ATP-sensitive K+ channels in an insulin-secreting cell line are inhibited byd-glyceraldehyde and activated by membrane permeabilization

Summary The control of K⁺ channels in the insulin-secreting cell line RINm5F has been investigated by patch-clamp singlechannel current recording experiments. The unitary current events recorded from cell-attached patches are due to large and small inwardly rectifying ATP-sensitive K⁺ channels with conductance properties similar to the two channels previously identified in primary cultured rat islet cells (Findlay, I., Dunne, M.J., & Petersen, O. H.J. Membrane Biol. 88:165–172, 1985). Cell permeabilization through brief exposure to 10 m digitonin or 0.05% saponin (outside the isolated membrane patch area) results in a dramatic increase in current through the cell-attached patch due to opening of many large and small K⁺-selective channels. These channels are inhibited in a dose-dependent manner by ATP applied to the bath (near-complete inhibition by 5mm ATP). During prolonged ATP exposure (1–5 min) the initial inhibition is followed by partial recovery of channel activity, although further activation does occur when ATP is subsequently removed. From the maximal number of coincident channel openings in the permeabilized cells (in the absence of ATP), it is estimated that there are on average 12 large ATP-sensitive K⁺ channels per membrane patch, but in the intact cells less than 5% of the membrane patches exhibited three or more coincident K⁺ channel openings, indicating the degree to which the channels are inhibited in the resting condition by endogenous ATP. Stimulation of RINm5F cells to secrete insulin was carried out by challenging intact cells with 10mm d-glyceraldehyde.d-glyceraldehyde induced depolarization of the membrane from about –70 to –20 mV and evoked a marked reduction in the open-state probability of both the large and small ATP-sensitive channels.d-glyceraldehyde also induced action potentials in a number of cases. All effects of stimulation were largely transient, lasting about 100 sec. The two ATP-sensitive K⁺ channels are probably responsible for the resting potential and play a crucial role in coupling metabolism to membrane depolarization.     

Transport of K+ and other cations across phospholipid membranes by nonesterified fatty acids

The rate of change of internal pH and transmembrane potential has been monitored in liposomes following the external addition of various cation salts. Oleic acid increases the transmembrane movement of H⁺ following the imposition of a K⁺ gradient. An initial fast change in internal pH is seen followed by a slower rate of alkalinization. High concentrations of the fatty acid enhance the rate comparable to that seen in the presence of nigericin in contrast to the effect of FCCP (carbonyl cyanide p-(tri-fluoromethoxy)phenyl hydrazone) which saturates at an intermediate value. The ability of nonesterified fatty acids to catalyze the movement of cations across the liposome membrane increases with the degree of unsaturation and decreases with increasing chain length. Li and Na salts cause a similar initial fast pH change but have less effect on the subsequent slower rate. Similarly, the main effect of divalent cation salts is on the initial fast change. The membrane potential can enhance or inhibit cation transport depending on its polarity with respect to the cation gradient. It is concluded that nonesterified fatty acids have the capability to complex with, and transport, a variety of cations across phospholipid bilayers. However, they do not act simply as proton/cation exchangers analogous to nigericin nor as protonophores analogous to FCCP. The full cycle of ionophoric action involves a combination of both functions.The authors would like to thank P. Nicholts (Brock University, Canada) for helpful discussions. M.A.S. received a Science and Engineering Research Council studentship and C.E.C. acknowledges the award of a King's College London fellowship followed by a Medical Research Council Training Fellowship.     

Activation and inhibition of ATP-sensitive K+ channels by fluorescein derivatives.

