ATP-sensitive K+ channels in pancreatic, cardiac, and vascular smooth muscle cells

ATP-sensitiveK⁺(K_ATP) channels are therapeutictargets for several diseases, including angina, hypertension, anddiabetes. This is because stimulation ofK_ATP channels is thought toproduce vasorelaxation and myocardial protection against ischemia,whereas inhibition facilitates insulin secretion. It is well known that native K_ATP channels are inhibitedby ATP and sulfonylurea (SU) compounds and stimulated by nucleotidediphosphates and K⁺channel-opening drugs (KCOs). Although these characteristics can beshared with K_ATP channels indifferent tissues, differences in properties among pancreatic, cardiac,and vascular smooth muscle (VSM) cells do exist in terms of the actionsproduced by such regulators. Recent molecular biology andelectrophysiological studies have provided useful information towardthe better understanding of K_ATPchannels. For example, native K_ATPchannels appear to be a complex of a regulatory protein containing theSU-binding site [sulfonylurea receptor (SUR)] and aninward-rectifying K⁺ channel(K_ir) serving as a pore-formingsubunit. Three isoforms of SUR (SUR1, SUR2A, and SUR2B) have beencloned and found to have two nucleotide-binding folds (NBFs). It seemsthat these NBFs play an essential role in conferring the MgADP and KCOsensitivity to the channel, whereas theK_ir channel subunit itselfpossesses the ATP-sensing mechanism as an intrinsic property. Themolecular structure of K_ATPchannels is thought to be a heteromultimeric (tetrameric) assembly ofthese complexes: K_ir6.2 with SUR1(SUR1/K_ir6.2, pancreatic type),K_ir6.2 with SUR2A(SUR2A/K_ir6.2, cardiac type), andK_ir6.1 with SUR2B(SUR2B/K_ir6.1, VSM type)[i.e.,(SUR/K_ir6.x)₄]. It remains to be determined what are the molecular connections betweenthe SUR and K_ir subunits thatenable this unique complex to work as a functionalK_ATP channel.

     

Dual regulation of the ATP-sensitive potassium channel by caffeine

ATP-sensitive potassium (K_ATP) channels couple cellular metabolic status to changes in membrane electrical properties. Caffeine (1,2,7-trimethylxanthine) has been shown to inhibit several ion channels; however, how caffeine regulates K_ATP channels was not well understood. By performing single-channel recordings in the cell-attached configuration, we found that bath application of caffeine significantly enhanced the currents of Kir6.2/SUR1 channels, a neuronal/pancreatic K_ATP channel isoform, expressed in transfected human embryonic kidney (HEK)293 cells in a concentration-dependent manner. Application of nonselective and selective phosphodiesterase (PDE) inhibitors led to significant enhancement of Kir6.2/SUR1 channel currents. Moreover, the stimulatory action of caffeine was significantly attenuated by KT5823, a specific PKG inhibitor, and, to a weaker extent, by BAPTA/AM, a membrane-permeable Ca²⁺ chelator, but not by H-89, a selective PKA inhibitor. Furthermore, the stimulatory effect was completely abrogated when KT5823 and BAPTA/AM were co-applied with caffeine. In contrast, the activity of Kir6.2/SUR1 channels was decreased rather than increased by caffeine in cell-free inside-out patches, while tetrameric Kir6.2LRKR368/369/370/371AAAA channels were suppressed regardless of patch configurations. Caffeine also enhanced the single-channel currents of recombinant Kir6.2/SUR2B channels, a nonvascular smooth muscle K_ATP channel isoform, although the increase was smaller. Moreover, bidirectional effects of caffeine were reproduced on the K_ATP channel present in the Cambridge rat insulinoma G1 (CRI-G1) cell line. Taken together, our data suggest that caffeine exerts dual regulation on the function of K_ATP channels: an inhibitory regulation that acts directly on Kir6.2 or some closely associated regulatory protein(s), and a sulfonylurea receptor (SUR)-dependent stimulatory regulation that requires cGMP-PKG and intracellular Ca²⁺-dependent signaling. phosphodiesterase; protein kinase; calcium; single channel; patch clamp     

