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PubMed1998

PIP2 and PIP as determinants for ATP inhibition of KATP channels.

Adenosine triphosphate (ATP)-sensitive potassium (KATP) channels couple electrical activity to cellular metabolism through their inhibition by intracellular ATP. ATP inhibition of KATP channels varies among tissues and is affected by the metabolic and regulatory state of individual cells, suggesting involvement of endogenous factors. It is reported here that phosphatidylinositol-4, 5-bisphosphate (PIP2) and phosphatidylinositol-4-phosphate (PIP) controlled ATP inhibition of cloned KATP channels (Kir6.2 and SUR1). These phospholipids acted on the Kir6.2 subunit and shifted ATP sensitivity by several orders of magnitude. Receptor-mediated activation of phospholipase C resulted in inhibition of KATP-mediated currents. These results represent a mechanism for control of excitability through phospholipids.

ATP-Binding Cassette Transporters↗
PubMed1998

Membrane phospholipid control of nucleotide sensitivity of KATP channels.

Adenosine triphosphate (ATP)-sensitive potassium (KATP) channels couple cell metabolism to electrical activity. Phosphatidylinositol phosphates (PIPs) profoundly antagonized ATP inhibition of KATP channels when applied to inside-out membrane patches. It is proposed that membrane-incorporated PIPs can bind to positive charges in the cytoplasmic region of the channel's Kir6.2 subunit, stabilizing the open state of the channel and antagonizing the inhibitory effect of ATP. The tremendous effect of PIPs on ATP sensitivity suggests that in vivo alterations of membrane PIP levels will have substantial effects on KATP channel activity and hence on the gain of metabolism-excitation coupling.

ATP-Binding Cassette Transporters↗
PubMed2003

Absence of ischemic preconditioning protection in diabetic sheep hearts: role of sarcolemmal KATP channel dysfunction.

Sarcolemmal ATP-sensitive potassium (KATP) channels have been mentioned to participate in preconditioning protection. Since these channels are altered in diabetes, it would be possible that preconditioning does not develop in diabetic (D) hearts. The purpose of this study was to assess whether early (EP) and late (LP) ischemic preconditioning protect diabetic hearts against stunning in a conscious diabetic sheep model and whether diabetes might have altered KATP channel functioning. Sheep received alloxan monohydrate (1 g) and were ascribed to three experimental groups: control (DC, 12 min of ischemia (I) followed by 2 h of reperfusion (R)), early preconditioning (DEP, six 5 min I-5 min R periods were performed before the 12 min I) and late preconditioning (DLP, same as DEP except that the preconditioning stimulus was performed 24 h before the 12 min I). Regional mechanics during reperfusion was evaluated as the percent recovery of wall thickening fraction (%WTH) expressed as percentage of basal values (100%) and KATP behaviour was indirectly assessed by monophasic action potential duration (MAPD) and sensitivity to glibenclamide blockade (0.1 and 0.4 mg/Kg). The results were compared to those obtained in normal (N) sheep. EP and LP protected against stunning in normal sheep (%WTH: NC = 63 +/- 3.7, NLP = 80 +/- 5**, NEP = 78 +/- 3*, *p < 0.05 and **p < 0.01 against NC) whereas contrary results occurred in diabetic ones, where DLP (%WTH = 60 +/- 4) afforded a similar recovery to DC (%WTH = 54 +/- 5) and DEP surprisingly worsened instead of improving mechanical function (%WTH = 38 +/- 6, p < 0.01 against DC). KATP channel behaviour appeared altered in diabetic hearts as shown by MAPD during ischemia in normal sheep (153 +/- 9 msec) compared to diabetic ones (128 +/- 11 msec, p < 0.05) and by the sensitivity to glibenclamide (while 0.4 mg/Kg blocked action potential shortening in normal and diabetic animals, 0.1 mg/Kg completely blocked KATP in diabetic but not in normal hearts, p < 0.05). A sarcolemmal KATP channel dysfunction might afford a primary approach to explain the absence of ischemic preconditioning protection against stunning in diabetic sheep.

