Aging augments interstitial K+ concentrations in active muscle of rats.

Skeletal muscle performance declines with advancing age, and the underlying mechanism is not completely understood. A large body of convincing evidence has demonstrated a crucial role for interstitial K+ concentration ([K+]o) in modulating contractile function of skeletal muscle. The present study tested the hypothesis that during muscle contraction there is a greater accumulation of [K+]o in aged compared with adult skeletal muscle. Twitch muscle contraction was induced by electrical stimulation of the sciatic nerves of 8- and 32-mo-old Fischer 344 x Brown Norway rats. Levels of [K+]o were measured continuously by a microdialysis technique with the probes inserted into the gastrocnemius muscle. Stimulation at 1, 3, and 5 Hz elevated muscle [K+]o by 52, 64, and 88% in adult rats, and by 78, 98, and 104% in aged rats, respectively, and the increase was significantly higher in aged than in adult rats. Recovery for [K+]o, as measured by the time for [K+]o to recover by 20 and 50% from peak response after stimulation, was slower in aged rats. Ouabain (5 mM), a specific inhibitor of the Na+-K+ pump, was added in the perfusate to inhibit the reuptake of K+ into the cells to assess the role of the pump in the overall K+ balance. Ouabain elevated muscle [K+]o at rest, and the effect was significantly attenuated in aged animals. The present data demonstrated an augmented [K+]o in aged skeletal muscle compared with adult skeletal muscle, and the data suggested that an alteration in the function of the Na+-K+ pump may contribute, in part, to the deficiency in K+ balance in skeletal muscle of aged rats.     

Cellular mechanisms underlying cutaneous pressure-induced vasodilation: in vivo involvement of potassium channels

In the skin of humans and rodents, local pressure induces localized cutaneous vasodilation, which may be protective against pressure-induced microvascular dysfunction and lesion formation. Once activated by the local pressure application, capsaicin-sensitive nerve fibers release neuropeptides that act on the endothelium to synthesize and release nitric oxide (NO) and prostaglandins, leading to the development of the cutaneous pressure-induced vasodilation (PIV). The present study was undertaken to test in vivo the hypothesis that PIV is mediated or modulated by differential activation of K+ channels in anesthetized rats using pharmacological methods. Local pressure was applied at 11.1 Pa/s. Endothelium-independent and -dependent vasodilation were tested using iontophoretic delivery of sodium nitroprusside (SNP) and acetylcholine (ACh), respectively, and was correlated with PIV response. PIV was reduced after systemic administration of tetraethylammonium (a nonspecific K+ channel blocker), iberiotoxin [a specific large-conductance Ca2+-activated K+ (BKCa) channel blocker], and glibenclamide [a specific ATP-sensitive K+ (KATP) channel blocker], whereas PIV was unchanged by apamin (a specific small-conductance Ca2+-activated K+ channel blocker) and 4-aminopyridine (a specific voltage-sensitive K+ channel blocker). The responses to SNP and ACh were reduced by iberiotoxin but were unchanged by glibenclamide. We conclude that the cellular mechanism of PIV in skin involves BKCa and KATP channels. We suggest that the opening of BKCa and KATP channels contributes to the hyperpolarization of vascular smooth muscle cells to produce PIV development mainly via the NO and prostaglandin pathways, respectively.     

Coronary blood flow regulation in exercising swine involves parallel rather than redundant vasodilator pathways

In dogs, only combined blockade of vasodilator pathways [via adenosine receptors, nitric oxide synthase (NOS) and ATP-sensitive K+ (KATP) channels] results in impairment of metabolic vasodilation, which suggests a redundancy design of coronary flow regulation. Conversely, in swine and humans, blocking KATP channels, adenosine receptors, or NOS each impairs coronary blood flow (CBF) at rest and during exercise. Consequently, we hypothesized that these vasodilators act in parallel rather than in redundancy to regulate CBF in swine. Swine exercised on a treadmill (0-5 km/h), during control and after blockade of KATP channels (with glibenclamide), adenosine receptors [with 8-phenyltheophylline (8-PT)], and/or NOS [with Nomega-nitro-l-arginine (l-NNA)]. l-NNA, 8-PT, and glibenclamide each reduced myocardial O2 delivery and coronary venous O2 tension. These effects of l-NNA, 8-PT, and glibenclamide were not modified by simultaneous blockade of the other vasodilators. Combined blockade of KATP channels and adenosine receptors with or without NOS inhibition was associated with increased H+ production and impaired myocardial function. However, despite an increase in O2 extraction to >90% during administration of l-NNA + 8-PT + glibenclamide, vasodilator reserve could still be recruited during exercise. Thus in awake swine, loss of KATP channels, adenosine, or NO is not compensated for by increased participation of the other two vasodilator mechanisms. These findings suggest a parallel rather than a redundancy design of CBF regulation in the porcine circulation.     