Lung endothelial cell proliferation with decreased shear stress is mediated by reactive oxygen species

Acute cessation of flow (ischemia) leads to depolarization of the endothelial cell (EC) membrane mediated by K_ATP channels and followed by production of reactive oxygen species (ROS) from NADPH oxidase. We postulated that ROS are a signal for initiating EC proliferation associated with the loss of shear stress. Flow cytometry was used to identify proliferating CD31-positive pulmonary microvascular endothelial cells (mPMVECs) from wild-type, Kir6.2^–/–, and gp91^phox–/– mice. mPMVECs were labeled with PKH26 and cultured in artificial capillaries for 72 h at 5 dyn/cm² (flow adaptation), followed by 24 h of stop flow or continued flow. ROS production during the first hour of ischemia was markedly diminished compared with wild-type mice in both types of gene-targeted mPMVECs. Cell proliferation was defined as the proliferation index (PI). After 72 h of flow, >98% of PKH26-labeled wild-type mPMVECs were at a single peak (PI 1.0) and the proportion of cells in the S+G₂/M phases were at 5.8% on the basis of cell cycle analysis. With ischemia (24 h), PI increased to 2.5 and the ratio of cells in S+G₂/M phases were at 35%. Catalase, diphenyleneiodonium, and cromakalim markedly inhibited ROS production and cell proliferation in flow-adapted wild-type mPMVECs. Significant effects of ischemia were not observed in Kir6.2^–/– and gp91^phox–/– cells. ANG II activation of NADPH oxidase was unaffected by K_ATP gene deletion. Thus loss of shear stress in flow-adapted mPMVECs results in cell division associated with ROS generated by NADPH oxidase. This effect requires a functioning cell membrane K_ATP channel. cell signaling; ischemia; mechanotransduction; K_ATP channels; NADPH oxidase     

Role of Hsp90 in Biogenesis of the β-Cell ATP-sensitive Potassium Channel Complex

The pancreatic β-cell ATP-sensitive potassium (K_ATP) channel is a multimeric protein complex composed of four inwardly rectifying potassium channel (Kir6.2) and four sulfonylurea receptor 1 (SUR1) subunits. K_ATP channels play a key role in glucose-stimulated insulin secretion by linking glucose metabolism to membrane excitability. Many SUR1 and Kir6.2 mutations reduce channel function by disrupting channel biogenesis and processing, resulting in insulin secretion disease. To better understand the mechanisms governing K_ATP channel biogenesis, a proteomics approach was used to identify chaperone proteins associated with K_ATP channels. We report that chaperone proteins heat-shock protein (Hsp)90, heat-shock cognate protein (Hsc)70, and Hsp40 are associated with β-cell K_ATP channels. Pharmacologic inhibition of Hsp90 function by geldanamycin reduces, whereas overexpression of Hsp90 increases surface expression of wild-type K_ATP channels. Coimmunoprecipitation data indicate that channel association with the Hsp90 complex is mediated through SUR1. Accordingly, manipulation of Hsp90 protein expression or function has significant effects on the biogenesis efficiency of SUR1, but not Kir6.2, expressed alone. Interestingly, overexpression of Hsp90 selectively improved surface expression of mutant channels harboring a subset of disease-causing SUR1 processing mutations. Our study demonstrates that Hsp90 regulates biogenesis efficiency of heteromeric K_ATP channels via SUR1, thereby affecting functional expression of the channel in β-cell membrane.     

