Redox regulation of the mitochondrial KATP channel in cardioprotection

The mitochondrial ATP-sensitive potassium channel (mK_ATP) is important in the protective mechanism of ischemic preconditioning (IPC). The channel is reportedly sensitive to reactive oxygen and nitrogen species, and the aim of this study was to compare such species in parallel, to build a more comprehensive picture of mK_ATP regulation. mK_ATP activity was measured by both osmotic swelling and Tl⁺ flux assays, in isolated rat heart mitochondria. An isolated adult rat cardiomyocyte model of ischemia-reperfusion (IR) injury was also used to determine the role of mK_ATP in cardioprotection by nitroxyl. Key findings were as follows: (i) mK_ATP was activated by O₂⁻ and H₂O₂ but not other peroxides. (ii) mK_ATP was inhibited by NADPH. (iii) mK_ATP was activated by S-nitrosothiols, nitroxyl, and nitrolinoleate. The latter two species also inhibited mitochondrial complex II. (iv) Nitroxyl protected cardiomyocytes against IR injury in an mK_ATP-dependent manner. Overall, these results suggest that the mK_ATP channel is activated by specific reactive oxygen and nitrogen species, and inhibited by NADPH. The redox modulation of mK_ATP may be an underlying mechanism for its regulation in the context of IPC. This article is part of a Special Issue entitled: Mitochondria and Cardioprotection.     

大鼠海马CA1区锥体细胞上一种Ca2+依赖性KATP通道

K_ATP通道在细胞的新陈代谢与膜兴奋性的耦联中起重要作用.采用膜片钳的内面向外式记录方法,在成年大鼠海马CA1区锥体细胞上记录到一种被胞浆侧ATP和甲糖宁（tolbutamide,一种K_ATP通道阻断剂）抑制的Ca²⁺依赖性钾离子通道.在细胞膜内外的K⁺浓度均为140 mmol/L时,通道的电导为(204±21) pS,翻转电位为(3.57±1.13) mV,通道无整流性.通道开放概率及ATP对通道的抑制作用均呈现电压依赖性.该K_ATP通道与以往报道的“经典”K_ATP通道有显著不同,其活动受膜电位、胞内Ca²⁺和ATP三重调节,表明这是一种新型的K_ATP通道.上述结果表明在海马神经元上至少有两种性质不同的K_ATP通道,提示神经元可能通过不同性质的K_ATP通道感受细胞内的代谢状态,进而调节细胞膜的兴奋性.     

Potential role of potassium channel openers in the treatment of asthma and chronic obstructive pulmonary disease

Airway smooth muscle (ASM) cells express various types of potassium (K+) channels which play a key role in determining the resting membrane potential, a relative electrical stability and the responsiveness to both contractile and relaxant agents. In addition, K+ channels are also involved in modulation of neurotransmitter release from airway nerves. The most important K+ channels identified in airways include large and small Ca2+-activated, delayed-rectifier, and ATP-sensitive channels. These K+ channels are structurally and functionally different, thus playing distinct roles in airway electrophysiology and pharmacology. Many in vitro and in vivo studies, performed in both animals and humans, have shown that K+ channel openers are able to induce hyperpolarization of ASM cells, bronchodilation, suppression of airway hyperresponsiveness (AHR), and inhibition of neural reflexes. Therefore, airway K+ channels represent a suitable pharmacological target for the development of new effective therapeutic options in the treatment of asthma and chronic obstructive pulmonary disease (COPD).     

Identification of two types of ATP-sensitive K+ channels in rat ventricular myocytes

The ATP-sensitive K(+) (K(ATP)) channels are known to provide a functional linkage between the electrical activity of the cell membrane and metabolism. Two types of inwardly rectifying K(+) channel subunits (i.e., Kir6.1 and Kir6.2) with which sulfonylurea receptors are associated were reported to constitute the K(ATP) channels. In this study, we provide evidence to show two types of K(ATP) channels with different biophysical properties functionally expressed in isolated rat ventricular myocytes. Using patch-clamp technique, we found that single-channel conductance for the different two types of K(ATP) channels in these cells was 57 and 21 pS. The kinetic properties, including mean open time and bursting kinetics, did not differ between these two types of K(ATP) channels. Diazoxide only activated the small-conductance K(ATP) channel, while pinacidil and dinitrophenol stimulated both channels. Both of these K(ATP) channels were sensitive to block by glibenclamide. Additionally, western blotting, immunochemistry, and RT-PCR revealed two types of Kir6.X channels, i.e., Kir6.1 and Kir6.2, in rat ventricular myocytes. Single-cell Ca(2+) imaging also revealed that similar to dinitrophenol, diazoxide reduced the concentration of intracellular Ca(2+). The present results suggest that these two types of K(ATP) channels may functionally be related to the activity of heart cells.     

