Evidence against functional heteromultimerization of the KATP channel subunits Kir6.1 and Kir6.2

K(ATP) channels consist of pore-forming potassium inward rectifier (Kir6.x) subunits and sulfonylurea receptors (SURs). Although Kir6.1 or Kir6.2 coassemble with different SUR isoforms to form heteromultimeric functional K(ATP) channels, it is not known whether Kir6.1 and Kir6.2 coassemble with each other. To define the molecular identity of K(ATP) channels, we used adenoviral gene transfer to express wild-type and dominant-negative constructs of Kir6.1 and Kir6.2 in a heterologous expression system (A549 cells) and in native cells (rabbit ventricular myocytes). Dominant-negative (DN) Kir6.2 gene transfer suppressed current through heterologously expressed SUR2A + Kir6.2 channels. Conversely, DN Kir6.1 suppressed SUR2B + Kir6.1 current but had no effect on coexpressed SUR2A + Kir6. 2. We next probed the ability of Kir6.1 and Kir6.2 to affect endogenous K(ATP) channels in adult rabbit ventricular myocytes, using adenoviral vectors to achieve efficient gene transfer. Infection with the DN Kir6.2 virus for 72 h suppressed pinacidil-inducible K(ATP) current density measured by whole-cell patch clamp. However, there was no effect of infection with the DN Kir6.1 on the K(ATP) current. Based on these functional assays, we conclude that Kir6.1 and Kir6.2 do not heteromultimerize with each other and that Kir6.2 is the sole K(ATP) pore-forming subunit in the surface membrane of heart cells.     

Functional analysis of a structural model of the ATP-binding site of the KATP channel Kir6.2 subunit

ATP-sensitive potassium (KATP) channels couple cell metabolism to electrical activity by regulating K+ flux across the plasma membrane. Channel closure is mediated by ATP, which binds to the pore-forming subunit (Kir6.2). Here we use homology modelling and ligand docking to construct a model of the Kir6.2 tetramer and identify the ATP-binding site. The model is consistent with a large amount of functional data and was further tested by mutagenesis. Ligand binding occurs at the interface between two subunits. The phosphate tail of ATP interacts with R201 and K185 in the C-terminus of one subunit, and with R50 in the N-terminus of another; the N6 atom of the adenine ring interacts with E179 and R301 in the same subunit. Mutation of residues lining the binding pocket reduced ATP-dependent channel inhibition. The model also suggests that interactions between the C-terminus of one subunit and the 'slide helix' of the adjacent subunit may be involved in ATP-dependent gating. Consistent with a role in gating, mutations in the slide helix bias the intrinsic channel conformation towards the open state.     

Characterization of low-affinity binding sites for glibenclamide on the Kir6.2 subunit of the beta-cell KATP channel

The ATP-sensitive K+ channel, an octameric complex of two structurally unrelated types of subunits, SUR1 and Kir6.2, plays a central role in the physiological regulation of insulin secretion. The sulfonylurea glibenclamide, which trigger insulin secretion by blocking the ATP-sensitive K+ channel, interacts with both high and low affinity binding sites present on beta-cells. The high affinity binding site has been localized on SUR1 but the molecular nature of the low affinity site is still uncertain. In this study, we analyzed the pharmacology of glibenclamide in a transformed COS-7 cell line expressing the rat Kir6.2 cDNA and compared with that of the MIN6 beta cell line expressing natively both the Kir6.2 and the SUR1 subunits. Binding studies and Scatchard analysis revealed the presence of a single class of low affinity binding sites for glibenclamide on the COS/Kir6.2 cells with characteristics similar to that observed for the low affinity site of the MIN6 beta cells.     

3-D structural and functional characterization of the purified KATP channel complex Kir6.2-SUR1

ATP-sensitive potassium (K(ATP)) channels conduct potassium ions across cell membranes and thereby couple cellular energy metabolism to membrane electrical activity. Here, we report the heterologous expression and purification of a functionally active K(ATP) channel complex composed of pore-forming Kir6.2 and regulatory SUR1 subunits, and determination of its structure at 18 A resolution by single-particle electron microscopy. The purified channel shows ATP-ase activity similar to that of ATP-binding cassette proteins related to SUR1, and supports Rb(+) fluxes when reconstituted into liposomes. It has a compact structure, with four SUR1 subunits embracing a central Kir6.2 tetramer in both transmembrane and cytosolic domains. A cleft between adjacent SUR1s provides a route by which ATP may access its binding site on Kir6.2. The nucleotide-binding domains of adjacent SUR1 appear to interact, and form a large docking platform for cytosolic proteins. The structure, in combination with molecular modelling, suggests how SUR1 interacts with Kir6.2.     