Four novel splice variants of sulfonylurea receptor 1

ATP-sensitiveK⁺ (K_ATP) channels are composed of pore-formingKir6.x subunits and regulatory sulfonylurea receptor (SUR) subunits. SURs are ATP-binding cassette proteins with two nucleotide-binding folds (NBFs) and binding sites for sulfonylureas, like glibenclamide, and for channel openers. Here we report the identification and functional characterization of four novel splice forms of guinea pigSUR1. Three splice forms originate from alternative splicing of theregion coding for NBF1 and lack exons 17 (SUR117), 19 (SUR119),or both (SUR11719). The fourth (SUR1C) is a COOH-terminal SUR1-fragment formed by exons 31-39 containing the last twotransmembrane segments and the COOH terminus of SUR1. RT-PCR analysisshowed that these splice forms are expressed in several tissues with strong expression of SUR1C in cardiomyocytes. Confocal microscopy usingenhanced green fluorescent protein-tagged SUR or Kir6.x did not provideany evidence for involvement of these splice forms in themitochondrial K_ATP channel. Only SUR1 and SUR117 showed high-affinity binding of glibenclamide (K_d 2 nM in the presence of 1 mM ATP) and formed functional K_ATPchannels upon coexpression with Kir6.2.

     

KATP channels in focus: Progress toward a structural understanding of ligand regulation

K_ATP channels are hetero-octameric complexes of four inward rectifying potassium channels, Kir6.1 or Kir6.2, and four sulfonylurea receptors, SUR1, SUR2A, or SUR2B from the ABC transporter family. This unique combination enables K_ATP channels to couple intracellular ATP/ADP ratios, through gating, with membrane excitability, thus regulating a broad range of cellular activities. The prominence of K_ATP channels in human physiology, disease, and pharmacology has long attracted research interest. Since 2017, a steady flow of high-resolution K_ATP cryoEM structures has revealed complex and dynamic interactions between channel subunits and their ligands. Here, we highlight insights from recent structures that begin to provide mechanistic explanations for decades of experimental data and discuss the remaining knowledge gaps in our understanding of K_ATP channel regulation.     

Evidence for Direct Physical Association between a K+ Channel (Kir6.2) and an ATP-Binding Cassette Protein (SUR1) Which Affects Cellular Distribution and Kinetic Behavior of an ATP-Sensitive K+ Channel

Structurally unique among ion channels, ATP-sensitive K⁺ (K_ATP) channels are essential in coupling cellular metabolism with membrane excitability, and their activity can be reconstituted by coexpression of an inwardly rectifying K⁺ channel, Kir6.2, with an ATP-binding cassette protein, SUR1. To determine if constitutive channel subunits form a physical complex, we developed antibodies to specifically label and immunoprecipitate Kir6.2. From a mixture of Kir6.2 and SUR1 in vitro-translated proteins, and from COS cells transfected with both channel subunits, the Kir6.2-specific antibody coimmunoprecipitated 38- and 140-kDa proteins corresponding to Kir6.2 and SUR1, respectively. Since previous reports suggest that the carboxy-truncated Kir6.2 can form a channel independent of SUR, we deleted 114 nucleotides from the carboxy terminus of the Kir6.2 open reading frame (Kir6.2ΔC37). Kir6.2ΔC37 still coimmunoprecipitated with SUR1, suggesting that the distal carboxy terminus of Kir6.2 is unnecessary for subunit association. Confocal microscopic images of COS cells transfected with Kir6.2 or Kir6.2ΔC37 and labeled with fluorescent antibodies revealed unique honeycomb patterns unlike the diffuse immunostaining observed when cells were cotransfected with Kir6.2-SUR1 or Kir6.2ΔC37-SUR1. Membrane patches excised from COS cells cotransfected with Kir6.2-SUR1 or Kir6.2ΔC37-SUR1 exhibited single-channel activity characteristic of pancreatic K_ATP channels. Kir6.2ΔC37 alone formed functional channels with single-channel conductance and intraburst kinetic properties similar to those of Kir6.2-SUR1 or Kir6.2ΔC37-SUR1 but with reduced burst duration. This study provides direct evidence that an inwardly rectifying K⁺ channel and an ATP-binding cassette protein physically associate, which affects the cellular distribution and kinetic behavior of a K_ATP channel.     