Induction of anhydrobiosis in fat body tissue from an insect

The larva of the African chironomid Polypedilum vanderplanki can withstand complete desiccation. Our previous reports revealed that even when the larva is dehydrated without a brain, it accumulated a great amount of trehalose and successfully went into anhydrobiosis. In this paper we determined the viability after rehydration in tissues from the larvae followed by complete dehydration. Only fat-body tissues that were the main producer of trehalose could be preserved in a dry state at room temperature for an extended period of more than 18 months in a viable form. Thus we have confirmed that the central nervous system is not involved in the induction of anhydrobiosis, even in this complex multicellular organism.     

Effects of pyridine nucleotides on the gating of ATP-sensitive potassium channels in insulin-secreting cells

Summary The single-channel current recording technique has been used to study the influences that the pyridine nucleotides NAD, NADH, NADP and NADPH have on the gating of ATP-sensitive K⁺ channels in an insulin-secreting cell line (RINm5F). The effects of the nucleotides were studied at the intracellular surface using either excised inside-out membrane patches or permeabilized cells. All four pyridine nucleotides were found to evoke similar effects. At low concentrations, 100 m and less, each promoted channel opening whereas high concentrations, 500 m and above, evoked channel closure. The degree of K⁺ channel activation by pyridine nucleotides (low conc.) was found to be similar to that evoked by the same concentrations of ADP or GTP, whereas the degree of K⁺ channel inhibition (high conc.) was less marked than that evoked by the same concentrations of ATP, and never resulted in refreshment of K⁺ channels following removal. The effects of NAD, NADH, NADP and NADPH seemed to interact with those of ATP and ADP. In the presence of 1mm ADP and 4mm ATP, 10 to 100 m concentrations of the pyridine nucleotides could not evoke channel opening, whereas concentrations of 500 m and above were found to evoke channel closure. In the presence of 2mm ATP and 0.5mm ADP, however, 10 to 100 m concentrations of the pyridine nucleotides were able to activate K⁺ channels.     

Separation of heteromeric potassium channel Kcv towards probing subunit composition-regulated ion permeation and gating

The chlorella virus-encoded Kcv can form a homo-tetrameric potassium channel in lipid membranes. This miniature peptide can be synthesized in vitro, and the tetramer purified from the SDS-polyacrylamide gel retains the K⁺ channel functionality. Combining this capability with the mass-tagging method, we propose a simple, straightforward approach that can generically manipulate individual subunits in the tetramer, thereby enabling the detection of contribution from individual subunits to the channel functions. Using this approach, we showed that the structural change in the selectivity filter from only one subunit is sufficient to cause permanent channel inactivation (“all-or-none” mechanism), whereas the mutation near the extracellular entrance additively modifies the ion permeation with the number of mutant subunits in the tetramer (“additive” mechanism).     

IP3 decreases coronary artery tone via activating the BKCa channel of coronary artery smooth muscle cells in pigs

Large conductance Ca²⁺-activated K⁺ channel (BK_Ca) is a potential target for coronary artery-relaxing medication, but its functional regulation is largely unknown. Here, we report that inositol trisphosphate (IP3) activated BK_Ca channels in isolated porcine coronary artery smooth muscle cells and by which decreased the coronary artery tone. Both endogenous and exogenous IP3 increased the spontaneous transient outward K⁺ currents (STOC, a component pattern of BK_Ca currents) in perforated and regular whole-cell recordings, which was dependent on the activity of IP3 receptors. IP3 also increased the macroscopic currents (MC, another component pattern of BK_Ca currents) via an IP3 receptor- and sarcoplasmic Ca²⁺ mobilization-independent pathway. In inside-out patch recordings, direct application of IP3 to the cytosolic side increased the open probability of single BK_Ca channel in an IP3 receptor-independent manner. We conclude that IP3 is an activator of BK_Ca channels in porcine coronary smooth muscle cells and exerts a coronary artery-relaxing effect. The activation of BK_Ca channels by IP3 involves the enhancement of STOCs via IP3 receptors and stimulation of MC by increasing the Ca²⁺ sensitivity of the channels.     