Hydrogen sulfide-induced relaxation of resistance mesenteric artery beds of rats

Hydrogen sulfide (H2S) has been shown recently to function as an important gasotransmitter. The present study investigated the vascular effects of H2S, both exogenously applied and endogenously generated, on resistance mesenteric arteries of rats and the underlying mechanisms. Both H2S and NaHS evoked concentration-dependent relaxation of in vitro perfused rat mesenteric artery beds (MAB). The sensitivity of MAB to H2S (EC50, 25.2 +/- 3.6 microM) was about fivefold higher than that of rat aortic tissues. Removal of endothelium or coapplication of charybdotoxin and apamin to endothelium-intact MAB significantly reduced the vasorelaxation effects of H2S. The H2S-induced relaxation of MAB was partially mediated by ATP-sensitive K+ (KATP) channel activity in vascular smooth muscle cells. Pinacidil (EC50, 1.7 +/- 0.1 microM, n=6) mimicked, but glibenclamide (10 microM, n=6) suppressed, the vasorelaxant effect of H2S. KATP channel currents in isolated mesenteric artery smooth muscle cells were significantly augmented by H2S. L-cysteine, a substrate of cystathionine-gamma-lyase (CSE), at 1 mM increased endogenous H2S production by sixfold in rat mesenteric artery tissues and decreased contractility of MAB. DL-propargylglycine (a blocker of CSE) at 10 microM abolished L-cysteine-dependent increase in H2S production and relaxation of MAB. Our results demonstrated a tissue-specific relaxant response of resistance arteries to H2S. The stimulation of KATP channels in vascular smooth muscle cells and charybdotoxin/apamin-sensitive K+ channels in vascular endothelium by H2S represents important cellular mechanisms for H2S effect on MAB. Our study also demonstrated that endogenous CSE can generate sufficient H2S from exogenous L-cysteine to cause vasodilation. Future studies are merited to investigate direct contribution of endogenous H2S to regulation of vascular tone.     

Ankyrin regulates KATP channel membrane trafficking and gating in excitable cells

KATP channels play critical roles in many cellular functions by coupling cell metabolic status to electrical activity. First discovered in cardiomyocytes 1, KATP channels (comprised of Kir6.x and SUR subunits) have since been found in many other tissues, including pancreatic beta cells, skeletal muscle, smooth muscle, brain, pituitary, and kidney. By linking cellular metabolic state with membrane potential, KATP channels are able to regulate a number of cellular functions such as hormone secretion, vascular tone, and excitability. Specifically, a reduction in metabolism causes a decrease in the ATP:ADP ratio, opening of KATP channels and allowing K+ efflux, membrane hyperpolarization, and suppression of electrical activity. Conversely, increased cellular metabolism causes a decrease in the ATP:ADP ratio that leads to closure of the KATP channel, membrane depolarization, and stimulation of cell electrical activity.     

Microdialysis and the measurement of muscle interstitial K+ during rest and exercise in humans.

The purpose of this study was to examine whether microdialysis and the internal reference thallium-201 ((201)Tl) could accurately measure muscle interstitial K+ (Ki+) before, during, and after exercise. The relative loss of (201)Tl and simultaneous relative recovery of K+ were measured in vitro for 12 microdialysis probes that were bathed in Ringer acetate medium and perfused at various flows (3-10 microl/min). (201)Tl loss was linearly related to K+ recovery, and their level of agreement was not different from zero. Microdialysis and (201)Tl were then used to measure Ki+ in the gastrocnemius medialis muscle of four humans during rest and static plantar flexion exercise. At rest, Ki+ was 3.9-4.3 mmol/l when the perfusate flow was 2 or 5 microl/min. During exercise, Ki+ increased from 6.9 +/- 0.4 to 7.5 +/- 0.3 mmol/l at low to high intensity and declined to 5.2 +/- 0.3 mmol/l after exercise. These results suggest that large changes in Ki+ in human skeletal muscle can be accurately measured by using microdialysis and (201)Tl.     