Action Potentials↗
PubMed1996

Direct preconditioning of cardiac ventricular myocytes via adenosine A1 receptor and KATP channel.

Both adenosine receptor and ATP-sensitive K (KATP) channel mediate the protective effect of ischemic preconditioning in the intact heart. The objective of the present study was to determine the role of adenosine receptor and KATP channel as well as their interaction in simulating and mediating preconditioning of the cardiac myocyte. Cardiac ventricular myocytes cultured from chick embryos 14 days in ovo were developed as a myocyte model of preconditioning. Myocytes were preconditioned by exposing them to 5-min hypoxia, termed preconditioning hypoxia, before a second 90-min hypoxia. Preconditioning resulted in a 64 +/- 3% decrease in the amount of creatine kinase released and a 66 +/- 2% reduction in the percentage of myocytes (+/-SE, n = 11) killed. Glibenclamide or 5-hydroxydecanoic acid (5-HD), when present during the preconditioning hypoxia, blocked the preconditioning effect. Prior exposure of the myocytes to pinacidil also led to a decrease in the injury sustained during the 90-min hypoxia. The protective effect of pinacidil was blocked by glibenclamide or 5-HD, suggesting that KATP channel activation can mimic as well as mediate preconditioning. Adenosine receptor antagonist 8-sulfophenyltheophylline (8-SPT) blocked the protective effect of preconditioning hypoxia. Adenosine or the A1 receptor agonist 2-chloro-N6-cyclopentyladenosine (CCPA) can replace preconditioning hypoxia and mimic preconditioning; this effect was fully antagonized by 8-SPT, glibenclamide, or 5-HD. Adenosine A1-receptor activation caused a glibenclamide-sensitive inhibition of the basal 45Ca influx and basal myocyte contractile amplitude, consistent with coupling of A1 receptor to stimulation of KATP channel in the myocytes. The data provide direct evidence that myocyte KATP channel is the effector downstream from adenosine A1 receptor in mediating the direct preconditioning of cardiac myocytes.

Adenosine Triphosphate↗
PubMed2004

Long-chain CoA esters activate human pancreatic beta-cell KATP channels: potential role in Type 2 diabetes.

AIMS/HYPOTHESIS: The ATP-regulated potassium (KATP) channel in the pancreatic beta cell couples the metabolic state to electrical activity. The primary regulator of the KATP channel is generally accepted to be changes in ATP/ADP ratio, where ATP inhibits and ADP activates channel activity. Recently, we showed that long-chain CoA (LC-CoA) esters form a new class of potent KATP channel activators in rodents, as studied in inside-out patches. METHODS: In this study we have investigated the effects of LC-CoA esters in human pancreatic beta cells using the inside-out and whole-cell configurations of the patch clamp technique. RESULTS: Human KATP channels were potently activated by acyl-CoA esters with a chain length exceeding 12 carbons. Activation by LC-CoA esters did not require the presence of Mg2+ or adenine nucleotides. A detailed characterization of the concentration-dependent relationship showed an EC50 of 0.7+/-0.1 micromol/l. Furthermore, in the presence of an ATP/ADP ratio of 10 (1.1 mmol/l total adenine nucleotides), whole-cell KATP channel currents increased approximately six-fold following addition of 1 micro mol/l LC-CoA ester. The presence of 1 micro mol/l LC-CoA in the recording pipette solution increased beta-cell input conductance, from 0.5+/-0.2 nS to 2.5+/-1.3 nS. CONCLUSION/INTERPRETATION: Taken together, these results show that LC-CoA esters are potent activators of the KATP channel in human pancreatic beta cells. The fact that LC-CoA esters also stimulate KATP channel activity recorded in the whole-cell configuration, points to the ability of these compounds to have an important modulatory role of human beta-cell electrical activity under both physiological and pathophysiological conditions.

Acyl Coenzyme A↗
PubMed1997

Control of rectification and gating of cloned KATP channels by the Kir6.2 subunit.

