Open Probability Of KATP Channels Research Articles

Calcium dynamics control K-ATP channel-mediated bursting in substantia nigra dopamine neurons: a combined experimental and modeling study.

Burst firing in medial substantia nigra (mSN) dopamine (DA) neurons has been selectively linked to novelty-induced exploration behavior in mice. Burst firing in mSN DA neurons, in contrast to lateral SN DA neurons, requires functional ATP-sensitive potassium (K-ATP) channels both in vitro and in vivo. However, the precise role of K-ATP channels in promoting burst firing is unknown. We show experimentally that L-type calcium channel activity in mSN DA neurons enhances open probability of K-ATP channels. We then generate a mathematical model to study the role of Ca2+ dynamics driving K-ATP channel function in mSN DA neurons during bursting. In our model, Ca2+ influx leads to local accumulation of ADP due to Ca-ATPase activity, which in turn activates K-ATP channels. If K-ATP channel activation reaches levels sufficient to terminate spiking, rhythmic bursting occurs. The model explains the experimental observation that, in vitro, coapplication of NMDA and a selective K-ATP channel opener, NN414, is required to elicit bursting as follows. Simulated NMDA receptor activation increases the firing rate and the rate of Ca2+ influx, which increases the activation of K-ATP. The model suggests that additional sources of hyperpolarization, such as GABAergic synaptic input, are recruited in vivo for burst termination or rebound burst discharge. The model predicts that NN414 increases the sensitivity of the K-ATP channel to ADP, promoting burst firing in vitro, and that that high levels of Ca2+ buffering, as might be expected in the calbindin-positive SN DA neuron subpopulation, promote rhythmic bursting pattern, consistent with experimental observations in vivo. NEW & NOTEWORTHY Recently identified distinct subpopulations of midbrain dopamine neurons exhibit differences in their two primary activity patterns in vivo: tonic (single spike) firing and phasic bursting. This study elucidates the biophysical basis of bursts specific to dopamine neurons in the medial substantia nigra, enabled by ATP-sensitive K+ channels and necessary for novelty-induced exploration. A better understanding of how dopaminergic signaling differs between subpopulations may lead to therapeutic strategies selectively targeted to specific subpopulations.

Cardiac KATP channel modulation by 16Hz magnetic fields - A theoretical study.

Heart exposure to 16Hz magnetic fields (MFs) was shown to be cardio-protective for diseased hearts; still, the mechanism of this effect is unknown. We hypothesize that a possible one mechanism is an increased trans-membrane KATP channel open probability due to modulation of the degree of dissociation between K+ ions, having a resonance frequency of 16Hz, and the channel selectivity filter. The Fan-Makielski Markovian KATP channel model was adopted, and the MF bio-effect was manifested by modulating the open probability of the channel using the predictive MF bio-effect parameter based on Binhi's quantum mechanics model. The model was integrated in a ventricular single cell model and the MF effect on the calcium transients [Ca2+] was assessed. Periodic pacing (Cycle Length CL=1sec) was applied and a 16Hz or 32Hz MF was turned on at t=0 for 10min. MF exposure gradually decreased [Ca2+] due to KATP channel opening, more strongly at 16Hz. Additionally, a small negative diastolic shift was observed. These numerical results demonstrated similarity to published experimental data using similar 16Hz MF exposure. We conclude that 16Hz MF exposure increases the KATP channel open probability, lowering the cellular calcium load. Our model could be integrated in a tissue model to predict optimal MF parameters for future cardiac therapy devices.

