A transmembrane domain of the sulfonylurea receptor mediates activation of ATP-sensitive K(+) channels by K(+) channel openers.

ATP-sensitive K(+) (K(ATP)) channels are a complex of an ATP-binding cassette transporter, the sulfonylurea receptor (SUR), and an inward rectifier K(+) channel subunit, Kir6.2. The diverse pharmacological responsiveness of K(ATP) channels from various tissues are thought to arise from distinct SUR isoforms. Thus, when assembled with Kir6. 2, the pancreatic beta cell isoform SUR1 is activated by the hyperglycemic drug diazoxide but not by hypotensive drugs like cromakalim, whereas the cardiac muscle isoform SUR2A is activated by cromakalim and not by diazoxide. We exploited these differences between SUR1 and SUR2A to pursue a chimeric approach designed to identify the structural determinants of SUR involved in the pharmacological activation of K(ATP) channels. Wild-type and chimeric SUR were coexpressed with Kir6.2 in Xenopus oocytes, and we studied the resulting channels with the patch-clamp technique in the excised inside-out configuration. The third transmembrane domain of SUR is found to be an important determinant of the response to cromakalim, which possibly harbors at least part of its binding site. Contrary to expectations, diazoxide sensitivity could not be linked specifically to the carboxyl-terminal end (nucleotide-binding domain 2) of SUR but appeared to involve complex allosteric interactions between transmembrane and nucleotide-binding domains. In addition to providing direct evidence for the structure-function relationship governing K(ATP) channel activation by potassium channel-opening drugs, a family of drugs of the highest therapeutic interest, these findings delineate the determinants of ligand specificity within the modular ATP-binding cassette-transporter architecture of SUR.     

Inhibition of ATP-sensitive K+ channels by taurine through a benzamido-binding site on sulfonylurea receptor 1

ATP-sensitive potassium (K(ATP)) channels in pancreatic beta-cells comprise sulfonylurea receptor (SUR) 1 and inwardly-rectifying potassium channel (Kir) 6.2 subunits. We have evaluated the effect of intracellular taurine on K(ATP) channel activity in rat pancreatic beta-cells using the patch-clamp technique. The mechanism of taurine action was also examined using recombinant K(ATP) channels. The islets and single beta-cells from male Sprague-Dawley rats were collected by collagenase digestion technique. Single K(ATP) channel currents were recorded by the inside-out mode at a membrane potential of -60mV. Cytosolic free-Ca(2+) concentration ([Ca(2+)](c)) and insulin secretory capacity were measured by the dual-excitation fluorimetry and radioimmunoassay, respectively. The native beta-cell K(ATP) channel was directly inhibited by taurine in a dose-dependent manner. Taurine did not influence ATP-mediated inhibition or MgADP-induced activation of the channel activity. The sensitivity of the K(ATP) channel to glybenclamide, but not gliclazide, was enhanced by taurine. Glybenclamide elicited a greater increase in [Ca(2+)](c) and increased insulin secretion in the beta-cells when pretreated with taurine. Taurine did not inhibit Kir6.2DeltaC36 currents, a truncated form of Kir6.2, expressed in Xenopus oocytes without SUR. These results demonstrate that taurine inhibits the K(ATP) channel activity in the beta-cells, interacting with a benzamido-binding site on SUR1, but not Kir6.2.     

ATP敏感性钾通道的阻断剂与开放剂研究进展

ATP敏感性钾通道是高血压、心绞痛、糖尿病及缺血性脑损伤等多种疾病的治疗靶点之一。通过对调节KATP通道药物的选择性、可逆性、作用位点以及相互调节的研究，可以对一些临床用药进行重新评估，并指导开发高选择性的KATP通道阻断剂和开放剂。     

ATP-sensitive K+ channel openers: old drugs with new clinical benefits for the heart