KATP channels are a functional complex of sulphonylurea receptor (SUR1, SUR2) and inward rectifier K+ (Kir6.1, Kir6.2) channel subunits. We have studied the role of the putative pore forming subunit (Kir6.2) in regulation of rectification and gating of KATP channels generated by transfection of SUR1 and Kir6.2 cDNAs in COSm6 cells. In the absence of internal polyvalent cations, the current-voltage relationship is sigmoidal. Mg2+ or spermine4+ (spm) each induces a mild inward rectification. Mutation of the asparagine at position 160 in Kir6.2 to aspartate (N160D) or glutamate (N160E) increases the degree of rectification induced by Mg2+ or spermine4+, whereas wild-type rectification is still observed after mutation to other neutral residues (alanine-N160A, glutamine-N160Q). These results are consistent with this residue lining the pore of the channel and contributing to the binding of these cations, as demonstrated for the equivalent site in homomeric ROMK1 (Kir1.1) channels. Since Kir6.2 contains no consensus ATP binding site, whereas SUR1 does, inhibition by ATP has been assumed to depend on interactions with SUR1. However, we found that the [ATP] causing half-maximal inhibition of current (Ki) was affected by mutation of N160. Channels formed from N160D or N160Q mutant subunits had lower apparent sensitivity to ATP (Ki,N160D = 46.1 microM; Ki,N160Q = 62.9 microM) than wild-type, N160E, or N160A channels (Ki = 10.4, 17.7, 6.4 microM, respectively). This might suggest that ATP binding to the channel complex was altered, although examination of channel open probabilities indicates instead that the residue at position 160 alters the ATP-independent open probability, i.e., it controls the free energy of the open state, thereby affecting the "coupling" of ATP binding to channel inhibition. The results can be interpreted in terms of a kinetic scheme whereby the residue at Kir6.2 position 160 controls the rate constants governing transitions to and from the open state, without directly affecting ATP binding or unbinding transitions.

Adenosine Triphosphate↗
PubMed2006

Cardiac and vascular KATP channels in rats are activated by endogenous epoxyeicosatrienoic acids through different mechanisms.

We have reported that epoxyeicosatrienoic acids (EETs), the cytochrome P450 (CYP) epoxygenase metabolites of arachidonic acid (AA), are potent sarcolemmal ATP-sensitive K+ (KATP) channel activators. However, activation of cardiac and vascular KATP channels by endogenously produced EETs under physiological intracellular conditions has not been demonstrated and direct comparison of the mechanisms whereby EETs activate the KATP channels in cardiac myocytes versus vascular smooth muscle cells has not been made. In this study, we examined the effects of AA on KATP channels in freshly isolated cardiac myocytes from rats, wild-type (WT) and transgenic mice overexpressing CYP2J2 cDNA, and mesenteric arterial smooth muscle cells from rats. We also compared the activation of cardiac and vascular KATP channels by extracellularly and intracellularly applied 11,12-EET. We found that 1 microm AA enhanced KATP channel activities in both cardiac and vascular smooth muscle cells, and the AA effects were inhibited by preincubation with CYP epoxygenase inhibitors. Baseline cardiac KATP current densities in CYP2J2 transgenic mice were 190% higher than those of WT mice, and both were reduced to similar levels by CYP epoxygenase inhibition. Western blot analysis showed that expression of Kir6.2 and SUR2A was similar between WT and CYP2J2 transgenic hearts. 11,12-EET (5 microm) applied intracellularly enhanced the KATP currents by 850% in cardiac myocytes, but had no effect in vascular smooth muscle cells. In contrast, 11,12-EET (5 microm) applied extracellularly increased KATP currents by 520% in mesenteric arterial smooth muscle cells, but by only 209% in cardiac myocytes. Preincubation with 100 microm m-iodobenzylguanidine or 5 microm myristoylated PKI amide did not alter the activation of cardiac KATP channels by 5 microm 11,12-EET, but significantly inhibited activation of vascular KATP channels. Moreover, EET only enhanced the inward component of cardiac KATP currents, but activated both the inward and outward components of vascular KATP currents. Our results indicate that endogenously derived CYP metabolites of AA potently activate cardiac and vascular KATP channels. EETs regulate cardiac electrophysiology and vascular tone by KATP channel activation, albeit through different mechanisms: the cardiac KATP channels are directly activated by EETs, whereas activation of the vascular KATP channels by EETs is protein kinase A dependent.