Carbamazepine promotes surface expression of mutant Kir6.2-A28V ATP-sensitive potassium channels by modulating Golgi retention and autophagy

Pancreatic β-cells express ATP-sensitive potassium (K_ATP) channels, consisting of octamer complexes containing four sulfonylurea receptor 1 (SUR1) and four Kir6.2 subunits. Loss of K_ATP channel function causes persistent hyperinsulinemic hypoglycemia of infancy (PHHI), a rare but debilitating condition if not treated. We previously showed that the sodium-channel blocker carbamazepine (Carb) corrects K_ATP channel surface expression defects induced by PHHI-causing mutations in SUR1. In this study, we show that Carb treatment can also ameliorate the trafficking deficits associated with a recently discovered PHHI-causing mutation in Kir6.2 (Kir6.2-A28V). In human embryonic kidney 293 or INS-1 cells expressing this mutant K_ATP channel (SUR1 and Kir6.2-A28V), biotinylation and immunostaining assays revealed that Carb can increase surface expression of the mutant K_ATP channels. We further examined the subcellular distributions of mutant K_ATP channels before and after Carb treatment; without Carb treatment, we found that mutant K_ATP channels were aberrantly accumulated in the Golgi apparatus. However, after Carb treatment, coimmunoprecipitation of mutant K_ATP channels and Golgi marker GM130 was diminished, and K_ATP staining was also reduced in lysosomes. Intriguingly, Carb treatment also simultaneously increased autophagic flux and p62 accumulation, suggesting that autophagy-dependent degradation of the mutant channel was not only stimulated but also interrupted. In summary, our data suggest that surface expression of Kir6.2-A28V K_ATP channels is rescued by Carb treatment via promotion of mutant K_ATP channel exit from the Golgi apparatus and reduction of autophagy-mediated protein degradation.     

Octameric Stoichiometry of the KATP Channel Complex

ATP-sensitive potassium (K_ATP) channels link cellular metabolism to electrical activity in nerve, muscle, and endocrine tissues. They are formed as a functional complex of two unrelated subunits—a member of the Kir inward rectifier potassium channel family, and a sulfonylurea receptor (SUR), a member of the ATP-binding cassette transporter family, which includes cystic fibrosis transmembrane conductance regulators and multidrug resistance protein, regulators of chloride channel activity. This recent discovery has brought together proteins from two very distinct superfamilies in a novel functional complex. The pancreatic K_ATP channel is probably formed specifically of Kir6.2 and SUR1 isoforms. The relationship between SUR1 and Kir6.2 must be determined to understand how SUR1 and Kir6.2 interact to form this unique channel. We have used mutant Kir6.2 subunits and dimeric (SUR1-Kir6.2) constructs to examine the functional stoichiometry of the K_ATP channel. The data indicate that the K_ATP channel pore is lined by four Kir6.2 subunits, and that each Kir6.2 subunit requires one SUR1 subunit to generate a functional channel in an octameric or tetradimeric structure.     

KATP channels depress force by reducing action potential amplitude in mouse EDL and soleus muscle

Although ATP-sensitive K⁺ (K_ATP) channel openers depress force, channel blockers have no effect. Furthermore, the effects of channel openers on single action potentials are quite small. These facts raise questions as to whether 1) channel openers reduce force via an activation of K_ATP channels or via some nonspecific effects and 2) the reduction in force by K_ATP channels operates by changes in amplitude and duration of the action potential. To answer the first question we tested the hypothesis that pinacidil, a channel opener, does not affect force during fatigue in muscles of Kir6.2^-/- mice that have no cell membrane K_ATP channel activity. When wild-type extensor digitorum longus (EDL) and soleus muscles were stimulated to fatigue with one tetanus per second, pinacidil increased the rate at which force decreased, prevented a rise in resting tension, and improved force recovery. Pinacidil had none of these effects in Kir6.2^-/- muscles. To answer the second question, we tested the hypothesis that the effects of K_ATP channels on membrane excitability are greater during action potential trains than on single action potentials, especially during metabolic stress such as fatigue. During fatigue, M wave areas of control soleus remained constant for 90 s, suggesting no change in action potential amplitude for half of the fatigue period. In the presence of pinacidil, the decrease in M wave areas became significant within 30 s, during which time the rate of fatigue also became significantly faster compared with control muscles. It is therefore concluded that, once activated, K_ATP channels depress force and that this depression involves a reduction in action potential amplitude. Kir6.2^-/- mice; pinacidil; action potential train; M wave     