The Molecular Basis for Heme Modulation of KATP Channels

Heme, iron protoporphyrin IX, is a vital prosthetic component of a number of functional hemoproteins playing essential roles in a diverse range of biological actions including oxygen transport, electron transport, catalysis and gene regulation. An emerging role of heme is its ability to regulate the activity of ion channels including voltage gated K+ (Kv), large conductance Ca2+-activated K+ (BK) and epithelial Na+ (ENaC) channels. Here we report heme regulation of the ATP-sensitive K+ (KATP) channel in both cardiac myocytes and HEK293 cells heterologously expressing Kir6.2 and SUR2A subunits. The KATP channel is sensitive to the intracellular nucleotides ATP and ADP. The KATP channel links cellular metabolic state and excitability most notably during ischaemic stress. ATP acts with high affinity upon the cytosolic face to inhibit opening, thus the channel opens during periods of depleted cellular energy.Whole-cell KATP currents of both ventricular myocytes and HEK293 cells expressing KATP channels were increased upon application of 500 nM hemin extracellularly. In inside-out patches, KATP channel activity was reduced in the presence of ATP (500 µM), but on subsequent addition of hemin (500 nM) KATP channel open probability significantly increased from 0.024 ± 0.013 to 0.110 ± 0.028 (n = 6, P < 0.01).Sequence alignments with known heme binding regions revealed a structurally unresolved region located on an intracellular linker in the non-pore forming SUR2A subunit containing the residues C628XXH631 and H648, which was analogous to the reported Kv1.4 heme binding sequence. Mutating these residues (C628S, H631A and H648A) led to reduced sensitivity of the resulting KATP channels to heme. Mutagenesis of each residue revealed C628 and H648 to have the greatest effect at reducing the agonistic effects of heme. Here we provide evidence for heme binding and regulation of KATP

Imaging energy status in live cells with a fluorescent biosensor of the intracellular ATP-to-ADP ratio

The ATP:ADP ratio is a critical parameter of cellular energy status that regulates many metabolic activities. Here we report an optimized genetically-encoded fluorescent biosensor, PercevalHR, that senses the ATP:ADP ratio. PercevalHR is tuned to the range of intracellular ATP:ADP expected in mammalian cells, and it can be used with one- or two-photon microscopy in live samples. We use PercevalHR to visualize activity-dependent changes in ATP:ADP when neurons are exposed to multiple stimuli, demonstrating that it is a sensitive reporter of physiological changes in energy consumption and production. We also use PercevalHR to visualize intracellular ATP:ADP while simultaneously recording currents from ATP-sensitive potassium (KATP) channels in single cells, showing that PercevalHR enables the study of coordinated variation in ATP:ADP and KATP channel open probability in intact cells. With its ability to monitor changes in cellular energetics within seconds, PercevalHR should be a versatile tool for metabolic research.

Insulin Hypersecretion in Islets From Diet-Induced Hyperinsulinemic Obese Female Mice Is Associated With Several Functional Adaptations in Individual β-Cells

Insulin resistance and hyperinsulinemia are generally associated with obesity. Obese nondiabetic individuals develop a compensatory β-cell response to adjust insulin levels to the increased demand, maintaining euglycemia. Although several studies indicate that this compensation relies on structural changes, the existence of β-cell functional adaptations is incompletely understood. Here, we fed female mice with a high-fat diet (HFD) for 12 weeks. These animals became obese, hyperinsulinemic, insulin-resistant, and mildly glucose-intolerant while fed, and fasting glycemia was comparable in HFD and control mice. Islets from HFD animals exhibited increased β-cell mass and hypertrophy. Additionally, they had enhanced insulin gene expression and content and augmented glucose-induced insulin secretion. Electrophysiological examination of β-cells from both groups showed no differences in KATP channel open probability and conductance. However, action potentials elicited by glucose had larger amplitude in obese mice. Glucose-induced Ca²⁺ signals in intact islets, in isolated β-cells, and individual β-cells within islets were also increased in HFD mice. Additionally, a higher proportion of glucose-responsive cells was present in obese mice. In contrast, whole-cell Ca²⁺ current densities were similar in both groups. Capacitance measurements showed that depolarization-evoked exocytosis was enhanced in HFD β-cells compared with controls. Although this augment was not significant when capacitance increases of the whole β-cell population were normalized to cell size, the exocytotic output varied significantly when β-cells were distributed by size ranges. All these findings indicate that β-cell functional adaptations are present in the islet compensatory response to obesity.

Enhanced cardiac KATP channel activity after acclimation to hypoxia in goldfish (Carassius auratus L.)