Na(+) current through KATP channels: consequences for Na(+) and K(+) fluxes during early myocardial ischemia

During early myocardial ischemia, the myocytes are loaded with Na(+), which in turn leads to Ca(2+) overload and cell death. The pathway of the Na(+) influx has not been fully elucidated. The aim of the study was to quantify the Na(+) inward current through sarcolemmal KATP channels (IKATP,Na) in anoxic isolated cardiomyocytes at the actual reversal potential (Vrev) and to estimate the contribution of this current to the Na(+) influx in the ischemic myocardium. IKATP,Na was determined in excised single channel patches of mouse ventricular myocytes and macropatches of Xenopus laevis oocytes expressing SUR2A/Kir6.2 channels. In the presence of K+ ions, the respective permeability ratios for Na(+) to K(+) ions, PNa/PK, were close to 0.01. Only in the presence of Na(+) ions on both sides of the membrane was IKATP,Na similarly large to that calculated from the permeability ratio PNa/PK, indicative of a Na(+) influx that is largely independent of the K+ efflux at Vrev. With the use of a peak KATP channel conductance in anoxic cardiomyocytes of 410 nS, model simulations for a myocyte within the ischemic myocardium showed that the amplitude of the Na(+) influx and K(+) efflux is even larger than the respective fluxes by the Na(+) - K(+) pump and all other background fluxes. These results suggest that during early ischemia the Na(+) influx through KATP channels essentially contributes to the total Na+ influx and that it also balances the K(+) efflux through KATP channels.     

Syntaxin-1A inhibits cardiac KATP channels by its actions on nucleotide binding folds 1 and 2 of sulfonylurea receptor 2A

ATP-sensitive potassium (KATP) channels couple the metabolic status of the cell to its membrane potential to regulate a number of cell actions, including secretion (neurons and neuroendocrine cells) and muscle contractility (skeletal, cardiac, and vascular smooth muscle). KATP channels consist of regulatory sulfonylurea receptors (SUR) and pore-forming (Kir6.X) subunits. We recently reported (Pasyk, E. A., Kang, Y., Huang, X., Cui, N., Sheu, L., and Gaisano, H. Y. (2004) J. Biol. Chem. 279, 4234-4240) that syntaxin-1A (Syn-1A), known to mediate exocytotic fusion, was capable of binding the nucleotide binding folds (NBF1 and C-terminal NBF2) of SUR1 to inhibit the KATP channels in insulin-secreting pancreatic islet beta cells. This prompted us to examine whether Syn-1A might modulate cardiac SUR2A/KATP channels. Here, we show that Syn-1A is present in the plasma membrane of rat cardiac myocytes and binds the SUR2A protein (of rat brain, heart, and human embryonic kidney 293 cells expressing SUR2A/Kir6. 2) at its NBF1 and NBF2 domains to decrease KATP channel activation. Unlike islet beta cells, in which Syn-1A inhibition of the channel activity was apparently mediated only via NBF1 and not NBF2 of SUR1, both exogenous recombinant NBF1 and NBF2 of SUR2A were found to abolish the inhibitory actions of Syn-1A on K(ATP) channels in rat cardiac myocytes and HEK293 cells expressing SUR2A/Kir6.2. Together with our recent report, this study suggests that Syn-1A binds both NBFs of SUR1 and SUR2A but appears to exhibit distinct interactions with NBF2 of these SUR proteins in modulating the KATP channels in islet beta cells and cardiac myocytes.     

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

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

Kinetic differences between Ca(2+)-dependent K+ channels in smooth muscle cells isolated from normal and atherosclerotic human aorta.

The properties of large conductance Ca(2+)-dependent K+ channels in smooth muscle cells (SMC) isolated from normal and atherosclerotic human aorta were studied using the patch-clamp technique. It was shown that SMC from normal human aorta possess a homogeneous population of normal Ca(2+)-dependent K+ channels. In atherosclerotic aorta two kinetically different types of these channels could be distinguished: along with normal 'long' (L)-type channels there appeared channels of 'short' (s)-type. Under similar conditions s-type channels had about a four times shorter mean open time. About five times higher [Ca2+]in was necessary for s-type channels to reach the probability of the channels being open equal to L-type channels. No differences in conductance and voltage dependency were found between the two channel types. Channels of the s-type resembled those previously described in SMC isolated from foetal human aorta. Thus, it can be suggested that during the development of atherosclerosis a population of SMC with s-type Ca(2+)-dependent K+ channels appears in human aorta.     

Activation of ATP-dependent K+ currents in intact skeletal muscle fibres by reduced intracellular pH.