Fluorescein derivatives are known to bind to nucleotide-binding sites on transport ATPases. In this study, they have been used as ligands to nucleotide-binding sites on ATP-sensitive K+ channels in insulinoma cells. Their effect on channel activity has been studied using 86Rb+ efflux and patch-clamp techniques. Fluorescein derivatives have two opposite effects. First, like ATP, they can inhibit active ATP-sensitive K+ channels. Second, they are able to reactivate ATP-sensitive K+ channels subjected to inactivation or "run-down" in the absence of cytoplasmic ATP. Therefore reactivation of the inactivated ATP-sensitive K+ channel clearly does not require channel phosphorylation as is commonly believed. The results indicate the existence of two binding sites for nucleotides, one activator site and one inhibitor site. Irreversible binding at either the inhibitor or the activator site on the channel was obtained with eosin-5-maleimide, resulting in irreversible inhibition or activation of the ATP-sensitive K+ channel respectively. The irreversibly activated channel could still be inhibited by 2 mM ATP. After activation by fluorescein derivatives, ATP-sensitive K+ channels become resistant to the classical blocker of this channel, the sulfonylurea glibenclamide. Negative allosteric interactions between fluorescein/nucleotide receptors and sulfonylurea-binding sites were suggested by results obtained in [3H]glibenclamide-binding experiments.     

Cardiac ATP-sensitive K+ channels. Evidence for preferential regulation by glycolysis

The ability of glycolysis, oxidative phosphorylation, the creatine kinase system, and exogenous ATP to suppress ATP-sensitive K+ channels and prevent cell shortening were compared in patch-clamped single guinea pig ventricular myocytes. In cell-attached patches on myocytes permeabilized at one end with saponin, ATP-sensitive K+ channels were activated by removing ATP from the bath, and could be closed equally well by exogenous ATP or substrates for endogenous ATP production by glycolysis (with the mitochondrial inhibitor FCCP present), mitochondrial oxidative phosphorylation, or the creatine kinase system. In the presence of an exogenous ATP-consuming system, however, glycolytic substrates (with FCCP present) were superior to substrates for either oxidative phosphorylation or the creatine kinase system at suppressing ATP-sensitive K+ channels. All three groups of substrates were equally effective at preventing cell shortening. In 6 of 38 excised inside-out membrane patches, ATP-sensitive K+ channels activated by removing ATP from the bath were suppressed by a complete set of substrates for the ATP-producing steps of glycolysis but not by individual glycolytic substrates, which is consistent with the presence of key glycolytic enzymes located near the channels in these patches. Under whole-cell voltage-clamp conditions, inclusion of 15 mM ATP in the patch electrode solution dialyzing the interior of the cell did not prevent activation of the ATP-sensitive K+ current under control conditions or during exposure to complete metabolic inhibition. In isolated arterially perfused rabbit interventricular septa, selective inhibition of glycolysis caused an immediate increase in 42K+ efflux rate, which was prevented by 100 microM glyburide, a known blocker of ATP-sensitive K+ channels. These observations suggest that key glycolytic enzymes are associated with cardiac. ATP-sensitive K+ channels and under conditions in which intracellular competition for ATP is high (e.g., in beating heart) that act as a preferential source of ATP for these channels.     

Modification of ATP-sensitive K+ channels by proteolysis in smooth muscle cells from pig urethra

Patch-clamp experiments have been performed to investigate the effects of endoproteases (such as trypsin, carboxypeptidase B) on both membrane currents and unitary currents in isolated smooth muscle cells from pig proximal urethra (conventional whole-cell configuration, cell-attached configuration, and inside-out patches). Application of either trypsin (1 mg/mL) or carboxypeptidase B (0.1 mg/mL) to the intracellular surface of the excised membrane patches stimulated the activity of a 2.1 pA K+ channel (in symmetrical 140 mM K+ conditions) at a holding potential of -50 mV. The trypsin-induced K+ channels in inside-out configuration exhibited the same amplitude and similar channel opening kinetics to the levcromakalim-induced ATP-sensitive K+ channel (i.e. K ATP channel) in cell-attached patches of the same membrane; however, the sensitivity of the channels to glibenclamide was greatly reduced after the trypsin-treatment. The activity of the trypsin-induced K+ channel was reversibly inhibited by cibenzoline in an inside-out configuration (Ki = 5 microM). It is concluded that trypsin and carboxypeptidase B reactivate the channel with an intact pore activity but the different pharmacological properties of the channels may reflect some change in the conformation in channel proteins after proteolysis.     