ATP-sensitive potassium channel (K<Subscript>ATP</Subscript>channel) expression in the normal canine pancreas and in canine insulinomas

Background

Pancreatic beta cells express ATP-sensitive potassium (K_ATP) channels that are needed for normal insulin secretion and are targets for drugs that modulate insulin secretion. The K_ATP channel is composed of two subunits: a sulfonylurea receptor (SUR 1) and an inward rectifying potassium channel (Kir_6.2). K_ATP channel activity is influenced by the metabolic state of the cell and initiates the ionic events that precede insulin exocytosis. Although drugs that target the K_ATP channel have the expected effects on insulin secretion in dogs, little is known about molecular aspects of this potassium channel. To learn more about canine beta cell K_ATP channels, we studied K_ATP channel expression by the normal canine pancreas and by insulin-secreting tumors of dogs.

Results

Pancreatic tissue from normal dogs and tumor tissue from three dogs with histologically-confirmed insulinomas was examined for expression of K_ATP channel subunits (SUR1 and Kir_6.2) using RT-PCR. Normal canine pancreas expressed SUR1 and Kir_6.2 subunits of the K_ATP channel. The partial nucleotide sequences for SUR1 and Kir_6.2 obtained from the normal pancreas showed a high degree of homology to published sequences for other mammalian species. SUR1 and Kir_6.2 expression was observed in each of the three canine insulinomas examined. Comparison of short sequences from insulinomas with those obtained from normal pancreas did not reveal any mutations in either SUR1 or Kir_6.2 in any of the insulinomas.

Conclusion

Canine pancreatic K_ATP channels have the same subunit composition as those found in the endocrine pancreases of humans, rats, and mice, suggesting that the canine channel is regulated in a similar fashion as in other species. SUR1 and Kir_6.2 expression was found in the three insulinomas examined indicating that unregulated insulin secretion by these tumors does not result from failure to express one or both K_ATP channel subunits.

     

Carbamazepine as a Novel Small Molecule Corrector of Trafficking-impaired ATP-sensitive Potassium Channels Identified in Congenital Hyperinsulinism

ATP-sensitive potassium (K_ATP) channels consisting of sulfonylurea receptor 1 (SUR1) and the potassium channel Kir6.2 play a key role in insulin secretion by coupling metabolic signals to β-cell membrane potential. Mutations in SUR1 and Kir6.2 that impair channel trafficking to the cell surface lead to loss of channel function and congenital hyperinsulinism. We report that carbamazepine, an anticonvulsant, corrects the trafficking defects of mutant K_ATP channels previously identified in congenital hyperinsulinism. Strikingly, of the 19 SUR1 mutations examined, only those located in the first transmembrane domain of SUR1 responded to the drug. We show that unlike that reported for several other protein misfolding diseases, carbamazepine did not correct K_ATP channel trafficking defects by activating autophagy; rather, it directly improved the biogenesis efficiency of mutant channels along the secretory pathway. In addition to its effect on channel trafficking, carbamazepine also inhibited K_ATP channel activity. Upon subsequent removal of carbamazepine, however, the function of rescued channels was recovered. Importantly, combination of the K_ATP channel opener diazoxide and carbamazepine led to enhanced mutant channel function without carbamazepine washout. The corrector effect of carbamazepine on mutant K_ATP channels was also demonstrated in rat and human β-cells with an accompanying increase in channel activity. Our findings identify carbamazepine as a novel small molecule corrector that may be used to restore K_ATP channel expression and function in a subset of congenital hyperinsulinism patients.     