Simultaneous influx of ammonium and potassium into maize roots: kinetics and interactions

The interaction between ammonium and potassium during influx was examined in roots of dark-grown decapitated corn seedlings (Zea mays L., cv. Pioneer 3369A). Influx was measured during a 10-min exposure to either (¹⁵NH₄)₂SO₄ ranging from 10 to 200 M NH ₄ ⁺ with and without 200 M K(⁸⁶Rb)Cl or to K(⁸⁶Rb)Cl ranging from 10 to 200 M K⁺ with and without 200 M NH ₄ ⁺ as (¹⁵NH₄)₂SO₄. The simple Michaelis-Menten model described the data well only for potassium influx in the presence of ambient ammonium. For the other three instances, the data were improved by assuming that a second influx mechanism became operative as the low-concentration phase approached saturation. Two distinct mechanisms are thus indicated for both ammonium and potassium influx within the range of 10 to 200 M.The influx mechanism operating at low concentrations showed greater affinity for potassium than for ammonium, even though the capacity for ammonium transport was twice as large as that for potassium. It is suggested that this phase involved a common transport system for the two ions and that localized low acidity next to the internal surface, following H⁺ extrusion, favored ammonium deprotonation and dissociation from the transport system-ammonium complex. Parallel decreases in V _max and increases in K_m of the low-concentration saturable phase occurred for ammonium influx when ambient potassium was present and for potassium influx when ambient ammonium was present. The data support a mixed-type inhibition in each case. Simultaneous measurement of potassium and ammonium influx showed that they were highly negatively correlated at the lower concentrations, indicating that the extent to which influx of the inhibited ion was restricted was associated with influx of the inhibitor ion. Presence of ambient ammonium eliminated the second phase of potassium influx. In contrast, the presence of ambient potassium decreased the concentration at which the second phase of ammonium influx was initiated but did not restrict the rate.Paper no. 11131 of the Journal Series of the North Carolina Agricultural Research ServiceThe use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Research Service of the products named, nor criticism of similar ones not mentioned     

Reactions of the fac-[Re(CO)3] and [ReO] moieties with substituted benzothiazoles

The reaction of 2-(2-aminophenyl)benzothiazole (Habt) with [Re(CO)₅Br] led to the isolation of the rhenium(I) complex fac-[Re(Habt)(CO)₃Br] (1). With trans-[ReOCl₃(PPh₃)₂], the ligand Habt decomposed to form the oxofree rhenium(V) complex [Re(itp)₂Cl(PPh₃)] (2) (itp = 2-amidophenylthiolate). From the reaction of trans-[ReOBr₃(PPh₃)₂] with 2-(2-hydroxyphenyl)benzothiazole (Hhpd) the complex [Re^VOBr₂(hpd)(PPh₃)] (3) was obtained. Complexes 1-3 are stable and lipophilic. ¹H NMR and infrared assignments, as well as the X-ray crystal structures, of the complexes are reported.     

PKA-dependent activation of the vascular smooth muscle isoform of KATP channels by vasoactive intestinal polypeptide and its effect on relaxation of the mesenteric resistance artery

Vasoactive intestinal polypeptide (VIP) is a potent vasodilator and has been successfully used to alleviate hypertension. Consistently, disruption of VIP gene in mice leads to hypertension. However, its downstream targets in the vascular regulation are still not well demonstrated. To test the hypothesis that the vascular smooth muscle isoform of K_ATP channels is a downstream target of the VIP signaling, we performed the studies on the Kir6.1/SUR2B channel expressed in HEK293 cells. We found that the channel was strongly activated by VIP. Through endogenous VIP receptors, the channel activation was reversible and dependent on VIP concentrations with the midpoint-activation concentration ∼ 10 nM. The channel activation was voltage-independent and could be blocked by K_ATP channel blocker glibenclamide. In cell-attached patches, VIP augmented the channel open-state probability with modest suppression of the single channel conductance. The VIP-induced Kir6.1/SUR2B channel activation was blocked by PKA inhibitor RP-cAMP. Forskolin, an adenylyl cyclase activator, activated the channel similarly as VIP. The effect of VIP was further evident in the native tissues. In acutely dissociated mesenteric vascular smooth myocytes, VIP activated the K_ATP currents in a similar manner as in HEK293 cells. In endothelium-free mesenteric artery rings, VIP produced concentration-dependent vasorelaxation that was attenuated by glibenclamide. These results therefore indicate that the vascular isoform (Kir6.1/SUR2B) of K_ATP channels is a target of VIP. The channel activation relies on the PKA pathway and produces mesenteric arterial relaxation.     