8,11,14-Eicosatrienoic Acid↗
PubMed1996

Diadenosine polyphosphate-induced inhibition of cardiac KATP channels: operative state-dependent regulation by a nucleoside diphosphate.

It has been proposed that the regulatory action of mononucleotides, such as ATP and UDP, on cardiac ATP-sensitive K+ (KATP) channels is determined by the state of the channel. Recently, dinucleotides, such as diadenosine tetraphosphate (Ap4A) and diadenosine pentaphosphate (Ap5A), have been recognized as novel intracellular ligands of cardiac KATP channels. However, it is not known whether the state of KATP channels also determines the response of the channel to dinucleotides. Therefore, we examined the action of diadenosine polyphosphates on KATP channel activity during different operative channel states, using the inside-out patch clamp technique applied to patches excised from guinea-pig ventricular myocytes. Spontaneous openings of KATP channels (or operative condition 1) were inhibited by Ap4A and Ap5A. Addition of UDP, which on its own did not affect spontaneous openings of KATP channels, prevented Ap4A and Ap5A to inhibit spontaneous KATP channel activity. In contrast, following "run-down" of spontaneous channel activity, UDP induced KATP channel openings (or operative condition 2), but could no longer antagonize the inhibitory effect of Ap4A and Ap5A. We conclude that the inhibitory action of diadenosine polyphosphates on KATP channels can be blocked by UDP only when KATP channels are in operative condition 1, but not in operative condition 2. Thus, the operative condition of KATP channels determines the UDP-mediated regulation of the diadenosine polyphosphate-dependent inhibitory channel gating. This finding further suggests that the operative state of the cardiac KATP channel protein is a critical determinant of the regulatory action of an intracellular ligand.

Animals↗
PubMed2003

Myocardial oxygenation and high-energy phosphate levels during KATP channel blockade.

Inhibition of ATP-sensitive K+ (KATP) channel activity has previously been demonstrated to result in coronary vasoconstriction with decreased myocardial blood flow and loss of phosphocreatine (PCr). This study was performed to determine whether the high-energy phosphate abnormality during KATP channel blockade can be ascribed to oxygen insufficiency. Myocardial blood flow and oxygen extraction were measured in open-chest dogs during KATP channel blockade with intracoronary glibenclamide, whereas high-energy phosphates were examined with 31P magnetic resonance spectroscopy (MRS), and myocardial deoxymyoglobin (Mb-delta) was determined with 1H MRS. Glibenclamide resulted in a 20 +/- 8% decrease of myocardial blood flow that was associated with a loss of phosphocreatine (PCr) and accumulation of inorganic phosphate. Mb-delta was undetectable during basal conditions but increased to 58 +/- 5% of total myoglobin during glibenclamide administration. This degree of myoglobin desaturation during glibenclamide was far greater than we previously observed during a similar reduction of blood flow produced by a coronary stenosis (22% of myoglobin deoxygenated during stenosis). The findings suggest that reduction of coronary blood flow with an arterial stenosis was associated with a decrease of myocardial energy demands and that this response to hypoperfusion was inhibited by KATP channel blockade.

Adenosine Triphosphate↗
PubMed1994

Effects of the KATP channel opener bimakalim on coronary blood flow, monophasic action potential duration, and infarct size in dogs.