The effects of E23K polymorphism in Kir6.2 subunit on insulin sensitivity in skeletal muscle cells by long-chain fatty acyl CoA

ATP-sensitive K⁺ (K_ATP) channels couple intermediary metabolism to cellular activity. Genetic disruption of these channels impairs glucose homeostasis. Similar effects occur from a single-nucleotide polymorphism of the Kir6.2 subunit seen in greater than 50% of the human population, which causes a point mutation of Glu23 to lysine. This E23K variant shows higher susceptibility to diabetes due to mechanisms that are not fully understood. This study was designed to examine the dysregulation of E23K on insulin sensitivity in the presence of long-chain fatty acyl CoA (LC-CoA), a major active form of free fatty acids. Physiological concentrations of LC-CoA decreased insulin sensitivity in E23K-transfected L6 muscle cells by increasing the activation of negative regulators in the insulin signaling pathway. LC-CoA also reduced IRS-1 and Akt phosphorylation and glucose transport. This effect was not due to the expression of the E23K mutant on cell membrane. Our results indicate that E23K could impair insulin sensitivity, thus predisposing E23K carriers to insulin resistance.     

Regulation of ATP-sensitive potassium channel subunit Kir6.2 expression in rat intestinal insulin-producing progenitor cells

We have reported that the combined expression of Pdx-1 (pancreatic duodenal homeobox 1) and Isl-1 (islet 1) enables immature rat enterocytes (IEC-6) to produce and release insulin. A key component regulating the release of insulin is the ATP-sensitive potassium channel subunit Kir6.2. To investigate the regulation of Kir6.2 gene expression, we assessed Kir6.2 expression in IEC-6 cells expressing Pdx-1 and/or Isl-1. We observed that Kir6.2 protein was expressed de novo in IEC-6 cells expressing both Pdx-1 and Isl-1 but not in cells expressing Pdx-1 alone. Next, we analyzed the regions of the Kir6.2 promoter (-1677/-45) by performing a luciferase assay and electrophoretic mobility shift assay. The results have demonstrated that Kir6.2 promoter possesses two regions regulating the promoter activity: a Foxa2-binding site (-1364 to -1210) and an Sp1/Sp3-binding site (-1035 to -939). The additional expression of Isl-1 in IEC-6 cells expressing Pdx-1 attenuated overexpression of Foxa2 protein and enhanced Kir6.2 expression. Finally, knockdown of Isl-1 using the iRNA technique resulted in decreased expression of Kir6.2 protein in a rat pancreatic beta-cell line (RIN-5F cells). These results indicate that expression of Kir6.2 in the rat intestine is moderated by Isl-1.     

The Antiepileptic Effect of the Glycolytic Inhibitor 2-Deoxy-d-Glucose is Mediated by Upregulation of KATP Channel Subunits Kir6.1 and Kir6.2

Metabolic modulation of neuronal excitability is becoming increasingly important as an antiepileptic therapy. It was reported that the glycolytic inhibitor 2-deoxy-d-glucose (2-DG) and the activation of the ATP-sensitive potassium ion channel (K_ATP channel) had an antiepileptic effect in models of epilepsy. To explore whether 2-DG exerts an antiepileptic effect through upregulation of the K_ATP channel subunits Kir6.1 and Kir6.2, the expression of these subunits in hippocampus of five groups of mice with pilocarpine-induced status epilepticus (SE) was evaluated. A seizure group with pilocarpine-kindling convulsions (EP) was compared to similar groups treated with high, medium, and low 2-DG concentrations (100–500 mg/kg) and a normal control group (Con). Kir6.1 and Kir6.2 mRNAs and proteins were analyzed at 4 h, 1 days (acute period), 7 days (latent period), 30, and 60 days (chronic period) following SE. In the seizure group (compared to the Con group), hippocampal expression of Kir6.1 and Kir6.2 increased dramatically at 1, 7, and 30 days, and was further increased after treatment with medium and high dose 2-DG (all P < 0.05). Our results suggest that 2-DG may exert an antiepileptic effect through up-regulation of mRNAs and protein levels of Kir6.1 and Kir6.2, which may therefore be used as molecular targets in the treatment of epilepsy with 2-DG.     