We have used three-microelectrode voltage clamp in conjunction with the ammonium prepulse method to investigate the effects of lowered intracellular pH (pHi) on resting potassium currents of frog skeletal muscle fibres. Potassium currents were recorded in 40 mM K+, Cl(-)-free solution in response either to voltage steps or ramps. An ammonium prepulse (2 h) reduced pHi to 6.45 from a control value of 7.19. The intracellular ATP concentration, measured with high-pressure liquid chromatography (HPLC), was unchanged by this procedure. Mean outward potassium currents were larger in low pHi than in control fibres, being about twice as large at +40 mV, whereas mean inward currents were very similar in control and low-pHi fibres. The sulphonylurea glibenclamide blocked single KATP channels in excised patches with a Kd of 3 microM. In intact fibres 50 microM glibenclamide had no effect on K+ currents in controls but reduced currents in low-pHi fibres. In the presence of glibenclamide, K+ currents in low-pHi fibres were not significantly different from those in control fibres. We suggest that reduced pHi in intact skeletal muscle fibres opens ATP-dependent potassium channels (KATP channels), as has been shown to occur in excised patches of membrane.     

Surface charge and properties of cardiac ATP-sensitive K+ channels

ATP-sensitive K+ (KATP) channels are present in a wide variety of tissues. The sensitivity of these channels to closure by cytosolic ATP (ATPi) varies significantly among different tissues and even within the same tissue. The purpose of this study was to test the hypothesis that negative surface charges modulate the sensitivity of the KATP channels to ATPi by influencing surface potential in the vicinity of the ATP- binding site(s) of the channel. Unitary currents through KATP channels were measured in inside-out membrane patches excised from rabbit ventricular myocytes using the patch-clamp technique. Agents known to be effective at screening negative surface charges were applied to the cytosolic surface of the patches, and their effects on ATP sensitivity were examined. These agents included Mg2+ (2-15 mM), Ba2+ (2-10 mM), and the polycations protamine (0.01-10 microM), poly-L-lysine (500 microM), and poly-L-arginine (0.5 microM). The divalent cations and the various polycations all dramatically reduced the concentration of ATPi required to half-maximally suppress current through KATP channels (Kd), from approximately 100 microM in the absence of these agents to 1.6-8 microM in their presence. The effects were dose dependent. Protamine also reduced the sensitivity of KATP channels to block by cytosolic ADP. The sensitivity of KATP channels to block by ATP was independent of membrane potential, suggesting that the ATP-binding site is not located within the transmembrane voltage field. The effects of the polycation poly-L-lysine on ATP sensitivity were also independent of membrane potential or the direction (inward or outward) of current through KATP channels. In addition to increasing ATP sensitivity, Mg2+, Ba2+, and the polycations all caused dose-dependent block of inward and outward currents through KATP channels over similar concentration ranges as their effects on ATP sensitivity. The block of inward current by polycations was not associated with reduction of single-channel conductance or evidence of fast open channel block. However, the polycations did cause a modest reduction in single-channel conductance of outward current. These results are consistent with the presence of negative surface charges that reduce the local ATP concentration at the ATP-binding site(s) on the channel, relative to the bulk cytosolic ATP concentration. Screening these negative surface charges with divalent cations or polycations decreases the local ATP gradient, resulting in a decrease in the apparent Kd for ATP.(ABSTRACT TRUNCATED AT 400 WORDS)     

Simultaneous recordings of glucose dependent electrical activity and ATP-regulated K(+)-currents in isolated mouse pancreatic beta-cells

Membrane potential and membrane currents were recorded from single mouse pancreatic beta-cells using the perforated patch whole-cell recording technique at 30 degrees C. Single beta-cells maintained in primary tissue culture exhibited glucose-dependent electrical activity similar to that reported for freshly isolated intact islets. The resting input conductance (5.1 +/- 0.9 nS) was determined by ATP-regulated K+ (KATP) channels as it was blocked by 1 mM tolbutamide. 8 mM glucose decreased the input conductance by 80%. The input conductance at -70 mV was of a similar value during the plateau phase and during the silent phase of electrical activity in 8 mM glucose. This suggests that oscillations of KATP channel activity do not underlie the slow waves.     

Gene delivery of Kir6.2/SUR2A in conjunction with pinacidil handles intracellular Ca2+ homeostasis under metabolic stress.