BACKGROUND: The major purpose of the present study was to determine the effect of the potassium channel opener bimakalim, administered intracoronary only during the initial 10 minutes of ischemia, on myocardial infarct size in anesthetized dogs. A second aim was to test the possibility that bimakalim mediates its cardioprotective effects by accelerating the rate of myocyte action potential shortening during early ischemia. A third aim was to determine the relative potency of bimakalim to open coronary vascular ATP-regulated potassium (KATP) channels versus myocyte KATP channels. METHODS AND RESULTS: Barbital-anesthetized open-chest dogs were used. In the initial studies, bimakalim (0.1 to 10 micrograms/min) was infused into the left anterior descending coronary artery (LAD), and changes in coronary blood flow and monophasic action potential duration (MAPD) were used as indexes of coronary vascular KATP channel and myocyte KATP channel activity, respectively. In subsequent infarct studies, dogs were subjected to 60 minutes of LAD occlusion followed by 4 hours of reperfusion. Based on preliminary studies, two doses of bimakalim that did not shorten MAPD during nonischemic conditions (0.1 and 0.3 microgram/min) and one that markedly shortened MAPD during nonischemic conditions (3.0 micrograms/min) or an equal volume of vehicle were infused into the LAD during the initial 10 minutes of coronary artery occlusion. Transmural myocardial blood flow was measured at 5 and 30 minutes of occlusion by the radioactive microsphere technique, and infarct size was determined at the end of 4 hours of reperfusion by triphenyltetrazolium staining. The monophasic action potential duration at 50% repolarization (MAPD50) was measured by an epicardial probe placed in the center of the ischemic area. Bimakalim had an approximately 10-fold greater affinity for the coronary vascular than the myocardial KATP channel (ED50 coronary, approximately 0.3 microgram/min; ED50 myocyte, approximately 3.0 micrograms/min). Three doses of bimakalim (0.1, 0.3, and 3.0 micrograms/min) all markedly reduced infarct size expressed as percent of the area at risk (12.6 +/- 3.3%, 14.5 +/- 2.2%, and 14.2 +/- 5.3%, respectively, versus 27.2 +/- 5.7% in controls) to nearly equal extents. Subsequently, we found that the two higher doses of bimakalim (0.3 and 3.0 micrograms/min) markedly accelerated yet the 0.1-microgram/min dose of bimakalim did not significantly affect the ischemia-related shortening of the action potential during the initial 5 minutes of occlusion. In addition, 0.1 and 0.3 microgram/min bimakalim did not increase the incidence of ventricular fibrillation during the 60 minutes of occlusion (0 of 7 and 0 of 8 dogs, respectively), whereas 3.0 micrograms/min bimakalim had a profibrillatory effect (6 of 6) compared with the control group (1 of 8). There were no significant differences between groups in systemic hemodynamics, myocardial oxygen demand, ischemic bed size, or collateral blood flow to the ischemic region. CONCLUSIONS: The results of the present study clearly reveal that a small dose (0.1 or 0.3 microgram/min) of the KATP channel opener bimakalim administered only during the initial 10-minute period of ischemia markedly reduced myocardial infarct size to an extent equal to that of a higher profibrillatory dose in barbital-anesthetized dogs. These data also suggest that bimakalim and other potassium channel openers may partially exert their cardioprotective effects by accelerating KATP channel activation during early ischemia as evidenced by an enhanced rate of ischemic myocyte action potential shortening. However, the results also suggest that other cellular mechanisms may be involved in mediating the cardioprotection produced by a low dose of bimakalim (0.1 microgram/min) because it did not significantly accelerate the shortening of the action potential duration, yet it had an efficacy to redu BACKGROUND: The major purpose of the present study was to de

Action Potentials↗
PubMed1998

Toward understanding the assembly and structure of KATP channels.

Adenosine 5'-triphosphate-sensitive potassium (KATP) channels couple metabolic events to membrane electrical activity in a variety of cell types. The cloning and reconstitution of the subunits of these channels demonstrate they are heteromultimers of inwardly rectifying potassium channel subunits (KIR6.x) and sulfonylurea receptors (SUR), members of the ATP-binding cassette (ABC) superfamily. Recent studies indicate that SUR and KIR6.x associate with 1:1 stoichiometry to assemble a large tetrameric channel, (SUR/KIR6.x)4. The KIR6.x subunits form the channel pore, whereas SUR is required for activation and regulation. Two KIR6.x genes and two SUR genes have been identified, and combinations of subunits give rise to KATP channel subtypes found in pancreatic beta-cells, neurons, and cardiac, skeletal, and smooth muscle. Mutations in both the SUR1 and KIR6.2 genes have been shown to cause familial hyperinsulinism, indicating the importance of the pancreatic beta-cell channel in the regulation of insulin secretion. The availability of cloned KATP channel genes opens the way for characterization of this family of ion channels and identification of additional genetic defects.