Freshwater fishes of the genus Carassius are known for their capacity to survive environmental hypoxia. Further, individual animals may exhibit enhanced tolerance of low oxygen in response to prior acclimation. It has been suggested that the hypoxia‐induced activation of ATP‐sensitive potassium (KATP) channels in critical tissues serves a protective role, increasing tolerance of low oxygen. In goldfish, we have previously shown that mRNA transcription of genes relevant to cardiac KATP channel structure and regulation are altered in response to acclimation under moderately hypoxic conditions (2.6 mg O2/l for seven days at 22°C). In the present study, we used standard intracellular and patch clamp recording techniques to investigate the possibility that this altered transcription is accompanied by functional changes that increase overall KATP channels activity. Intracellular action potential duration in excised ventricles from hypoxia‐acclimated animals was significantly (p&lt;0.05) reduced at both 50% and 90% of full repolarization relative to those from normoxia‐acclimated fish. In cell‐attached ventricular membrane patches from hypoxia‐acclimated goldfish, sarcolemmal KATP channel open probability (NPo) was significantly (p&lt;0.05) enhanced vs. control. These data indicate that prior whole‐animal acclimation to moderate hypoxia enhances overall cardiac KATP channel activity in this species.

Cardiac KATP channel alterations associated with acclimation to hypoxia in goldfish (Carassius auratus L.)

Goldfish (Carassius auratus L.) are highly tolerant of environmental hypoxia, and with appropriate acclimation may survive and remain active for several days in the complete absence of oxygen. Previous work suggests that the hypoxia-induced activation of cardiac ATP-sensitive potassium (KATP) channels serves to increase tolerance of low oxygen in many species. For goldfish, we have previously characterized a nitric oxide (NO)- and cGMP-dependent pathway by which this channel activation occurs in acute hypoxia. The purpose of the present study was to resolve alterations in KATP channel activity and relevant gene expression in response to acclimation under moderately hypoxic conditions (2.6mg O2/L for seven days at 22°C). Intracellular action potential duration in excised ventricles from hypoxia-acclimated animals was significantly (p<0.05) reduced at both 50% and 90% of full repolarization relative to those from normoxia-acclimated fish. In cell-attached ventricular membrane patches from hypoxia-acclimated goldfish, sarcolemmal KATP channel open probability (NPo) was significantly enhanced vs. control. Of the two genes coding for the pore-forming subunits of cardiac KATP channels (Kir6.1 and Kir6.2), mRNA transcription of kcnj8 (revealed by quantitative real-time PCR) was unchanged while kcnj11 was downregulated in response to chronic low oxygen. The mRNA levels for hif1a (hypoxia inducible factor 1α) in the hearts of hypoxia-acclimated fish were significantly enhanced, as was nitric oxide synthase (nos2) and the sulfonylurea receptor regulatory subunit (sur2, abcc9). These data suggest that prior whole-animal acclimation to chronic hypoxia enhances cardioprotective sarcolemmal KATP currents by altering transcription of regulatory proteins.

Ankyrin-B Regulates Kir6.2 Membrane Expression and Function in Heart

Ankyrin polypeptides are critical for normal membrane protein expression in diverse cell types, including neurons, myocytes, epithelia, and erythrocytes. Ankyrin dysfunction results in defects in membrane expression of ankyrin-binding partners (including ion channels, transporters, and cell adhesion molecules), resulting in aberrant cellular function and disease. Here, we identify a new role for ankyrin-B in cardiac cell biology. We demonstrate that cardiac sarcolemmal K(ATP) channels directly associate with ankyrin-B in heart via the K(ATP) channel alpha-subunit Kir6.2. We demonstrate that primary myocytes lacking ankyrin-B display defects in Kir6.2 protein expression, membrane expression, and function. Moreover, we demonstrate a secondary role for ankyrin-B in regulating K(ATP) channel gating. Finally, we demonstrate that ankyrin-B forms a membrane complex with K(ATP) channels and the cardiac Na/K-ATPase, a second key membrane transporter involved in the cardiac ischemia response. Collectively, our new findings define a new role for cardiac ankyrin polypeptides in regulation of ion channel membrane expression in heart.