Metabolic injury is a complex process affecting various tissues, with intracellular Ca2+ loading recognized as a common precipitating event leading to cell death. We have recently observed that cells overexpressing recombinant ATP-sensitive K+ (KATP) channel subunits may acquire resistance against metabolic stress. To examine whether, under metabolic challenge, intracellular Ca2+ homeostasis can be maintained by an activator of channel proteins, we delivered Kir6.2 and SUR2A genes, which encode KATP channel subunits, into a somatic cell line lacking native KATP channels. Hypoxia-reoxygenation was simulated by application and removal of the mitochondrial poison 2,4 dinitrophenol. Under such metabolic stress, Ca2+ loading was induced by Ca2+ influx during hypoxia and release of Ca2+ from intracellular stores during reoxygenation. Delivery of Kir6.2/SUR2A genes, in conjunction with the KATP channel activator pinacidil, prevented intracellular Ca2+ loading irrespective of whether the channel opener was applied throughout the duration of hypoxia-reoxygenation or transiently during the hypoxic or reoxygenation stage. In all stages of injury, the effect of pinacidil was inhibited by the selective antagonist of KATP channel, 5-hydroxydecanoate. The present study provides evidence that combined use of gene delivery and pharmacological targeting of recombinant proteins can handle intracellular Ca2+ homeostasis under hypoxia-reoxygenation irrespective of the stage of the metabolic insult.     

A novel crystallization method for visualizing the membrane localization of potassium channels.

The high permeability of K+ channels to monovalent thallium (Tl+) ions and the low solubility of thallium bromide salt were used to develop a simple yet very sensitive approach to the study of membrane localization of potassium channels. K+ channels (Kir1.1, Kir2.1, Kir2.3, Kv2.1), were expressed in Xenopus oocytes and loaded with Br ions by microinjection. Oocytes were then exposed to extracellular thallium. Under conditions favoring influx of Tl+ ions (negative membrane potential under voltage clamp, or high concentration of extracellular Tl+), crystals of TlBr, visible under low-power microscopy, formed under the membrane in places of high density of K+ channels. Crystals were not formed in uninjected oocytes, but were formed in oocytes expressing as little as 5 microS K+ conductance. The number of observed crystals was much lower than the estimated number of functional channels. Based on the pattern of crystal formation, K+ channels appear to be expressed mostly around the point of cRNA injection when injected either into the animal or vegetal hemisphere. In addition to this pseudopolarized distribution of K+ channels due to localized microinjection of cRNA, a naturally polarized (animal/vegetal side) distribution of K+ channels was also frequently observed when K+ channel cRNA was injected at the equator. A second novel "agarose-hemiclamp" technique was developed to permit direct measurements of K+ currents from different hemispheres of oocytes under two-microelectrode voltage clamp. This technique, together with direct patch-clamping of patches of membrane in regions of high crystal density, confirmed that the localization of TlBr crystals corresponded to the localization of functional K+ channels and suggested a clustered organization of functional channels. With appropriate permeant ion/counterion pairs, this approach may be applicable to the visualization of the membrane distribution of any functional ion channel.     

Membrane proteins involved in potassium shifts during muscle activity and fatigue

Muscle activity is associated with potassium displacements, which may cause fatigue. It was reported previously that the density of the large-conductance Ca2+-dependent K+ (BK(Ca)) channel is higher in the T tubule membrane than in the sarcolemmal membrane and that the opposite is the case for the ATP-sensitive K+ (K(ATP)) channel. In the present experiments, we investigated the subcellular localizations of the strong inward rectifier 2.1 K+ (Kir2.1) channel and the Na+-K+-2Cl- (NKCC)1 cotransporter with Western blot analysis of different muscle fractions. Furthermore, muscle function was studied while trying to manipulate the opening probability or transport capacity of these proteins during electrical stimulation of isolated soleus muscles. All experiments were made with excised muscle from male Wistar rats. Kir2.1 channels were almost undetectable in the sarcolemmal membrane but present in the T tubule membrane, whereas NKCC1 cotransporters were present in the sarcolemmal membrane. For muscles incubated in a buffer containing pinacidil, NS1619, Ba2+, or bumetanide, there was a faster reduction in peak force (P < 0.05). Furthermore, bumetanide incubation reduced the peak force at the onset of electrical stimulation (P < 0.05). Thus the effects on muscle force indicate that these drugs can affect K+-transporting proteins and thereby influence K+ accumulation, especially in the T tubules, suggesting that K(ATP) and BK(Ca) channels are responsible for K+ release and decrease in force during repeated muscle contractions, whereas Kir2.1 and NKCC1 may have a role in K+ reuptake.     