ATP-Binding Cassette Transporters↗
PubMed1996

Effect of adenosine and intracellular GTP on KATP channels of mammalian skeletal muscle.

We investigated the action of adenosine and GTP on KATP channels, using inside-out patch clamp recordings from dissociated single fibers of rat flexor digitorum brevis (FDB) skeletal muscle. In excised patches, KATP channels could be activated by a combination of an extracellular adenosine agonist and intracellular Mg2+-ATP and GTP or GTP-gamma-S. The activation required hydrolyzable ATP and could be partially reversed with Mg2+, suggesting that it may involve a G-protein dependent phosphorylation of KATP channels. We found that KATP channels of the rat FDB could not be activated by Mg2+-ATP alone or by Mg2+-ATP in the presence of extracellular adenosine. Patches whose channel activity had been 'rundown' by Ca2+ could not be recovered by adenosine, GTP or Mg2+-ATP. KATP channels activated by adenosine receptor agonists had a similar ATP sensitivity to those under control conditions; but adenosine appears to be able to switch these KATP channels from an inactive to an active mode.

Adenosine↗
PubMed1996

Modulation of vascular KATP channels in hypothyroidism.

The role of vascular KATP channels in hypothyroidism-induced decrease in myogenic activity of rat portal vein was examined by using pharmacologically relevant concentrations of K+ channel ligands. As compared to controls, a significant decrease in the myogenic tone and noradrenaline (10(-9)-10(-5) M)-induced contractions was observed in portal veins from hypothyroid rats. In both euthyroid and hypothyroid states, pinacidil (10(-9)-10(-5) M) and cromakalim (10(-9)-10(-5) M) caused concentration-related inhibition of the myogenic tone (frequency and amplitude). However, hypothyroidism caused a leftward shift in the concentration-response curves of the K+ channel openers with a corresponding decrease in their IC50 values both in the absence and presence of the KATP channel blocker, glibenclamide (10(-7) M). Further, concentration-dependent increase in the frequency of myogenic tone by glibenclamide (10(-8)-3 x 10(-6) M) was greater in tissues from hypothyroid rats (EC50 = 2.07 x 10(-7) M; 95% CL, 1.06-4.05 x 10(-7) M) in comparison to controls (EC50 = 8.07 x 10(-7) M; 95% CL, 0.53-1.22 x 10(-6) M). These results suggest that a decrease in the myogenic tone of rat portal vein may possibly be related to an enhanced opening of the KATP channels in hypothyroidism.

Animals↗
PubMed1999

Altered functional properties of KATP channel conferred by a novel splice variant of SUR1.