Exercise responsive genes measured in peripheral blood of women with Chronic Fatigue Syndrome and matched control subjects

Background

Electrophysiological data suggest that cardiac K_ATP channels consist of Kir6.2 and SUR2A subunits, but the distribution of these (and other K_ATP channel subunits) is poorly defined. We examined the localization of each of the K_ATP channel subunits in the mouse and rat heart.

Results

Immunohistochemistry of cardiac cryosections demonstrate Kir6.1 protein to be expressed in ventricular myocytes, as well as in the smooth muscle and endothelial cells of coronary resistance vessels. Endothelial capillaries also stained positive for Kir6.1 protein. Kir6.2 protein expression was found predominantly in ventricular myocytes and also in endothelial cells, but not in smooth muscle cells. SUR1 subunits are strongly expressed at the sarcolemmal surface of ventricular myocytes (but not in the coronary vasculature), whereas SUR2 protein was found to be localized predominantly in cardiac myocytes and coronary vessels (mostly in smaller vessels). Immunocytochemistry of isolated ventricular myocytes shows co-localization of Kir6.2 and SUR2 proteins in a striated sarcomeric pattern, suggesting t-tubular expression of these proteins. Both Kir6.1 and SUR1 subunits were found to express strongly at the sarcolemma. The role(s) of these subunits in cardiomyocytes remain to be defined and may require a reassessment of the molecular nature of ventricular K_ATP channels.

Conclusions

Collectively, our data demonstrate unique cellular and subcellular K_ATP channel subunit expression patterns in the heart. These results suggest distinct roles for K_ATP channel subunits in diverse cardiac structures.     

Sulfonylureas suppress the stimulatory action of Mg-nucleotides on Kir6.2/SUR1 but not Kir6.2/SUR2A KATP channels: A mechanistic study

Sulfonylureas, which stimulate insulin secretion from pancreatic β-cells, are widely used to treat both type 2 diabetes and neonatal diabetes. These drugs mediate their effects by binding to the sulfonylurea receptor subunit (SUR) of the ATP-sensitive K⁺ (K_ATP) channel and inducing channel closure. The mechanism of channel inhibition is unusually complex. First, sulfonylureas act as partial antagonists of channel activity, and second, their effect is modulated by MgADP. We analyzed the molecular basis of the interactions between the sulfonylurea gliclazide and Mg-nucleotides on β-cell and cardiac types of K_ATP channel (Kir6.2/SUR1 and Kir6.2/SUR2A, respectively) heterologously expressed in Xenopus laevis oocytes. The SUR2A-Y1206S mutation was used to confer gliclazide sensitivity on SUR2A. We found that both MgATP and MgADP increased gliclazide inhibition of Kir6.2/SUR1 channels and reduced inhibition of Kir6.2/SUR2A-Y1206S. The latter effect can be attributed to stabilization of the cardiac channel open state by Mg-nucleotides. Using a Kir6.2 mutation that renders the K_ATP channel insensitive to nucleotide inhibition (Kir6.2-G334D), we showed that gliclazide abolishes the stimulatory effects of MgADP and MgATP on β-cell K_ATP channels. Detailed analysis suggests that the drug both reduces nucleotide binding to SUR1 and impairs the efficacy with which nucleotide binding is translated into pore opening. Mutation of one (or both) of the Walker A lysines in the catalytic site of the nucleotide-binding domains of SUR1 may have a similar effect to gliclazide on MgADP binding and transduction, but it does not appear to impair MgATP binding. Our results have implications for the therapeutic use of sulfonylureas.     

ATP-sensitive potassium channel (KATP channel) expression in the normal canine pancreas and in canine insulinomas

Background  

Pancreatic beta cells express ATP-sensitive potassium (K_ATP) channels that are needed for normal insulin secretion and are targets for drugs that modulate insulin secretion. The K_ATP channel is composed of two subunits: a sulfonylurea receptor (SUR 1) and an inward rectifying potassium channel (Kir_6.2). K_ATP channel activity is influenced by the metabolic state of the cell and initiates the ionic events that precede insulin exocytosis. Although drugs that target the K_ATP channel have the expected effects on insulin secretion in dogs, little is known about molecular aspects of this potassium channel. To learn more about canine beta cell K_ATP channels, we studied K_ATP channel expression by the normal canine pancreas and by insulin-secreting tumors of dogs.     