Acclimation of potassium influx in rye (Secale cereale) to low root temperatures

The influx of K⁺(⁸⁶Rb⁺) into intact roots of rye (Secale cereale L. cv. Rheidal) exposed to a differential temperature (DT) between the root (8° C) and shoot (20° C) is initially reduced compared with warm-grown (WG) controls with both shoot and root maintained at 20° C. Over a period of 3 d, however, K⁺-influx rates into DT plants are restored to levels similar to or greater than those of the WG controls, the absolute rates of K⁺ influx being strongly dependent upon the shoot/root ratio. Acclimation in DT plants results in a reduction of K⁺ influx into the apical (0–2 cm) region of the seminal root which is associated with a compensatory increase in K⁺ influx into the more mature, basal regions of the root. Values of V _max and apparent K _m for K⁺ influx into DT plants were similar to those for WG plants at assay temperatures of 8° C and 20° C except for an increase in the apparent K _m at 8° C. The influx of K⁺ from solutions containing 0.6 mol·m^-3 K⁺ into both WG and DT plants was found to be linearly related to assay temperature over the range 2–27° C, and the temperature sensitivity of K⁺ influx to be dependent upon shoot/root ratio. At high shoot/root ratios, the ratio of K⁺ influx at 20° C:K⁺ influx at 8° C for WG plants approached a minimum value of 1.9 whereas that for DT plants approached unity indicating that K⁺ influx into DT plants has a large temperature-insensitive component. Additionally, when plants were grown in solutions of low potassium concentration, K⁺ influx into DT plants was consistently greater than that into WG plants, in spite of having a greater root potassium concentration ([K⁺]int). This result indicates some change in the regulation of K⁺ influx by [K⁺]int in plants exposed to low root temperatures. We suggest that K⁺ influx into rye seedlings exposed to low root temperatures is regulated by the increased demand placed on the root system by a proportionally larger shoot and that the acclimation of K⁺ influx to low temperatures may be the result of an increased hydraulic conductivity of the root system.Abbreviations DT differential temperature pretreatment - [K⁺]int root potassium concentration - [K⁺]ext potassium concentration of nutrient medium - WG warm-grown pretreatment     

Cardiac mitochondrial ATP-sensitive potassium channel is activated by nitric oxide in vitro

Previous observations on the activation of the mitochondrial ATP-sensitive potassium channel (mitoK(ATP)) by nitric oxide (NO) in myocardial preconditioning were based on indirect evidence. In this study, we have investigated the direct effect of NO on the rat cardiac mitoK(ATP) after reconstitution of the inner mitochondrial membranes into lipid bilayers. We found that the mitoK(ATP) was activated by exogenous NO donor S-nitroso-N-acetyl penicillamine or PAPA NONOate. This activation was inhibited by mitoK(ATP) blockers 5-hydroxydecanoate or glibenclamide. Our observations confirm that NO can directly activate the cardiac mitoK(ATP), which may underlie its contribution to myocardial preconditioning.     

Closure of the yeast mitochondria unspecific channel (YMUC) unmasks a Mg2+ and quinine sensitive K+ uptake pathway in Saccharomyces cerevisiae

The K⁺ uptake pathways in yeast mitochondria are still undefined. Nonetheless, the K⁺-mediated mitochondrial swelling observed in the absence of phosphate (PO₄) and in the presence of a respiratory substrate has led to propose that large K⁺ movements occur in yeast mitochondria. Thus, the uptake of K⁺ by isolated yeast mitochondria was evaluated. Two parallel experiments were conducted to evaluate K⁺ transport; these were mitochondrial swelling and the uptake of the radioactive K⁺ analog ⁸⁶Rb⁺. The opening of the yeast mitochondrial unspecific channel (YMUC) was regulated by different PO₄ concentrations. The high protein concentrations used to measure ⁸⁶Rb⁺ uptake resulted in a slight stabilization of the transmembrane potential at 0.4 mM PO₄ but not at 0 or 4 mM PO₄. At 4 mM PO₄ swelling was inhibited while, in contrast, ⁸⁶Rb⁺ uptake was still observed. The results suggest that an energy-dependent K⁺ uptake mechanism was unmasked when the YMUC was closed. To further analyze the properties of this K⁺ uptake system, the Mg²⁺ and quinine sensitivity of both swelling and ⁸⁶Rb⁺ uptake were evaluated. Under the conditions where the unspecific pore was closed, K⁺ transport sensitivity to Mg²⁺ and quinine increased. In addition, when Zn²⁺ was added as an antiport inhibitor, uptake of ⁸⁶Rb⁺ increased. It is suggested that in yeast mitochondria, the K⁺ concentration is highly regulated by the equilibrium of uptake and exit of this cation through two specific transporters.     