DEND mutation in Kir6.2 (KCNJ11) reveals a flexible N-terminal region critical for ATP-sensing of the KATP channel

ATP-sensitive K⁺-channels link metabolism and excitability in neurons, myocytes, and pancreatic islets. Mutations in the pore-forming subunit (Kir6.2; KCNJ11) cause neonatal diabetes, developmental delay, and epilepsy by decreasing sensitivity to ATP inhibition and suppressing electrical activity. Mutations of residue G53 underlie both mild (G53R,S) and severe (G53D) forms of the disease. All examined substitutions (G53D,R,S,A,C,F) reduced ATP-sensitivity, indicating an intolerance of any amino acid other than glycine. Surprisingly, each mutation reduces ATP affinity, rather than intrinsic gating, although structural modeling places G53 at a significant distance from the ATP-binding pocket. We propose that glycine is required in this location for flexibility of the distal N-terminus, and for an induced fit of ATP at the binding site. Consistent with this hypothesis, glycine substitution of the adjacent residue (Q52G) partially rescues ATP affinity of reconstituted Q52G/G53D channels. The results reveal an important feature of the noncanonical ATP-sensing mechanism of K_ATP channels.     

The influence of denervation on DNA content in skeletal muscle fibers

In this paper we describe a significant reduction of nuclear DNA content in skeletal muscle fibers after denervation. Some properties of an endogenous DNAase activity in normal and denervated muscle are also reported.     

The diaphragm is better protected from oxidative stress than hindlimb skeletal muscle during CLP-induced sepsis

Objectives: The aim of this study was to determine whether non-lethal sepsis induced by cecal ligation and puncture (CLP) modulates oxidative damage and enzymatic antioxidant defenses in diaphragm and hindlimb skeletal muscles (soleus and Extensor Digitorus Longus (EDL)).

Methods: Female Wistar rats were divided into four experimental groups: (1) control animals, (2) animals sacrificed 2?hours or (3) 7 days after CLP, and (4) sham-operated animals. At the end of the experimental procedure, EDL, soleus, and diaphragm muscles were harvested and 4-hydroxynonenal (HNE)-protein adducts and protein carbonyl contents were examined in relation to superoxide dismutase and catalase expression and activities.

Results: We observed that both non-respiratory oxidative (i.e. soleus) and glycolytic skeletal muscles (i.e. EDL) are more susceptible to sepsis-induced oxidative stress than diaphragm, as attested by an increase in 4-HNE protein adducts and carbonylated proteins after 2?hours of CLP only in soleus and EDL.

Discussion: These differences could be explained by higher basal enzymatic antioxidant activities in diaphragm compared to hindlimb skeletal muscles. Together, these results demonstrate that diaphragm is better protected from oxidative stress than hindlimb skeletal muscles during CLP-induced sepsis.     

Engineered interaction between SUR1 and Kir6.2 that enhances ATP sensitivity in KATP channels

The ATP-sensitive potassium (K(ATP)) channel consisting of the inward rectifier Kir6.2 and SUR1 (sulfonylurea receptor 1) couples cell metabolism to membrane excitability and regulates insulin secretion. Inhibition by intracellular ATP is a hallmark feature of the channel. ATP sensitivity is conferred by Kir6.2 but enhanced by SUR1. The mechanism by which SUR1 increases channel ATP sensitivity is not understood. In this study, we report molecular interactions between SUR1 and Kir6.2 that markedly alter channel ATP sensitivity. Channels bearing an E203K mutation in SUR1 and a Q52E in Kir6.2 exhibit ATP sensitivity ～100-fold higher than wild-type channels. Cross-linking of E203C in SUR1 and Q52C in Kir6.2 locks the channel in a closed state and is reversible by reducing agents, demonstrating close proximity of the two residues. Our results reveal that ATP sensitivity in K(ATP) channels is a dynamic parameter dictated by interactions between SUR1 and Kir6.2.     