The Nucleotide-Binding Sites of SUR1: A Mechanistic Model

ATP-sensitive potassium (K_ATP) channels comprise four pore-forming Kir6.2 subunits and four modulatory sulfonylurea receptor (SUR) subunits. The latter belong to the ATP-binding cassette family of transporters. K_ATP channels are inhibited by ATP (or ADP) binding to Kir6.2 and activated by Mg-nucleotide interactions with SUR. This dual regulation enables the K_ATP channel to couple the metabolic state of a cell to its electrical excitability and is crucial for the K_ATP channel’s role in regulating insulin secretion, cardiac and neuronal excitability, and vascular tone. Here, we review the regulation of the K_ATP channel by adenine nucleotides and present an equilibrium allosteric model for nucleotide activation and inhibition. The model can account for many experimental observations in the literature and provides testable predictions for future experiments.     

Burst Kinetics of Co-expressed Kir6.2/SUR1 Clones: Comparison of Recombinant with Native ATP-sensitive K+ Channel Behavior

Co-expression of clones encoding Kir6.2, a K⁺ inward rectifier, and SUR1, a sulfonylurea receptor, reconstitutes elementary features of ATP-sensitive K⁺ (K_ATP) channels. However, the precise kinetic properties of Kir6.2/SUR1 clones remain unknown. Herein, intraburst kinetics of Kir6.2/SUR1 channel activity, heterologously co-expressed in COS cells, displayed mean closed times from 0.7 ± 0.1 to 0.4 ± 0.03 msec, and from 0.4 ± 0.1 to 2.0 ± 0.2 msec, and mean open times from 1.9 ± 0.4 to 4.5 ± 0.8 msec, and from 12.1 ± 2.4 to 5.0 ± 0.2 msec between −100 and −20 mV, and +20 to +80 mV, respectively. Burst duration for Kir6.2/SUR1 activity was 17.9 ± 1.8 msec with 5.6 ± 1.5 closings per burst. Burst kinetics of the Kir6.2/SUR1 activity could be fitted by a four-state kinetic model defining transitions between one open and three closed states with forward and backward rate constants of 1905 ± 77 and 322 ± 27 sec⁻¹ for intraburst, 61.8 ± 6.6 and 23.9 ± 5.8 sec⁻¹ for interburst, 12.4 ± 6.0 and 13.6 ± 2.9 sec⁻¹ for intercluster events, respectively. Intraburst kinetic properties of Kir6.2/SUR1 clones were essentially indistinguishable from pancreatic or cardiac K_ATP channel phenotypes, indicating that intraburst kinetics per se were insufficient to classify recombinant Kir6.2/SUR1 amongst native K_ATP channels. Yet, burst kinetic behavior of Kir6.2/SUR1 although similar to pancreatic, was different from that of cardiac K_ATP channels. Thus, expression of Kir6.2/SUR1 proteins away from the pancreatic micro-environment, confers the burst kinetic identity of pancreatic, but not cardiac K_ATP channels. This study reports the kinetic properties of Kir6.2/SUR1 clones which could serve in the further characterization of novel K_ATP channel clones. Received: 12 March 1997/Revised: 5 May 1997     