Interstitial K(+) in human skeletal muscle during and after dynamic graded exercise determined by microdialysis

Interstitial K(+) concentrations were measured during one-legged knee-extensor exercise by use of microdialysis with probes inserted in the vastus lateralis muscle of the subjects. K(+) in the dialysate was measured either by flame photometry or a K(+)-sensitive electrode placed in the perfusion outlet. The correction for fractional K(+) recovery was based on the assumption of identical fractional thallium loss. The interstitial K(+) was 4. 19 +/- 0.09 mM at rest and increased to 6.17 +/- 0.19, 7.48 +/- 1.18, and 9.04 +/- 0.74 mM at 10, 30, and 50 W exercise, respectively. The individual probes demonstrated large variations in interstitial K(+), and values >10 mM were obtained. The observed interstitial K(+) was markedly higher than previously found for venous K(+) concentrations at similar work intensities. The present data support a potential role for interstitial K(+) in regulation of blood flow and development of fatigue.     

ATPase activity of the sulfonylurea receptor: a catalytic function for the KATP channel complex.

ATP-sensitive K+ (KATP) channels are unique metabolic sensors formed by association of Kir6.2, an inwardly rectifying K+ channel, and the sulfonylurea receptor SUR, an ATP binding cassette protein. We identified an ATPase activity in immunoprecipitates of cardiac KATP channels and in purified fusion proteins containing nucleotide binding domains NBD1 and NBD2 of the cardiac SUR2A isoform. NBD2 hydrolyzed ATP with a twofold higher rate compared to NBD1. The ATPase required Mg2+ and was insensitive to ouabain, oligomycin, thapsigargin, or levamisole. K1348A and D1469N mutations in NBD2 reduced ATPase activity and produced channels with increased sensitivity to ATP. KATP channel openers, which bind to SUR, promoted ATPase activity in purified sarcolemma. At higher concentrations, openers reduced ATPase activity, possibly through stabilization of MgADP at the channel site. K1348A and D1469N mutations attenuated the effect of openers on KATP channel activity. Opener-induced channel activation was also inhibited by the creatine kinase/creatine phosphate system that removes ADP from the channel complex. Thus, the KATP channel complex functions not only as a K+ conductance, but also as an enzyme regulating nucleotide-dependent channel gating through an intrinsic ATPase activity of the SUR subunit. Modulation of the channel ATPase activity and/or scavenging the product of the ATPase reaction provide novel means to regulate cellular functions associated with KATP channel opening.     

The protein kinase A inhibitor, H-89, directly inhibits KATP and Kir channels in rabbit coronary arterial smooth muscle cells

The effects of the protein kinase A (PKA) inhibitor H-89 on ATP-sensitive K+ (KATP) and inward rectifier K+ (Kir) currents were examined in rabbit coronary arterial smooth muscle cells using the patch clamp technique. The H-89, in a dose-dependent manner, inhibited KATP and Kir currents with apparent Kd values of 1.19+/-0.18 and 3.78+/-0.37 microM, respectively. H-85, which is considered as an inactive form of H-89, inhibited KATP and Kir currents, similar to the result of H-89. KATP and Kir currents were not affected by either Rp-8-CPT-cAMPs, which is a membrane-permeable selective PKA inhibitor, or KT 5720, which is also known as a PKA inhibitor. Also, these two drugs did not significantly alter the effects of H-89 on the KATP and Kir currents. These results suggest that H-89 directly inhibits the KATP and Kir currents of rabbit coronary arterial smooth muscle cells independently of PKA inhibition.     

Role of exercise-induced potassium fluxes underlying muscle fatigue: a brief review

The site of exercise-induced muscle fatigue is suggested to be the muscle membrane, which includes the sarcolemma and T-tubule membrane; the excitability of the membrane is dependent on the membrane potential. Significant potassium flux from the intracellular space of contracting muscle may decrease the membrane potential to half its resting value. This is true for isolated muscle preparations as well as for the whole body exercise in humans. Specific K+ channels have been identified, that may account for the intracellular K+ loss. Calcium-sensitive K+ channels open when intracellular Ca2+ concentrations increase, as during excitation. ATP-sensitive K+ channels may be involved but may open only at ATP concentrations well below those attained at exhaustion. However, ATP may be compartmentalized and only the membrane-bound ATP concentration may be of significance. Ca2+ accumulation and ATP depletion cause cell destruction; these changes induce an increased K+ conductance, which may inactivate the membrane and consequently prevent tension development. It is hypothesized that such a safety mechanism is identical to the fatigue mechanism.