Satellite cell content is specifically reduced in type II skeletal muscle fibers in the elderly

Satellite cells (SC) are essential for skeletal muscle growth and repair. Because sarcopenia is associated with type II muscle fiber atrophy, we hypothesized that SC content is specifically reduced in the type II fibers in the elderly. A total of eight elderly (E; 76 +/- 1 yr) and eight young (Y; 20 +/- 1 yr) healthy males were selected. Muscle biopsies were collected from the vastus lateralis in both legs. ATPase staining and a pax7-antibody were used to determine fiber type-specific SC content (i.e., pax7-positive SC) on serial muscle cross sections. In contrast to the type I fibers, the proportion and mean cross-sectional area of the type II fibers were substantially reduced in E vs. Y. The number of SC per type I fiber was similar in E and Y. However, the number of SC per type II fiber was substantially lower in E vs. Y (0.044 +/- 0.003 vs. 0.080 +/- 0.007; P < 0.01). In addition, in the type II fibers, the number of SC relative to the total number of nuclei and the number of SC per fiber area were also significantly lower in E. This study is the first to show type II fiber atrophy in the elderly to be associated with a fiber type-specific decline in SC content. The latter is evident when SC content is expressed per fiber or per fiber area. The decline in SC content might be an important factor in the etiology of type II muscle fiber atrophy, which accompanies the loss of skeletal muscle with aging.     

KATP channels of mouse skeletal muscle: mechanism of channel blockage by AMP-PNP

Single ATP-sensitive potassium channels (K _ATP channels) were studied in inside-out membrane patches excised from mouse skeletal muscle. Channel blockage by the non-hydrolysable ATP analogue AMP-PNP was investigated in the absence or presence of 1 mM MgCl₂ with K⁺-rich solutions bathing the internal membrane surface. Currents through single. K _ATP channels were recorded at –40 and +40 mV AMP-PNP (5 to 500 M; Li salt) reduced the open-probability p_o of K _ATP channels and decreased the single-channel currents at high nucleotide concentrations by approximately 10%. Half maximal reduction of p_o at –40 mV was observed at nucleotide concentrations of 29 M in the absence and of 39 M in the presence of Mg²⁺. The steepness of the AMP-PNP concentration-response curves was strongly affected by Mg²⁺, the Hill coefficients of the curves were 0.6 in the absence and 1.6 in the presence of 1 mM MgCl₂. The efficacies of channel blockage by AMP-PNP at –40 and ⁺40 mV were not significantly different. The results indicate that a K _ATP channel can bind more divalent Mg²⁺-complexes of AMP-PNP than trivalent protonated forms of the nucleotide and that channel blockage is hardly affected by the membrane electric field. To estimate the contribution of lithium ions to the observed results, we studied the effects of LiCl (0.8 to 10 mM) in the Mg²⁺-free solution on the single channel current i. At a Li⁺ concentration of 10 mM, i was hardly affected at –40 mV but reduced by a factor of 0.75 at +40 mV. The results are interpreted by a fast, voltage-dependent blockage of K _ATP channels by internal Li⁺ ions. Correspondence to: B. Neumcke     

Evidence for a higher glycolytic than oxidative metabolic activity in white matter of rat brain

Different values exist for glucose metabolism in white matter; it appears higher when measured as accumulation of 2-deoxyglucose than when measured as formation of glutamate from isotopically labeled glucose, possibly because the two methods reflect glycolytic and tricarboxylic acid (TCA) cycle activities, respectively. We compared glycolytic and TCA cycle activity in rat white structures (corpus callosum, fimbria, and optic nerve) to activities in parietal cortex, which has a tight glycolytic-oxidative coupling. White structures had an uptake of [(3)H]2-deoxyglucose in vivo and activities of hexokinase, glucose-6-phosphate isomerase, and lactate dehydrogenase that were 40-50% of values in parietal cortex. In contrast, formation of aspartate from [U-(14)C]glucose in awake rats (which reflects the passage of (14)C through the whole TCA cycle) and activities of pyruvate dehydrogenase, citrate synthase, alpha-ketoglutarate dehydrogenase, and fumarase in white structures were 10-23% of cortical values, optic nerve showing the lowest values. The data suggest a higher glycolytic than oxidative metabolism in white matter, possibly leading to surplus formation of pyruvate or lactate. Phosphoglucomutase activity, which interconverts glucose-6-phosphate and glucose-1-phosphate, was similar in white structures and parietal cortex ( approximately 3 nmol/mg tissue/min), in spite of the lower glucose uptake in the former, suggesting that a larger fraction of glucose is converted into glucose-1-phosphate in white than in gray matter. However, the white matter glycogen synthase level was only 20-40% of that in cortex, suggesting that not all glucose-1-phosphate is destined for glycogen formation.     