Different types of ATP-sensitive K+ (K(ATP)) channels have been identified in cardiomyocytes, vascular smooth muscle cells, pancreatic beta-cells, neurons and mitochondria. Years before the discovery of the K(ATP) channel in cardiomyocytes, pharmacological openers of this channel had been developed for the treatment of angina pectoris and hypertension. The K(ATP) channel plays an important role not only in coronary blood flow regulation but also in protection of cardiovascular cells from ischemia/reperfusion injury. In animal models of myocardial ischemia/reperfusion, activation of the mitochondrial K(ATP) channels by their pharmacological openers has been shown to attenuate endothelial dysfunction and to reduce myocardial necrosis. Conversely, blockade of the K(ATP) channel aggravates microvascular necrosis and the no-reflow phenomenon after ischemia/reperfusion, resulting in augmentation of post-infarct ventricular dysfunction. Recent clinical studies have shown that a combination of coronary reperfusion therapy and infusion of nicorandil, a hybrid of K(ATP) channel opener and nitrate, improved left ventricular function in patients with acute myocardial infarction. Furthermore, chronic treatment with nicorandil has been shown to significantly improve prognosis of patients with high-risk stable angina pectoris. Both of these clinical benefits cannot be attributed to the nitrate property of nicorandil. However, a recent basic investigation has suggested that the protective function of K(ATP) channel openers is compromised by concurrent hypercholesterolemia and administration of sulfonylureas for diabetes mellitus. These interferences in the beneficial action of K(ATP) channel openers by concurrent illness and pharmacological agents need to be further investigated to allow a more effective use of K(ATP) channel openers in patients with coronary artery diseases.     

Hyperpolarisation of rat mesenteric endothelial cells by ATP-sensitive K(+) channel openers

Membrane potential responses of rat mesenteric endothelial cells were investigated in intact arteries using sharp electrodes. Levcromakalim, an activator of ATP-sensitive K(+) channels (K(ATP)) induced concentration-dependent hyperpolarisation of the endothelial cells, which was reversible by glibenclamide and ciclazindol, inhibitors of K(ATP). Another K(ATP) activator, diazoxide, also hyperpolarised the endothelial cells. Carbachol induced endothelial hyperpolarisation that was inhibited by combinations of apamin and charybdotoxin, but not Ba(2+) and ouabain. Prior stimulation with levcromakalim inhibited carbachol-induced responses, and this inhibitory effect was also sensitive to glibenclamide. 1, 3-dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl) -2H-benzimidazol-2-one (NS 1619), an activator of large conductance, Ca(2+)-activated K(+) channels (BK(Ca)), induced only small hyperpolarisations of the endothelial cells. Preincubation of tissues with 18 alpha- or 18 beta-glycyrrhetinic acid, inhibitors of gap junction communication, increased the input resistance and depolarised the endothelial cells, and inhibited the hyperpolarising effect of levcromakalim. It is concluded that activation of K(ATP) causes hyperpolarisation of rat mesenteric endothelial cells, probably through gap junctional transfer of smooth muscle hyperpolarisation, and that this may represent an important modulator of endothelial function.     

Taurine block of cloned ATP-sensitive K+ channels with different sulfonylurea receptor subunits expressed in Xenopus laevis oocytes

Taurine has been found to inhibit ATP-sensitive K+ (KATP) channels in rat pancreatic beta-cells [Park et al., Biochem Pharmacol 2004;67:1089-1096] which could be due to its interaction with a benzamido-binding site on SUR1. In present study, we further evaluated the mechanism of taurine action on the KATP-channel inhibition, using cloned KATP-channels with different types of SUR subunit expressed in Xenopus laevis oocytes. The oocytes were coinjected with Kir6.2 mRNA, and mRNA encoding SUR1, SUR2A or SUR2B. Macroscopic currents were recorded from giant excised inside-out patches. The binding of glibenclamide to SUR1 was assessed by using a glibenclamide-fluorescent probe. Intracellular taurine inhibited all three types of KATP-channels to a similar extent. They were fit to the Hill equation, showing IC50 of 11.0 mM for Kir6.2/SUR1, 10.9 mM for Kir6.2/SUR2A, and 9.0 mM for Kir6.2/SUR2B currents. Taurine at the concentration of 10 mM enhanced the high-affinity bindings of glibenclamide and repaglinide on all types of SUR, whereas the low-affinity binding on Kir6.2 was not affected. The intensity of glibenclamide fluorescence was higher in the plasma membrane of taurine-pretreated oocytes. The high-affinity binding of tolbutamide or gliclazide on SUR was not modified by taurine. These results suggest that the taurine inhibition of KATP-channels is mediated by an interaction with the site on SUR where the benzamido group is bound. Therefore, intracellular concentrations of taurine in different tissues may be more important in determining taurine modulation of the KATP-channel rather than distinct types of SUR subunit.     