Evidence for presence of ATP-sensitive K+ channels in rat colonic smooth muscle cells.

Coexpression of sulfonylurea receptor (SUR) and inward-rectifying K+ channel (Kir6.1 or 6.2) subunit yields ATP-sensitive K+ (K(ATP)) channels. Three subtypes of SUR have been cloned: pancreatic (SUR1), cardiac (SUR2A), and vascular smooth muscle (SUR2B). The distinct responses to K+ channel openers (KCOs) produced in different tissues may depend on the SUR isoform of K(ATP) channel. Therefore, we investigated the effects of pinacidil and diazoxide, two KCOs, on K(ATP) currents in intestinal smooth muscle cells of the rat colon (circular layer) using whole-cell voltage clamp. Pinacidil stimulated a time-independent K+ current evoked by various test potentials from a holding potential of -70 mV. The reversal potential of the stimulated current was about -75 mV, which is close to the equilibrium potential for K+ (E(K)). Both pinacidil and diazoxide dose-dependently stimulated K+ currents (evoked by ramp pulses), with EC50 values of 1.3 and 34.2 microM, respectively. The stimulated current was completely reversed by glybenclamide (3 microM). Since the EC50 values are close to those reported for vascular smooth muscle (VSM) cells, the SUR subtype may be similar to that in VSM cells, and could form the functional K(ATP) channel in rat colonic smooth muscle cells.     

Angiotensin II blocks ATP-sensitive K+ channels in porcine coronary artery smooth muscle cells.

ATP-sensitive K+ channels with small conductance (30 pS in symmetrical K(+)-rich solutions) in porcine coronary artery smooth muscle cells were highly active at physiological concentrations of Ca2+ (greater than 10(-4) M) even in the presence of physiological ATP levels, suggesting that these channels contribute to the generation of the resting membrane potential in vascular smooth muscle cells and their modulation is important in controlling vasomotor tone. Angiotensin II, applied from outside the membrane, blocked these channels in a concentration-dependent manner. This would be expected to cause depolarization and result in vasoconstriction.     

ATP-sensitive K+ channels in human isolated pancreatic B-cells

Glucose-stimulated insulin release from rodent pancreatic B-cells is thought to be initiated by the closing of ATP-sensitive K+ channels in the plasma membrane as a consequence of glucose metabolism. We have identified an ATP-sensitive K+ channel in membrane patches excised from human B-cells which is similar to that found in rodent B-cells in conductance, kinetics, ATP sensitivity and its inhibition by sulphonylureas. In man, the ATP-sensitive K+ channel may also have a central role in glucose-stimulated insulin secretion and may be (linked to) the receptor for the hypoglycemic sulphonylureas.     

State-dependent modification of ATP-sensitive K+ channels by phosphatidylinositol 4,5-bisphosphate

With inside-out patchrecordings in ventricular myocytes from the hearts of guinea pigs, westudied ATP-sensitive K⁺ (K_ATP) channelsactivated by phosphatidylinositol 4,5-bisphosphate (PIP₂)with respect to sensitivity to ATP when in either a rundown state (RS)or a non-rundown state (NRS). Rundown of K_ATP channels wasinduced by exposure either to ATP-free solution or to ATP-free solutioncontaining 19 µM Ca²⁺. Exposure of membrane patches to 10 µM PIP₂ reactivated channels with both types of rundown.The reactivation by PIP₂ did not require ATP in the bath.The IC₅₀ of channels recovered from RS and before therundown was 37.1 and 31.1 µM, respectively. PIP₂irreversibly increased the mean current when the channel was in theNRS. This was associated with a shift of IC₅₀ to 250.6 µMafter PIP₂ exposure. PIP₂ activates NRSK_ATP channels by decreasing their sensitivity to ATP,whereas PIP₂ reactivates RS-K_ATP channelsindependently of ATP without changing ATP sensitivity.