1. ATP-sensitive potassium (KATP) channels are composed of pore-forming (Kir6.x) and regulatory sulphonylurea receptor (SURx) subunits. We have isolated a novel SUR variant (SUR1bDelta33) from a hypothalamic cDNA library. This variant lacked exon 33 and introduced a frameshift that produced a truncated protein lacking the second nucleotide binding domain (NBD2). It was expressed at low levels in hypothalamus, midbrain, heart and the insulin-secreting beta-cell line MIN6. 2. We examined the properties of KATP channels composed of Kir6.2 and SUR1bDelta33 by recording macroscopic currents in membrane patches excised from Xenopus oocytes expressing these subunits. We also investigated the effect of truncating SUR1 at either the start (SUR1bT1) or end (SUR1bT2) of exon 33 on KATP channel properties. 3. Kir6.2/SUR1bDelta33 showed an enhanced open probability (Po = 0.6 at -60 mV) and a reduced ATP sensitivity (Ki, 86 microM), when compared with wild-type channels (Po = 0.3; Ki, 22 microM). However, Kir6.2/SUR1bT1 and Kir6.2/SUR1bT2 resembled the wild-type channel in their Po and ATP sensitivity. 4. Neither MgADP, nor the K+ channel opener diazoxide, enhanced Kir6.2/SUR1bDelta33, Kir6.2/SUR1bT1 or Kir6.2/SUR1bT2 currents, consistent with the idea that these agents require an intact NBD2 for their action. Sulphonylureas blocked KATP channels containing any of the three SUR variants, but in excised patches the extent of block was less than that for the wild-type channel. In intact cells, the extent of sulphonylurea block of Kir6.2/SUR1bDelta33 was greater than that in excised patches and was comparable to that found for wild-type channels. 5. Our results demonstrate that NBD2 is not essential for functional expression or sulphonylurea block, but is required for KATP channel activation by K+ channel openers and nucleotides. Some of the unusual properties of Kir6.2/SUR1bDelta33 resemble those reported for the KATP channel of ventromedial hypothalamic (VMH) neurones, but the fact that this mRNA is expressed at low levels in many other tissues makes it less likely that SUR1bDelta33 serves as the SUR subunit for the VMH KATP channel.

ATP-Binding Cassette Transporters↗
PubMed2006

A key role for the subunit SUR2B in the preferential activation of vascular KATP channels by isoflurane.

BACKGROUND AND PURPOSE: It has been postulated that isoflurane, a volatile anaesthetic, produces vasodilatation through activation of ATP-sensitive K+ (KATP) channels. However, there is no direct evidence for the activation of vascular KATP channels by isoflurane. This study was conducted to examine the effect of isoflurane on vascular KATP channels and compare it with that on cardiac KATP channels. EXPERIMENTAL APPROACH: Effects of isoflurane on KATP channels were examined in aortic smooth muscle cells and cardiomyocytes of the mouse using patch clamp techniques. Effects of the anaesthetic on the KATP channels with different combinations of the inward rectifier pore subunits (Kir6.1 and Kir6.2) and sulphonylurea receptor subunits (SUR2A and SUR2B) reconstituted in a heterologous expression system were also examined. KEY RESULTS: Isoflurane increased the coronary flow in Langendorff-perfused mouse hearts in a concentration-dependent manner, which was abolished by 10 microM glibenclamide. In enzymically-dissociated aortic smooth muscle cells, isoflurane evoked a glibenclamide-sensitive current (i.e. KATP current). In isolated mouse ventricular cells, however, isoflurane failed to evoke the KATP current unless the KATP current was preactivated by the K+ channel opener pinacidil. Although isoflurane readily activated the Kir6.1/SUR2B channels (vascular type), the volatile anesthetic could not activate the Kir6.2/SUR2A channels (cardiac type) expressed in HEK293 cells. Isoflurane activated a glibenclamide-sensitive current in HEK293 cells expressing Kir6.2/SUR2B channels. CONCLUSION AND IMPLICATIONS: Isoflurane activates KATP channels in vascular smooth muscle cells and produces coronary vasodilation in mouse hearts. SUR2B may be important for the activation of vascular-type KATP channels by isoflurane.

ATP-Binding Cassette Transporters↗
PubMed1997

Opening of cardiac sarcolemmal KATP channels by dinitrophenol separate from metabolic inhibition.

Opening of ATP-sensitive K+ (KATP) channels by the uncoupler of oxidative phosphorylation, 2,4 dinitrophenol (DNP), has been assumed to be secondary to metabolic inhibition and reduced intracellular ATP levels. Herein, we present data which show that DNP (200 microM) can induce opening of cardiac KATP channels, under whole-cell and inside-out conditions, despite millimolar concentrations of ATP (1-2. 5 mm). DNP-induced currents had a single channel conductance (71 pS), inward rectification, reversal potential, and intraburst kinetic properties (open time constant, tauopen: 4.8 msec; fast closed time constant, tauclosed(f): 0.33 msec) characteristic of KATP channels suggesting that DNP did not affect the pore region of the channel, but may have altered the functional coupling of the ATP-dependent channel gating. A DNP analogue, with the pH-titrable hydroxyl replaced by a methyl group, could not open KATP channels. The pH-dependence of the effect of DNP on channel opening under whole-cell, cell-attached, and inside-out conditions suggested that transfer of protonated DNP across the sarcolemma is essential for activation of KATP channels in the presence of ATP. We conclude that the use of DNP for metabolic stress-induced KATP channel opening should be reevaluated.