Structure-activity relationships of K+ channel openers

Seven groups of synthetic agent, distinguished by a combination of their chemical and pharmacological characteristics exert some or all of their effects by opening plasmalemmal K+ channels primarily in smooth muscle. Progress over the past two years now allows broad structure-activity relationships to be formulated within many of the individual groups of agent. Gillian Edwards and Arthur Weston review the historical basis of these discoveries and comment on the significance of new developments. They focus on the search for tissue and channel selectivity, two factors likely to be important for the successful clinical deployment of these substances as antihypertensive and bronchodilator agents.     

Mutation in nucleotide-binding domains of sulfonylurea receptor 2 evokes Na-ATP-dependent activation of ATP-sensitive K+ channels: implication for dimerization of nucleotide-binding domains to induce channel opening

The ATP-sensitive K+ (KATP) channel is composed of a sulfonylurea receptor (SUR) and a pore-forming subunit, Kir6.2. SUR is an ATP-binding cassette (ABC) protein with two nucleotide-binding domains (NBD1 and NBD2). Intracellular ATP inhibits KATP channels through Kir6.2 and activates them through NBDs. However, it is still unknown how ATP-bound NBDs activate KATP channels. A prokaryotic ABC protein, MJ0796, which is entirely NBD, forms a dimer in the presence of Na-ATP when its glutamate at position 171 is substituted with glutamine. Mg2+ or K+ destabilizes the dimer. We made the corresponding mutation in the NBD1 (D834N) and/or NBD2 (E1471Q) of SUR2A and SUR2B. As measured in the inside-out configuration of the patch-clamp method, SUR2x(D834N, E1471)/Kir6.2 channels mediated significantly larger currents in the presence of internal 1 mM Na-ATP than K-ATP alone or Mg-ATP. The response to Na-ATP resulted from an increase in the open probability but not single-channel amplitude of the channels and was abolished by glibenclamide (10(-5) M). In the presence of 1 mM Mg2+ -free ATP, Na+ increased the activity of the channels in a concentration-dependent manner. The Na-ATP-dependent activation was never observed with KATP channels including either the wild-type SUR2x, SUR2x(D834N), or SUR2x(E1471). Nicorandil activated SUR2x(D834N, E1471Q)/Kir6.2 channels more strongly in the presence of Na-ATP than K-ATP alone, whereas the reverse was true for wild-type SUR2x/Kir6.2 channels. Therefore, it is likely that NBDs of SUR2x dimerize in response to ATP and nicorandil. The dimerization induces the opening of the KATP channel, probably by causing a conformational change of SUR2x.     

K+ channel blockers: natriuretic potency correlates to vascular ATP-sensitive K+ channel blockade

Using analogs of known vascular ATP-sensitive K+ channel (KATP) blockers, we identified compounds with a wide range of potencies (over 500-fold) in their capacity to block the hypotensive response of 0.2 mg/kg pinacidil in rats. The most potent of these, U-97025E, belongs to a newly disclosed class of compounds, the cyanoguanidines. U-97025E at 0.04 mg/kg blocked 50% of the depressor response induced by 0.2 mg/kg pinacidil. The maximal natriuresis induced by U-97025E (0.4 mg/kg i.v.) increased Na+ excretion by approximately 60%. This natriuresis is of the same magnitude as that induced by thiazide without any effect on K+ excretion. We found a high degree of correlation between natriuretic potency and the capacity to block the blood pressure lowering effects of pinacidil, both among closely related analogs and dissimilar compounds. These findings imply an obligatory rather than incidental relationship between vascular KATP blockade and natriuresis. The exact molecular link of the vascular and renal effects remains to be determined.     