     

Lactate stimulates insulin secretion without blocking the K+ channels in HIT-T15 insulinoma cells.

To clarify the mechanism by which lactate affects insulin secretion, we investigated the effect of lactate on insulin secretion, cytosolic free Ca2+ ([Ca2+](i), the ATP sensitive K+ channel (K(ATP)) and the Ca2+-activated K+ channel (K(Ca)) in HIT-T15 cells, and the results were compared with those of glucose and glibenclamide. All three agents caused insulin secretion and increased [Ca2+](i), but the effects on the K+ channels were different. In cell-attached patch configurations, 10 mmol/l glucose blocked both the K(ATP) and KCa channels, while 100 nmol/l glibenclamide had no effect on KCa channels, but blocked K(ATP) channels. Lactate at a concentration of 10 mmol/l activated both the K(ATP) and KCa channels, not only in cell-attached, but also in inside-out patch configurations, indicating that the increase in [Ca2+](i) and secretion of insulin by lactate cannot be explained by the blocking of the K+ channels. Lactate, at concentrations of 10 mmol/l and 50 mmol/l decreased 45Ca2+ efflux, while glibenclamide increased the efflux. These results suggest that the lactate-induced Ca2+ increase is not due to the closing of K+ channels, but at least in part, to the suppression of Ca2+ efflux from HIT cells.     

Mitochondrial ATP-sensitive K+ channels are redox-sensitive pathways that control reactive oxygen species production

Pharmacological mitochondrial ATP-sensitive K(+) channel (mitoK(ATP)) opening protects against ischemic damage and mimics ischemic preconditioning. However, physiological and pathological signaling events that open this channel are still not fully understood. We found that catalase, which removes H(2)O(2), is capable of reversing the beneficial effects of ischemic preconditioning but not of mitoK(ATP) agonist diazoxide. On the other hand, 2-mercaptopropionylglycine prevented cardioprotection in both cases, suggesting that this compound may present effects other than scavenging of reactive oxygen species. Indeed, 2-mercaptopropionylglycine and a second thiol-reducing agent, dithiothreitol, impair diazoxide-mediated activation of mitoK(ATP) in isolated heart mitochondria. This demonstrates that mitoK(ATP) activity is regulated by thiol redox status. Furthermore, stimulating the generation of endogenous mitochondrial reactive oxygen species or treating samples with H(2)O(2) strongly enhances mitoK(ATP) activity, in a manner probably dependent on redox sensors located in the channel's sulfonylurea receptor. We also demonstrate that mitoK(ATP) channel activity effectively prevents mitochondrial reactive oxygen release. Collectively, our results suggest that mitoK(ATP) acts as a reactive oxygen sensor that decreases mitochondrial free radical generation in response to enhanced local levels of oxidants. As a result, these channels regulate mitochondrial redox state under physiological conditions and prevent oxidative stress under pathological conditions such as ischemia/reperfusion.     

Role of the mitochondrial ATP-sensitive K+ channels in cardioprotection

The mitochondrial ATP-sensitive K(+) (mitoK(ATP)) channel was discovered more than a decade ago. Since then, several pharmacological studies have identified agents that target this channel some of which selectively target mitoK(ATP). These and other studies have also suggested that mitoK(ATP) plays a key role in the process of ischemic preconditioning (IPC) and prevention of apoptosis. The mechanism by which mitoK(ATP) exerts its protective effects is unclear, however, changes in mitochondrial Ca(2+) uptake and levels of reactive oxygen species, and mitochondrial matrix swelling are believed to be involved. Despite major advances, several important issues regarding mitoK(ATP) remain unanswered. These questions include, but are not limited to: the molecular structure of mitoK(ATP), the downstream and upstream mechanisms that leads to IPC and cell death, and the pharmacological profile of the channel. This review attempts to provide an up-to-date overview of the role of mitoK(ATP) in cardioprotection.     