SUR1: a unique ATP-binding cassette protein that functions as an ion channel regulator

SUR1 is an ATP-binding cassette (ABC) transporter with a novel function. In contrast to other ABC proteins, it serves as the regulatory subunit of an ion channel. The ATP-sensitive (KATP) channel is an octameric complex of four pore-forming Kir6.2 subunits and four regulatory SUR1 subunits, and it links cell metabolism to electrical activity in many cell types. ATPase activity at the nucleotide-binding domains of SUR results in an increase in KATP channel open probability. Conversely, ATP binding to Kir6.2 closes the channel. Metabolic regulation is achieved by the balance between these two opposing effects. Precisely how SUR1 talks to Kir6.2 remains unclear, but recent studies have identified some residues and domains that are involved in both physical and functional interactions between the two proteins. The importance of these interactions is exemplified by the fact that impaired regulation of Kir6.2 by SUR1 results in human disease, with loss-of-function SUR1 mutations causing congenital hyperinsulinism and gain-of-function SUR1 mutations leading to neonatal diabetes. This paper reviews recent data on the regulation of Kir6.2 by SUR1 and considers the molecular mechanisms by which SUR1 mutations produce disease.

A Novel KCNJ11 Mutation Associated with Congenital Hyperinsulinism Reduces the Intrinsic Open Probability of β-Cell ATP-sensitive Potassium Channels

The beta-cell ATP-sensitive potassium (KATP) channel controls insulin secretion by linking glucose metabolism to membrane excitability. Loss of KATP channel function due to mutations in ABCC8 or KCNJ11, genes that encode the sulfonylurea receptor 1 or the inward rectifier Kir6.2 subunit of the channel, is a major cause of congenital hyperinsulinism. Here, we report identification of a novel KCNJ11 mutation associated with the disease that renders a missense mutation, F55L, in the Kir6.2 protein. Mutant channels reconstituted in COS cells exhibited a wild-type-like surface expression level and normal sensitivity to ATP, MgADP, and diazoxide. However, the intrinsic open probability of the mutant channel was greatly reduced, by approximately 10-fold. This low open probability defect could be reversed by application of phosphatidylinositol 4,5-bisphosphates or oleoyl-CoA to the cytoplasmic face of the channel, indicating that reduced channel response to membrane phospholipids and/or long chain acyl-CoAs underlies the low intrinsic open probability in the mutant. Our findings reveal a novel molecular mechanism for loss of KATP channel function and congenital hyperinsulinism and support the importance of phospholipids and/or long chain acyl-CoAs in setting the physiological activity of beta-cell KATP channels. The F55L mutation is located in the slide helix of Kir6.2. Several permanent neonatal diabetes-associated mutations found in the same structure have the opposite effect of increasing intrinsic channel open probability. Our results also highlight the critical role of the Kir6.2 slide helix in determining the intrinsic open probability of KATP channels.

Sulfonylurea stimulation of insulin secretion.

Sulfonylureas are widely used to treat type 2 diabetes because they stimulate insulin secretion from pancreatic beta-cells. They primarily act by binding to the SUR subunit of the ATP-sensitive potassium (K(ATP)) channel and inducing channel closure. However, the channel is still able to open to a limited extent when the drug is bound, so that high-affinity sulfonylurea inhibition is not complete, even at saturating drug concentrations. K(ATP) channels are also found in cardiac, skeletal, and smooth muscle, but in these tissues are composed of different SUR subunits that confer different drug sensitivities. Thus tolbutamide and gliclazide block channels containing SUR1 (beta-cell type), but not SUR2 (cardiac, smooth muscle types), whereas glibenclamide, glimepiride, repaglinide, and meglitinide block both types of channels. This difference has been exploited to determine residues contributing to the sulfonylurea-binding site. Sulfonylurea block is decreased by mutations or agents (e.g., phosphatidylinositol bisphosphate) that increase K(ATP) channel open probability. We now propose a kinetic model that explains this effect in terms of changes in the channel open probability and in the transduction between the drug-binding site and the channel gate. We also clarify the mechanism by which MgADP produces an apparent increase of sulfonylurea efficacy on channels containing SUR1 (but not SUR2).