The size of a single residue of the sulfonylurea receptor dictates the effectiveness of K ATP channel openers

K(ATP) channel openers are a diverse group of molecules able to activate ATP-sensitive K(+) channels in a tissue-dependent manner by binding to the channel regulatory subunit, the sulfonylurea receptor (SUR), an ATP-binding cassette protein. Residues crucial to this action were previously identified in the last transmembrane helix of SUR, transmembrane helix 17. This study examined the residue at the most important position, 1253 in the muscle isoform SUR2A and the matching 1290 in the pancreatic/neuronal isoform SUR1 (rat numbering). At this position in either isoform, a threonine enables action of openers, whereas a methionine prohibits it. Using single-point mutagenesis, we have examined the physicochemical basis of this phenomenon and discovered that it relied uniquely on side chain volume and not on shape, polarity, or hydrogen-bonding capacity of the residue. Moreover, the aromatic nature of neighboring residues conserved in SUR1 and SUR2A was found necessary for SUR2A to sustain the wild-type levels of channel activation by the openers tested, the cromakalim analog SR47063 [4-(2-cyanimino-1,2-dihydro-1-pyridyl)-2,2-dimethyl-6-nitrochromene] and the pinacidil analog P1075 [N-cyano-N'-(1,1-dimethylpropyl)-N'-3-pyridylguanidine]. These observations suggest that these residues can interact with openers via nonspecific stacking interactions provided that the adjacent 1253/1290 residue does not obstruct access. The smaller Thr1253 of SUR2A would permit activation, whereas the bulky Met1290 of SUR1 would not. This hypothesis is discussed in the context of a simple molecular model of transmembrane helix 17.     

The nucleotide-binding domains of sulfonylurea receptor 2A and 2B play different functional roles in nicorandil-induced activation of ATP-sensitive K+ channels

Nicorandil activates ATP-sensitive K(+) channels composed of Kir6.2 and either sulfonylurea receptor (SUR) 2A or 2B. Although SUR2A and SUR2B differ only in their C-terminal 42 amino acids (C42) and possess identical drug receptors and nucleotide-binding domains (NBDs), nicorandil more potently activates SUR2B/Kir6.2 than SUR2A/Kir6.2 channels. Here, we analyzed the roles of NBDs in these channels' response to nicorandil with the inside-out configuration of the patch-clamp method. Binding and hydrolysis of nucleotides by NBDs were impaired by mutations in the Walker A motif of NBD1 (K708A) and NBD2 (K1349A) and in the Walker B motif of NBD2 (D1470N). Experiments were done with internal ATP (1 mM). In SUR2A/Kir6.2 channels, the K708A mutation abolished, and the K1349A but not D1470N mutation reduced the sensitivity to nicorandil. ADP (100 microM) significantly increased the wild-type channels' sensitivity to nicorandil, which was abolished by the K1349A or D1470N mutation. Thus, the SUR2A/Kir6.2 channels' response to nicorandil critically depends on ATP-NBD1 interaction and is facilitated by interactions of ATP or ADP with NBD2. In SUR2B/Kir6.2 channels, either the K708A or K1349A mutation partially suppressed the response to nicorandil, and double mutations abolished it. The D1470N mutation also significantly impaired the response. ADP did not sensitize the channels. Thus, NBD2 hydrolyzes ATP, and NBD1 and NBD2 equally contribute to the response by interacting with ATP and ADP, accounting for the higher nicorandil sensitivity of SUR2B/Kir6.2 than SUR2A/Kir6.2 channels in the presence of ATP alone. Thus, C42 modulates the interaction of both NBDs with intracellular nucleotides.     

ATP-sensitive K+ channels of the cardiovascular system

ATP-sensitive K+ (KATP) channels are inhibited by intracellular ATP and activated by intracellular nucleoside diphosphates, and thus provide a link between cellular metabolism and excitability. They are widely distributed in various tissues including heart and vasculature, and thus may play essential regulatory roles in the cardiovascular system. Furthermore, KATP channels are the targets of two important classes of drugs, i.e., the antidiabetic sulfonylureas which block the channels, and a series of vasorelaxants called "K+ channel openers" which tend to maintain the channels in an open conformation. Recently, the molecular structure of KATP channels has been clarified in various tissues including cardiovascular system to be a complex of at least two subunits, i.e. SUR and Kir6.0. The KATP channels in heart and vascular smooth muscle now appear to be the complexes of SUR2A/Kir6.2 and SUR2B/Kir6.1, respectively. Further works are now in progress to understand the molecular mechanisms responsible for the control of KATP channel function by intracellular nucleotides and drugs.     

Regulation of ATP-sensitive K+ channels by chronic glyburide and pinacidil administration.