Cellular and tissue distribution of potassium: Physiological relevance,mechanisms and regulation

Potassium (K⁺) is the most important cationic nutrient for all living organisms. Its cellular levels are significant (typically around 100 mM) and are highly regulated. In plants K⁺ affects multiple aspects such as growth, tolerance to biotic and abiotic stress and movement of plant organs. These processes occur at the cell, organ and whole plant level and not surprisingly, plants have evolved sophisticated mechanisms for the uptake, efflux and distribution of K⁺ both within cells and between organs.     

The efficiency of (Na + K)-ATPase in tumorigenic cells

The efficiency of (Na⁺ + K⁺)-ATPase (i.e. the amount of K⁺ pumped per ATP hydrolyzed) in intact tumorigenic cells was estimated in this study. This was accomplished by simultaneously measuring the rate of ouabain-sensitive K⁺ uptake and oxygen consumption in tumorigenic cell suspensions during the reintroduction of K⁺ to K⁺-depleted cells. The ATP turnover was then estimated by assuming 5.6–6 ATP/O₂ as the stoichiometry of NADH-linked respiration in these cells. In the three cell lines tested (hamster and chick embryo cells transformed with Rous sarcoma virus and Ehrlich ascites cells), the K⁺/ATP ratio was approximately 2, the same value as that found in normal tissues. Furthermore, only 20% of the total ATP production of these cells was used by (Na⁺ + K⁺)-ATPase.     

Investigations of the inhibitory effect of propranolol,chlorpromazine, quinine,and dicyclohexylcarbodiimide on the swelling of yeast mitochondria in potassium acetate. Evidences for indirect effects mediated by the lipid phase

The mode of action of propranolol, chlorpromazine, and quinine, three cationic drugs inhibiting swelling of yeast mitochondria in potassium acetate, was investigated by looking at their effect on fluorescent probes of the polar heads and of the nonpolar moiety of the membranes, under inhibitory conditions of swelling. As expected, propranolol and chlorpromazine exhibited specificity for anionic phospholipids since they increased the binding of the anionic probe 1-anilino 8-naphthalenesulfonate (ANS). Although propranolol did not release 1,6-diphenyl-1,3,5-hexatriene (DPH) from the hydrophobic moiety of the membrane, it increased the excimer/ monomer fluorescence ratio of 10-(1-pyrene)decanoate, suggesting that it induced a limitation in the movements of the aliphatic chains of phospholipids. Opposite to propranolol, chlorpromazine removed DPH from the membrane, suggesting that it bound essentially to the hydrophobic moiety. However, chloramphenicol, which was also able to remove DPH but did not increase the binding of ANS, did not inhibit swelling. Inhibition by chlorpromazine therefore appeared to be related to its binding to the hydrophobic moiety of anionic phospholipids. Quinine had no effect on membrane properties: at inhibitory concentrations of swelling in potassium acetate, it did not inhibit swelling in ammonium phosphate (mediated by the phosphate/H⁺ cotransporter), whereas propranolol and chlorpromazine did, suggesting a more specific effect of quinine on (a) protein(s) involved in the K⁺/H⁺ exchange. Dicyclohexylcarbodiimide (DCCD), which irreversibly inhibits the swelling in potassium acetate, bound to ethanolamine heads; despite this effect, DCCD had no major consequences on the binding of the probes. Consequently, propranolol and chlorpromazine are of no help for characterizing protein(s) catalyzing the K⁺/H⁺ exchange, although their effect on lipids seems to involve limited zones of the inner mitochondrial membrane. Quinine and DCCD, although they also bind to lipids, may inhibit the activity by acting on a limited number of proteins.