Regulation of ATP-sensitive K(+) channels by protein kinase C in murine colonic myocytes.

We investigated the regulation of ATP-sensitive K(+) (K(ATP)) currents in murine colonic myocytes with patch-clamp techniques. Pinacidil (10(-5) M) activated inward currents in the presence of high external K(+) (90 mM) at a holding potential of -80 mV in dialyzed cells. Glibenclamide (10(-5) M) suppressed pinacidil-activated current. Phorbol 12,13-dibutyrate (PDBu; 2 x 10(-7) M) inhibited pinacidil-activated current. 4-alpha-Phorbol ester (5 x 10(-7) M), an inactive form of PDBu, had no effect on pinacidil-activated current. In cell-attached patches, the open probability of K(ATP) channels was increased by pinacidil, and PDBu suppressed openings of K(ATP) channels. When cells were pretreated with chelerythrine (10(-6) M) or calphostin C (10(-7) M), inhibition of the pinacidil-activated whole cell currents by PDBu was significantly reduced. In cells studied with the perforated patch technique, PDBu also inhibited pinacidil-activated current, and this inhibition was reduced by chelerythrine (10(-6) M). Acetylcholine (ACh; 10(-5) M) inhibited pinacidil-activated currents, and preincubation of cells with calphostin C (10(-7) M) decreased the effect of ACh. Cells dialyzed with protein kinase C epsilon-isoform (PKCepsilon) antibody had normal responses to pinacidil, but the effects of PDBu and ACh on K(ATP) were blocked in these cells. Immunofluorescence and Western blots showed expression of PKCepsilon in intact muscles and isolated smooth muscle cells of the murine proximal colon. These data suggest that PKC regulates K(ATP) in colonic muscle cells and that the effects of ACh on K(ATP) are largely mediated by PKC. PKCepsilon appears to be the major isozyme that regulates K(ATP) in murine colonic myocytes.

Molecular physiology of neuronal K-ATP channels

ATP sensitive potassium (K-ATP) channels are widely expressed in many cell types including neurons. K-ATP channels are heteromeric membrane proteins that consist of two very different subunits: the pore-forming, two-transmembrane spanning potassium channel subunit (Kir6) and the regulatory, 17 transmembrane spanning sulphonylurea receptor (SUR). This ensemble--joined together in a 4:4 stoichiometry--endows this channel with a unique combination of functional properties. The open probability of K-ATP channels directly depends on the intracellular ATP/ADP levels allowing the channels to directly couple the metabolic state of a cell to its electrical activity. Here, recent progress on the molecular composition and functional diversity of neuronal K-ATP channels is reviewed. One is particular concerned with single-cell mRNA expression studies that give insight to the coexpression patterns of Kir6 and SUR isoforms in identified neurons. In addition, the physiological roles of neuronal K-ATP channels in glucose sensing and adapting neuronal activity to metabolic demands are discussed, as well as their emerging pathophysiological functions in acute brain ischemia and chronic neurodegenerative diseases.

Blockade of adenosine triphosphate-sensitive potassium channels by thiamylal in rat ventricular myocytes.

The adenosine triphosphate (ATP)-sensitive potassium (KATP) channels protect myocytes during ischemia and reperfusion. This study investigated the effects of thiamylal on the activities of KATP channels in isolated rat ventricular myocytes during simulated ischemia. Male Wistar rats were anesthetized with ether. Single, quiescent ventricular myocytes were dispersed enzymatically. Membrane currents were recorded using patch-clamp techniques. In the cell-attached configuration, KATP channel currents were assessed before and during activation of these channels by 2,4-dinitrophenol and after administration of 25, 50, and 100 mg/l thiamylal. The open probability was determined from current-amplitude histograms. In the inside-out configuration, the current-voltage relation was obtained before and after the application of thiamylal (50 mg/1). In the cell-attached configuration, 2,4-dinitrophenol caused frequent channel opening. 2,4-Dinitrophenol-induced channel activities were reduced significantly by glibenclamide, suggesting that the channels studied were KATP channels. Open probability of KATP channels was reduced by thiamylal in a concentration-dependent manner. KATP channels could be activated in the inside-out configuration because of the absence of ATP. Thiamylal inhibited KATP channel activity without changing the single-channel conductance. The results obtained in this study indicate that thiamylal inhibits KATP channel activities in cell-attached and inside-out patches, suggesting a direct action of this drug on these channels.