Altered glycolytic and oxidative capacities of skeletal muscle contribute to insulin resistance in NIDDM

Simoneau, Jean-Aimé, and David E. Kelley. Alteredglycolytic and oxidative capacities of skeletal muscle contribute toinsulin resistance in NIDDM. J. Appl.Physiol. 83(1): 166-171, 1997.The insulinresistance of skeletal muscle in glucose-tolerant obese individuals isassociated with reduced activity of oxidative enzymes and adisproportionate increase in activity of glycolytic enzymes. Becausenon-insulin-dependent diabetes mellitus (NIDDM) is a disordercharacterized by even more severe insulin resistance of skeletal muscleand because many individuals with NIDDM are obese, the present studywas undertaken to examine whether decreased oxidative and increasedglycolytic enzyme activities are also present in NIDDM. Percutaneousbiopsy of vatus lateralis muscle was obtained in eight lean (L) andeight obese (O) nondiabetic subjects and in eight obese NIDDM subjectsand was assayed for marker enzymes of the glycolytic[phosphofructokinase, glyceraldehyde phosphate dehydrogenase,hexokinase (HK)] and oxidative pathways [citrate synthase(CS), cytochrome-c oxidase], aswell as for a glycogenolytic enzyme (glycogen phosphorylase) and amarker of anaerobic ATP resynthesis (creatine kinase). Insulinsensitivity was measured by using the euglycemic clamp technique.Activity for glycolytic enzymes (phosphofructokinase, glyceraldehyephosphate dehydrogenase, HK) was highest in subjects with subjects with NIDDM, following the order of NIDDM > O > L, whereas maximumvelocity for oxidative enzymes (CS,cytochrome-c oxidase) was lowest in subjects with NIDDM. The ratio between glycolytic andoxidative enzyme activities within skeletal muscle correlatednegatively with insulin sensitivity. The HK/CS ratio had the strongestcorrelation (r = 0.60, P < 0.01) with insulinsensitivity. In summary, an imbalance between glycolytic and oxidativeenzyme capacities is present in NIDDM subjects and is more severe thanin obese or lean glucose-tolerant subjects. The altered ratio betweenglycolytic and oxidative enzyme activities found in skeletal muscle ofindividuals with NIDDM suggests that a dysregulation betweenmitochondrial oxidative capacity and capacity for glycolysis is animportant component of the expression of insulin resistance.

     

Plasticity of skeletal muscle: regenerating fibers adapt more rapidly than surviving fibers

The properties of mammalian skeletal muscle demonstrate a high degree of structural and functional plasticity as evidenced by their adaptability to an atypical site after cross-transplantation and to atypical innervation after cross-innervation. We tested the hypothesis that, regardless of fiber type, skeletal muscles composed of regenerating fibers adapt more readily than muscles composed of surviving fibers when placed in an atypical site with atypical innervation. Fast muscles of rats were autografted into the site of slow muscles or vice versa with the donor muscle innervated by the motor nerve to the recipient site. Surviving fibers in donor muscles were obtained by grafting with vasculature intact (vascularized muscle graft), and regenerating fibers were obtained by grafting with vasculature severed (free muscle graft). Our hypothesis was supported because 60 days after grafting, transposed muscles with surviving fibers demonstrated only a slight change from the contractile properties and fiber typing of donor muscles, whereas transplanted muscles with regenerating fibers demonstrated almost complete change to those of the muscle formerly in the atypical site.     