Somatostatin activates glibenclamide-sensitive and ATP-regulated K+ channels in insulinoma cells via a G-protein

Somatostatin, an hyperglycemia-inducing hormone, was studied in rat insulinoma (RINm5F) cells using 86Rb+ efflux techniques. 86Rb+ efflux is stimulated by somatostatin in a dose-dependent manner. The half-maximum value of activation is 0.7 nM. Somatostatin-induced 86Rb+ efflux is abolished by the hypoglycemia-inducing sulfonylurea, glibenclamide, a known blocker of ATP-regulated K+ channels. Somatostatin activation is prevented by pretreatment of insulinoma cells with pertussis toxin. 86Rb+ efflux studies show that somatostatin activates an ATP-dependent K+ channel.     

Physiological and pathophysiological roles of ATP-sensitive K+ channels

ATP-sensitive potassium (K(ATP)) channels are present in many tissues, including pancreatic islet cells, heart, skeletal muscle, vascular smooth muscle, and brain, in which they couple the cell metabolic state to its membrane potential, playing a crucial role in various cellular functions. The K(ATP) channel is a hetero-octamer comprising two subunits: the pore-forming subunit Kir6.x (Kir6.1 or Kir6.2) and the regulatory subunit sulfonylurea receptor SUR (SUR1 or SUR2). Kir6.x belongs to the inward rectifier K(+) channel family; SUR belongs to the ATP-binding cassette protein superfamily. Heterologous expression of differing combinations of Kir6.1 or Kir6.2 and SUR1 or SUR2 variant (SUR2A or SUR2B) reconstitute different types of K(ATP) channels with distinct electrophysiological properties and nucleotide and pharmacological sensitivities corresponding to the various K(ATP) channels in native tissues. The physiological and pathophysiological roles of K(ATP) channels have been studied primarily using K(ATP) channel blockers and K(+) channel openers, but there is no direct evidence on the role of the K(ATP) channels in many important cellular responses. In addition to the analyses of naturally occurring mutations of the genes in humans, determination of the phenotypes of mice generated by genetic manipulation has been successful in clarifying the function of various gene products. Recently, various genetically engineered mice, including mice lacking K(ATP) channels (knockout mice) and mice expressing various mutant K(ATP) channels (transgenic mice), have been generated. In this review, we focus on the physiological and pathophysiological roles of K(ATP) channels learned from genetic manipulation of mice and naturally occurring mutations in humans.     

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.

     

ATP-sensitive inward rectifier and voltage- and calcium-activated K+ channels in cultured pancreatic islet cells

Summary K⁺ channels in cultured rat pancreatic islet cells have been studied using patch-clamp single-channel recording techniques in cell-attached and excised inside-out and outside-out membrane patches. Three different K⁺-selective channels have been found. Two inward rectifier K⁺ channels with slope conductances of about 4 and 17 pS recorded under quasi-physiological cation gradients (Na⁺ outside, K⁺ inside) and maximal conductances recorded in symmetrical K⁺-rich solutions of about 30 and 75 pS, respectively. A voltage- and calcium-activated K^– channel was recorded with a slope conductance of about 90 pS under the same conditions and a maximal conductance recorded in symmetrical K⁺-rich solutions of about 250 pS. Single-channel current recording in the cell-attached conformation revealed a continuous low level of activity in an apparently small number of both the inward rectifier K⁺ channels. But when membrane patches were excised from the intact cell a much larger number of inward rectifier K⁺ channels became transiently activated before showing an irreversible decline. In excised patches opening and closing of both the inward rectifier K⁺ channels were unaffected by voltage, internal Ca²⁺ or externally applied tetraethyl-ammonium (TEA) but the probability of opening of both inward rectifier K⁺ channels was reduced by internally applied 1–5mm adenosine-5-triphosphate (ATP). The large K⁺ channel was not operational in cell-attached membrane patches, but in excised patches it could be activated at negative membrane potentials by 10^–7 to 10^–6 m internal Ca²⁺ and blocked by 5–10mm external TEA.