Two types of K(+) channels at the basolateral membrane of proximal tubule: inhibitory effect of taurine.

The cell-attached configuration of the patch-clamp technique was used to investigate the effects of taurine on the basolateral potassium channels of rabbit proximal convoluted tubule. In the absence of taurine, the previously reported ATP-blockable channel, K(ATP), was observed in 51% of patches. It is characterized by an inwardly rectifying current-voltage curve with an inward slope conductance of 49 +/- 5 pS (n = 15) and an outward slope conductance of 13 +/- 6 pS (n = 15). The K(ATP) channel open probability (P(o)) is low, 0.15 +/- 0.06 (n = 15) at a -V(p) = -100 mV (V(p) is the pipette potential), and increases slightly with depolarization. The gating kinetics are characterized by one open time constant (tau(o) = 5.0 +/- 1.9 ms, n = 6) and two closed time constants (tau(C1) = 5. 2 +/- 1.5 ms, tau(C2) = 140 +/- 40 ms; n = 6). In 34% of patches, a second type of potassium channel, sK, with distinct properties was recorded. Its current-voltage curve is characterized by a sigmoidal shape, with an inward slope conductance of 12 +/- 2 pS (n = 4). Its P(o) is voltage independent and averages 0.67 +/- 0.03 (n = 4) at -V(p) = -80 mV. Both its open time and closed time distributions are described by a single time constant (tau(o) = 96 +/- 19 ms, tau(C) = 10.5 +/- 3.6 ms; n = 4). Extracellular perfusion of 40 mM taurine fails to affect sK channels, whereas K(ATP) channel P(o) decreases by 75% (from 0.17 +/- 0.06 to 0.04 +/- 0.02, n = 7, P < 0.05). In conclusion, the absolute basolateral potassium conductance of rabbit proximal tubules is the resulting combination of, at least, two types of potassium channels of roughly equal importance: a high-conductance low-open probability K(ATP) channel and a low-conductance high-open probability sK channel. The previously described decrease in the basolateral absolute potassium conductance by taurine is, however, mediated by a single type of K channel: the ATP-blockable K channel.

The effect of polyamines on KATP channels in guinea-pig ventricular myocytes.

1. The effect of natural polyamines on KATP channels was studied using inside-out patches from guinea-pig ventricular myocytes. 2. At a holding potential of +40 mV, spermine at the intracellular membrane surface reduced the KATP channel open probability (Popen) in a dose-dependent manner. Half-maximal inhibition occurred at 29 microM with a Hill coefficient of 1.2. 3. The effect of spermine on Popen was not greatly influenced by the membrane potential but there appeared to be a small reduction in unitary current amplitude during strong depolarizations. 4. Analysis of KATP single channel kinetics showed that spermine inhibited the channel by decreasing the mean open time and introducing transitions to a long closed state. 5. Spermidine (0.1 mM) was found to have a similar effect to spermine. Putrescine (10 mM) was found to block more effectively at positive membrane potentials. Up to 20 mM arginine had no significant effect on KATP channels. 6. Our results indicate that natural polyamines influence native KATP channel gating in cardiac myocytes.

Cross-talk between ATP-regulated K+ channels and Na+ transport via cellular metabolism in frog skin principal cells.