KATP channels in mesenchymal stromal stem cells: strong up-regulation of Kir6.2 subunits upon osteogenic differentiation

The promising use of mesenchymal stromal cells (MSC) in regenerative technologies accounts for necessity of detailed study of their physiology. Proliferation and differentiation of multipotent cells often involve changes in their metabolic state. In the present study, we analyzed the expression of ATP-sensitive potassium (K_ATP) channels in MSC and upon in vitro differentiation. K_ATP channels are present in many cells and regulate a variety of cellular functions by coupling cell metabolism with membrane potential. Kir6.1, Kir6.2 and SUR2A were expressed in undifferentiated MSC, whereas SUR2B and SUR1 were not detected on cDNA and protein level. Upon adipogenic differentiation Kir6.1 and SUR2A showed a significant reduction of the amount of mRNA by 84% and 95%, respectively, whereas Kir6.2 expression was unchanged. Osteogenic differentiation strongly up-regulated Kir6.2 mRNA (28-fold) whereas Kir6.1 and SUR2A showed no significant change in expression. Quantitative Western blot analysis and immunofluorescence staining confirmed the elevated expression of Kir6.2 upon osteogenic differentiation. Taken together, expression changes of K_ATP channels may contribute to in vitro differentiation of MSC and represent changes in the metabolic state of the developing tissue.     

Troponin subunit stoichiometry and content in rabbit skeletal muscle and myofibrils

The quantity and molar ratio of the three troponin subunits to actin were determined in rabbit psoas muscle, muscle homogenates (800 X g pellet), and purified myofibrils. Proteins were separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The quantities of the separated proteins were determined directly from the gel slices by amino acid analysis after correction for losses and background. The molar ratio of actin, troponin T, troponin I, and troponin C was found to be 6.99:1:05:1:04:0.92 in purified myofibrils and was not significantly different (p greater than 0.05) from those obtained from 800 X g pellets of muscle homogenates or intact muscle tissue. Isolated troponin purified by several different procedures also had a 1:1:1 subunit ratio although the variability was much greater than that found in myofibrils. The troponin content of rabbit psoas muscle and myofibrils was 91 +/- 16 and 770 +/- 110 pmol/mg, respectively.     

Regulation of the ATP-sensitive potassium channel subunit, Kir6.2, by a Ca2+-dependent protein kinase C

The activity of ATP-sensitive potassium (K(ATP)) channels is governed by the concentration of intracellular ATP and ADP and is thus responsive to the metabolic status of the cell. Phosphorylation of K(ATP) channels by protein kinase A (PKA) or protein kinase C (PKC) results in the modulation of channel activity and is particularly important in regulating smooth muscle tone. At the molecular level the smooth muscle channel is composed of a sulfonylurea subunit (SUR2B) and a pore-forming subunit Kir6.1 and/or Kir6.2. Previously, Kir6.1/SUR2B channels have been shown to be inhibited by PKC, and Kir6.2/SUR2B channels have been shown to be activated or have no response to PKC. In this study we have examined the modulation of channel complexes formed of the inward rectifier subunit, Kir6.2, and the sulfonylurea subunit, SUR2B. Using a combination of biochemical and electrophysiological techniques we show that this complex can be inhibited by protein kinase C in a Ca(2+)-dependent manner and that this inhibition is likely to be as a result of internalization. We identify a residue in the distal C terminus of Kir6.2 (Ser-372) whose phosphorylation leads to down-regulation of the channel complex. This inhibitory effect is distinct from activation which is seen with low levels of channel activity.     

Dihydropyridine binding of the calcium channel complex from skeletal muscle is modulated by subunit interaction.

The dihydropyridine-binding subunit alpha 1 of the calcium channel complex from rabbit skeletal muscle can be partially depleted from the alpha 2 delta beta-complex using wheat germ agglutinin-affinity chromatography. This depletion of the alpha 1 from the other subunits leads to a loss of dihydropyridine-binding, which can be fully reconstituted by repletion of the alpha 1 with the other subunits. Reassembly of these subunits results in an increase in the Kd and Bmax of the dihydropyridine-binding indicating that the non-dihydropyridine-binding subunits influence dihydropyridine-binding. The affinity of the alpha 1 subunit for the other subunits was determined to be approximately 35 nM. Since the free alpha 1 subunit will not bind to the beta subunit alone, there is evidence, given the selective partitioning of the beta subunit to the lectin-bound subunit pool, that either beta binds with higher affinity to the alpha 2 delta-complex than to the free alpha 1 subunit or that the bound alpha 1 creates or modulates beta-binding. This indicates a functional high affinity interaction between the dihydropyridine-binding alpha 1 subunit and the alpha 2 delta beta-complex.