Dexmedetomidine directly inhibits vascular ATP-sensitive potassium channels

AimsDexmedetomidine is reported to have an effect on peripheral vasoconstriction; however, the exact mechanisms underlying this process are unclear. In this study, we hypothesized that dexmedetomidine-induced inhibition of vascular ATP-sensitive K⁺ (K_ATP) channels may be associated with this vasoconstriction. To test this hypothesis, we investigated the effects of dexmedetomidine on vascular K_ATP-channel activity at the single-channel level.Main methodsWe used cell-attached and inside-out patch-clamp configurations to examine the effects of dexmedetomidine on the activities of native rat vascular K_ATP channels, recombinant K_ATP channels with different combinations of various inwardly rectifying potassium channels (Kir6.0 family: Kir6.1, 6.2) and sulfonylurea receptor subunits (SUR1, 2A, 2B), and SUR-deficient channels derived from a truncated isoform of Kir6.2 subunit, namely, Kir6.2ΔC36 channels.Key findingsDexmedetomidine was observed to inhibit the native rat vascular K_ATP channels in both cell-attached and inside-out configurations. This drug also inhibited the activity of all types of recombinant SUR/Kir6.0 K_ATP channels as well as Kir6.2ΔC36 channels with equivalent potency.SignificanceThese results indicate that dexmedetomidine directly inhibits K_ATP channels through the Kir6.0 subunit.     

ATP-sensitive potassium channels mediate hyperosmotic stimulation of NKCC in slow-twitch muscle

In mildly hyperosmotic medium, activation of the Na⁺-K⁺-2Cl^- cotransporter (NKCC) counteracts skeletal muscle cell water loss, and compounds that stimulate protein kinase A (PKA) activity inhibit the activation of the NKCC. The aim of this study was to determine the mechanism for PKA inhibition of NKCC activity in resting skeletal muscle. Incubation of rat slow-twitch soleus and fast-twitch plantaris muscles in isosmotic medium with the PKA inhibitors H-89 and KT-5720 caused activation of the NKCC only in the soleus muscle. NKCC activation caused by PKA inhibition was insensitive to MEK MAPK inhibitors and to insulin but was abolished by the PKA stimulators isoproterenol and forskolin. Furthermore, pinacidil [an ATP-sensitive potassium (K_ATP) channel opener] or inhibition of glycolysis increased NKCC activity in the soleus muscle but not in the plantaris muscle. Preincubation of the soleus muscle with glibenclamide (a K_ATP channel inhibitor) prevented the NKCC activation by hyperosmolarity, PKA inhibition, pinacidil, and glycolysis inhibitors. In contrast, glibenclamide stimulated NKCC activity in the plantaris muscle. In cells stably transfected with the Kir6.2 subunit of the of K_ATP channel, inhibition of glycolysis activated potassium current and NKCC activity. We conclude that activation of K_ATP channels in slow-twitch muscle is necessary for activation of the NKCC and cell volume restoration in hyperosmotic conditions. protein kinase A; glibenclamide; glycolysis; Na⁺-K⁺-2Cl^- cotransporter; Kir6.2     

Pinacidil suppresses contractility and preserves energy but glibenclamide has no effect during muscle fatigue

The effects of 10 µM glibenclamide, anATP-sensitive K⁺ (K_ATP) channelblocker, and 100 µM pinacidil, a channel opener, were studied todetermine how the K_ATP channel affects mouse extensor digitorum longus (EDL) and soleus muscle during fatigue. Fatigue waselicited with 200-ms-long tetanic contractions every second. Glibenclamide did not affect rate and extent of fatigue, force recovery, or ⁸⁶Rb⁺ fractional loss. The onlyeffects of glibenclamide during fatigue were: an increase in restingtension (EDL and soleus), a depolarization of the cell membrane, aprolongation of the repolarization phase of action potential, and agreater ATP depletion in soleus. Pinacidil, on the other hand,increased the rate but not the extent of fatigue, abolished the normalincrease in resting tension during fatigue, enhanced force recovery,and increased ⁸⁶Rb⁺ fractional loss in both theEDL and soleus. During fatigue, the decreases in ATP andphosphocreatine of soleus muscle were less in the presence ofpinacidil. The glibenclamide effects suggest that fatigue, elicitedwith intermittent contractions, activates few K_ATP channelsthat affect resting tension and membrane potentials but not tetanicforce, whereas opening the channel with pinacidil causes a fasterdecrease in tetanic force, improves force recovery, and helps inpreserving energy.