Isolated frog skin epithelium, mounted in an Ussing chamber and bathed in standard NaCl Ringer solution, recycles K+ across the basolateral membrane of principal cells through an inward-rectifier K+ channel (Kir) operating in parallel with a Na+-K+-ATPase pump. Here we report on the metabolic control of the Kir channel using patch clamping, short-circuit current measurement and enzymatic determination of cellular (ATP (ATPi). 2. The constitutively active Kir channel in the basolateral membrane has the characteristics of an ATP-regulated K+ channel and is now classed as a KATP channel. In excised inside-out patches the open probability (Po) of KATP channels was reduced by ATPi with half-maximum inhibition at an ATPi concentration of 50 microM. 3. ATPi measured (under normal Na+ transport conditions) with luciferin-luciferase was 1.50 +/- 0.23 mM (mean +/- S.E.M.; range, 0.4-3.3 mM n = 11). Thus the KATP channel would be expected to be inactive in intact cells if ATPi was the sole regulator of channel activity. KATP channels which were inactivated by 1 mM ATPi in excised patches could be reactivated by addition of 100 microM ADP on the cytosolic side. When added alone, ADP blocks this channel with half-maximal inhibition at [ADPi] > 5 mM. 4. Sulphonylureas inhibit single KATP channels in cell-attached patches as well as the total basolateral K+ current measured in frog skin epithelia perforated with nystatin on the apical side. 5. Na+-K+-ATPase activity is a major determinant of cytosolic ATP. Blocking the pump activity with ouabain produced a time-dependent increase in ATPi and reduced the open probability of KATP channels in cell-attached membranes. 6. We conclude that the ratio of ATP/ADP is an important metabolic coupling factor between the rate of Na+-K+ pumping and K+ recycling.

Modulation by Mg2+ and ADP of ATP-sensitive potassium channels in frog skeletal muscle.

The patch-clamp technique was used to examine the action of intracellular magnesium ions and ADP in the absence of ATP on skeletal muscle ATP-sensitive potassium channels (K-ATP channels). Inside-out patches were excised from the membrane of sarcolemmal blebs which arise spontaneously without enzymatic treatment after a frog muscle fiber is split in half. In the absence of nucleotides, K-ATP channel open probability was not significantly affected by intracellular magnesium even at a concentration (20 mM) which fully blocks cardiac and pancreatic K-ATP channels. On the other hand, Mg2+ ions (10-20 mM) decreased both inward and outward unitary currents. The percent reduction in inward currents (about 8%) was independent of voltage while the reduction in outward currents was larger at higher voltages, suggesting that the former effect resulted from cancellation of surface charges and the latter from rapid channel block. With or without Mg2+, intracellular ADP could either stimulate or inhibit K-ATP channel activity. Low concentrations (1-100 microM) of ADP rapidly and reversibly increased average activity by a factor of 2 to 3. This activation was seen in half of the patches tested and was greater in the presence of mM Mg2+. High concentrations (> 100 microM) of ADP inhibited activity with a half-block concentration of 450 microM in 0 Mg2+, i.e., more than an order of magnitude the value for ATP. ADP inhibition, like ATP inhibition, was partially relieved by mM Mg2+, suggesting that the Mg(2+)-bound ADP forms are less effective than free ADP forms. During exercise, free ADP levels rise and ATP declines while remaining high.(ABSTRACT TRUNCATED AT 250 WORDS)

Action potential duration and activation of ATP-sensitive potassium current in isolated guinea-pig ventricular myocytes

It is difficult to associate the ATP-sensitive potassium (K-ATP) channel of cardiac muscle with hypoxia/ischemia induced action potential shortening because this occurs before intracellular ATP falls to levels associated in vitro with channel opening. This leaves the cardiac K-ATP channel without any obvious physiological function. We have quantitatively examined the relationship between action potential duration and K-ATP channel activity in enzymatically isolated ventricular myocytes of the guinea-pig. In whole-cell voltage-clamp recording experiments when the K-ATP channel opener SR 44866 (2–10 μM) stimulated an outward membrane current greater than 50 pA at 0 mV membrane potential (the equivalent of 30 open K-ATP channels or 1% of the cell K-ATP channel population) action potential duration was reduced by more than 50%. In the majority of cell-attached membrane patch recordings metabolic inhibition stimulated K-ATP channel open probability of 1–2% which continued for long periods (7–25 min) before cell contracture and coincident major K-ATP channel activation (open probability 65%). Our quantitative analysis thus shows that physiologically relevant activity of K-ATP channels in cardiac muscle is confined to a very small percentage of the possible cell K-ATP current and thus intracellular ATP would not have to fall very far before the opening of K-ATP channels would influence cardiac excitability.