Treatment of rats with the K(ATP)+ channel antagonist sulfonylurea, glyburide (3 mg/kg/day, i.p., every 12 hr for 9 days), increased the Bmax value of [3H]glyburide binding to heart and whole brain total membranes by 30 and 24%, respectively. The ligand affinity was unaltered. Treatment with the K+ channel activator, pinacidil (20 mg/kg/day, i.p., every 12 hr for 9 days), did not alter the Bmax value for cardiac [3H]glyburide binding sites, but decreased the Bmax value in the brain by 21%. Chronic administration of hydralazine, which caused an acute reduction in systolic blood pressure equivalent to that of pinacidil, did not alter [3H]glyburide binding in either heart or brain. Treatment with glyburide, pinacidil or hydralazine did not alter L-type calcium channels, assessed by [3H]PN 200 110 binding, in cardiac and brain membranes or small size Ca(2+)-activated K+ channels in brain assessed by [125I]apamin binding. These studies show that the ATP-sensitive class of K+ channels can be regulated following chronic drug treatment in similar fashion to other receptor and channel systems.     

Activation of peripheral ATP-sensitive K+ channels mediates the antinociceptive effect of Crotalus durissus terrificus snake venom

The role of peripheral potassium channels on the antinociceptive effect of Crotalus durissus terrificus venom, a mixed delta- and kappa-opioid receptor agonist, was investigated in hyperalgesia induced by carrageenin or prostaglandin E(2). Rat paw pressure test was applied before and 3 h after the intraplantar (i.pl.) injection of the nociceptive stimuli. Oral administration of venom 2 h after carrageenin or prostaglandin E(2) induces antinociception. Local pretreatment with 4-aminopyridine and tetraethylammonium (blockers of voltage-dependent K(+) channel) or charybdotoxin and apamin (inhibitors of large- and small-conductance Ca(2+)-activated K(+) channel, respectively) did not modify venom effect. On the other hand, glybenclamide, an inhibitor of ATP-sensitive K(+) channel abolished antinociception induced by the venom. Glybenclamide also inhibited the antinociceptive effect of [D-Pen(2.5)] enkephalin (DPDPE), a delta opioid receptor agonist, but did not modify the effect of (+)-trans-(1R,2R)-U-50488 (U50488), a kappa opioid receptor agonist. Diazoxide and pinacidil, two ATP-sensitive K(+) channel openers, injected by intraplantar route, induced a long-lasting increment of pain threshold of the animals and produced antinociception in both models of hyperalgesia. These results suggest that the antinociceptive effect of crotalid venom is mediated by activation of ATP-sensitive K(+) channels at peripheral afferent neurons.     

Delta-opioid receptor agonist SNC80 induces peripheral antinociception via activation of ATP-sensitive K+ channels

We investigated the effect of several K+ channel blockers on the antinociception induced by delta-opioid receptor agonist SNC80 using the paw pressure test, in which pain sensitivity is increased by an intraplantar injection (2 microg) of prostaglandin E2 (PGE2). Administration of SNC80 (20, 40 and 80 microg/paw) caused a decrease in the hyperalgesia induced by PGE2, in a dose-dependent manner. The possibility of higher dose of SNC80 (80 microg) causing a central or systemic effect was excluded since administration of the drug into the contralateral paw did not elicit antinociception in the right paw. Specific blockers of ATP-sensitive K+ channels, glibenclamide (20, 40 and 80 microg/paw) and tolbutamide (40, 80 and 160 microg/paw), antagonized the peripheral antinociception induced by SNC80 (80 microg). On the other hand, charybdotoxin (2 microg/paw), a large-conductance blocker of Ca(2+)-activated K+ channels, and dequalinium (50 microg/paw), a small conductance selective blocker of Ca(2+)-activated K+ channels, did not modify the effect of SNC80. This effect also remained unaffected by intraplantar administration of the voltage-dependent K+ channel blockers tetraethylammonium (30 microg/paw) and 4-aminopyridine (10 microg/paw), and of a non-specific K+ channel blocker, cesium (500 microg/paw). This study provides evidence that the peripheral antinociceptive effect of SNC80 result from the activation of ATP-sensitive K+ channels, and the other K+ channels are not involved.