alpha-DTX or 200 nM ChTX were present. The effectsof TsTX-K alpha and ChTX on 86Rb efflux were also additive. When TsTX-K alphawas added in the presence of alpha-DTX, however, only the noninactivatingcomponent of the K(+)-stimulated efflux was blocked. The inactivating componentcould then be blocked by ChTX, which is structurally homologous to TsTX-K alpha.We conclude that TsTX-K alpha unblocks the voltage-gated inactivating K+channels in synaptosomes when they are blocked by alpha-DTX, but not when theyare blocked by ChTX. TsTX-K alpha binds to a site on the inactivating K+ channelthat does not occlude the pore; its binding apparently prevents alpha-DTX (7054Da), but not ChTX (4300 Da), from blocking the pore. The effects of TsTX-K alphaon 125I-alpha-DTX binding and 86Rb efflux are mimicked by noxiustoxin, which ishomologous to TsTX-K alpha and ChTX.     

Reduced sensitivity of dihydroxyacetone on ATP-sensitive K+ channels of pancreatic beta cells in GK rats

Summary In the GK (Goto-Kakizaki) rat, a genetic model of non-insulin-dependent diabetes mellitus, glucose-induced insulin secretion is selectively impaired. In addition, it has been suggested by previous studies that impaired glucose metabolism in beta cells of the GK rat results in insufficient closure of ATP-sensitive K⁺ channels (K_ATP channels) and a consequent decrease in depolarization, leading to a decreased insulin release. We have recently reported that the site of disturbed glucose metabolism is probably located in the early stages of glycolysis or in the glycerol phosphate shuttle. In the present study, in order to identify the impaired metabolic step in diabetic beta cells, we have investigated insulin secretory capacity by stimulation with dihydroxyacetone (DHA), which is known to be directly converted to DHA-phosphate and to preferentially enter the glycerol phosphate shuttle. In addition, using the patch-clamp technique, we also have studied the sensitivity of DHA on the K_ATP channels of beta cells in GK rats. The insulin secretion in response to 5 mmol/l DHA with 2.8 mmol/l glucose was impaired, and DHA sensitivity of the K_ATP channels was reduced in beta cells of GK rats. From these results, we suggest that the intracellular site responsible for impaired glucose metabolism in pancreatic beta cells of GK rats is located in the glycerol phosphate shuttle.Abbreviations DHA Dihydroxyacetone - K_ATP channel ATP-sensitive K⁺ channel - GK rat Goto-Kakizaki rat - KRBB Krebs Ringer bicarbonate buffer - BSA bovine serum albumin - NIDDM non-insulin-dependent diabetes     

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)     

Sulfonylureas, ATP-sensitive K+ channels, and cellular K+ loss during hypoxia, ischemia, and metabolic inhibition in mammalian ventricle

Sulfonylurea derivatives glibenclamide and tolbutamide are selective blockers of ATP-sensitive K+ (KATP) channels. However, their ability to prevent cellular K+ loss and shortening of action potential duration during ischemia or hypoxia in the intact heart is modest compared with their efficacy at blocking KATP channels in excised membrane patches. In the isolated arterially perfused rabbit interventricular septum, the increase in unidirectional K+ efflux and shortening of action potential duration during substrate-free hypoxia were effectively blocked by glibenclamide, but only by very high concentrations (100 microM); during hypoxia with glucose present, glibenclamide was only partially effective at reducing K+ loss. During total global ischemia (10 minutes), up to 100 microM glibenclamide or 1 mM tolbutamide attenuated shortening of action potential duration but only reduced [K+]0 accumulation by a maximum of 32 +/- 6%. In isolated patch-clamped guinea pig ventricular myocytes in which the whole-cell ATP-sensitive K+ current was activated by exposure to the metabolic inhibitors, glibenclamide (up to 100 microM) and tolbutamide (10 mM) were only partially effective at blocking the whole-cell ATP-sensitive K+ current (maximum block, 51 +/- 10% and 50 +/- 9%, respectively), especially when ADP was included in the patch electrode solution. In inside-out membrane patches excised from these myocytes, glibenclamide blocked unitary currents through KATP channels with a Kd of 0.5 microM and a Hill coefficient of 0.5 in the absence of ADP at the cytosolic membrane surface, but block was incomplete when 100 microM ADP (+2 mM free Mg2+) was present. ADP had a similar effect on block of KATP channels by tolbutamide. These findings suggest that free cytosolic [ADP], which rises rapidly to the 100 microM range during early myocardial ischemia and hypoxia, may account for the limited efficacy of sulfonylureas at blocking ischemic and hypoxic cellular K+ loss under these conditions.     

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.     

Role of cardiac ATP-sensitive K+ channels induced by HMG CoA reductase inhibitor in ischemic rabbit hearts.

Although 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors can protect the myocardium against ischemic injury, the mechanisms of their effect have not yet been characterized at the cellular level. Therefore, we investigated the role of cardiac ATP-sensitive K+ (K(ATP)) channels induced by the HMG-CoA reductase inhibitor known as pravastatin on the myocardial metabolism during ischemia by phosphorus 31-nuclear magnetic resonance (31P-NMR) in isolated rabbit hearts. Forty-five min of continuous normothermic global ischemia was carried out. Pravastatin with or without the K(ATP) channel blocker glibenclamide or the nitric oxide synthase inhibitor L-NAME was administered beginning 60 min prior to the global ischemia. Twenty-eight hearts were divided into 4 experimental groups consisting of 7 hearts each: the control group, the P group consisting of pravastatin treatment, the P+G group consisting of pravastatin treatment with glibenclamide, and the P+L group consisting of pravastatin treatment with L-NAME. During ischemia, the decreases in adenosine triphosphate (ATP) and intracellular pH (pHi) were significantly inhibited in the P group in comparison with Control group (at end of ischemia, respectively; both p<0.01), as was the increase in inorganic phosphate (Pi) (at end of ischemia, p<0.01). However, the decreases in ATP and pHi and the increase in Pi were not inhibited in the P+G group during ischemia. The P+L group also showed no inhibition of the aforementioned parameters during the same period. These results suggest that pravastatin has a significant beneficial effect for improving the myocardial energy metabolism, which is provided by K(ATP) channels and nitric oxide (NO), during myocardial ischemia. The cardioprotection of HMG-CoA reductase inhibitor may be caused by the K(ATP) channels that are mediated by the NO.