Adenosine Triphosphate↗
PubMed1998

Independent trafficking of KATP channel subunits to the plasma membrane.

KATP channels are unique in requiring two distinct subunits (Kir6.2, a potassium channel subunit) and SUR1 (an ABC protein) for generation of functional channels. To examine the cellular trafficking of KATP channel subunits, green fluorescent protein (GFP) was tagged to the cytoplasmic N or C terminus of SUR1 and Kir6. 2 subunits and to the C terminus of a dimeric fusion between SUR1 and Kir6.2 (SUR1-Kir6.2). All tagged constructs generated functional channels with essentially normal properties when coexpressed with the relevant other subunit. GFP-tagged Kir6.2 (Kir6.2-GFP) showed perinuclear and plasma membrane fluorescence patterns when expressed alone or with SUR1, and a very similar pattern was observed when channel-forming SUR1-Kir6.2-GFP was expressed on its own. In contrast, whereas SUR1 (SUR1-GFP) also showed a perinuclear and plasma membrane fluorescence pattern when expressed alone, an apparently cytoplasmic fluorescence was observed when coexpressed with Kir6.2 subunits. The results indicate that Kir6.2 subunits traffic to the plasma membrane in the presence or absence of SUR1, in contradiction to the hypothesis that homomeric Kir6.2 channels are not observed because SUR1 is required as a chaperone to guide Kir6.2 subunits through the secretory pathway.

Animals↗
PubMed1998

Reconstituted human cardiac KATP channels: functional identity with the native channels from the sarcolemma of human ventricular cells.

ATP-sensitive potassium (KATP) channels in striated myocytes are heteromultimers of KIR6.2, a weak potassium inward rectifier, plus SUR2A, a low-affinity sulfonylurea receptor. We have cloned human KIR6.2 (huKIR6.2) and a huSUR2A that corresponds to the major, full-length splice variant identified by polymerase chain reaction analysis of human cardiac poly A+ mRNA. ATP- and glibenclamide-sensitive K+ channels were produced when both subunits were coexpressed in COSm6 and Chinese hamster ovary cells lacking endogenous KATP channels, but not when huSUR2A or huKIR6.2 were transfected alone. Recombinant channels activated by metabolic inhibition in cell-attached configuration or in inside-out patches with ATP-free internal solution were compared with sarcolemmal KATP channels in human ventricular cells. The single-channel conductance of approximately 80 pS measured at -40 mV in quasi-symmetrical approximately 150 mmol/L K+ solutions, the intraburst kinetics that were dependent on K+ driving force, and the weak inward rectification were indistinguishable for both channels. Similar to the native channels, huSUR2A/huKIR6.2 recombinant channels were inhibited by ATP at quasi-physiological free Mg2+ ( approximately 0. 7 mmol/L) or in the absence of Mg2+, with an apparent IC50 of approximately 20 micromol/L and a pseudo-Hill coefficient of approximately 1. They were "refreshed" by MgATP and stimulated by ADP in the presence of Mg2+ when inhibited by ATP. The huSUR2A/huKIR6.2 channels were stimulated by cromakalim and pinacidil in the presence of ATP and Mg2+ but were insensitive to diazoxide. The results suggest that reconstituted huSUR2A/huKIR6.2 channels represent KATP channels in sarcolemma of human cardiomyocytes and are an adequate experimental model with which to examine structure-function relationships, molecular physiology, and pharmacology of these channels from human heart.

ATP-Binding Cassette Transporters↗