Identification of a voltage-gated potassium channel in gerbil hippocampal mitochondria

Transient cerebral ischemia is known to induce endogenous mechanisms that can prevent or delay neuronal injury, such as the activation of mitochondrial potassium channels. However, the molecular mechanism of this effect remains unclear. In this study, the single-channel activity was measured using the patch-clamp technique of the mitoplasts isolated from gerbil hippocampus. In 70% of all patches, a potassium-selective current with the properties of a voltage-gated Kv-type potassium channel was recorded with mean conductance 109 ± 6 pS in a symmetrical solution. The channel was blocked at negative voltages and irreversibly by margatoxin, a specific Kv1.3 channel inhibitor. The ATP/Mg²⁺ complex and Ca²⁺ ions had no effect on channel activity. Additionally, agitoxin-2, a potent inhibitor of voltage-gated potassium channels, had no effect on mitochondrial channel activity. This observation suggests that in contrast to surface membrane channels, the mitochondrial voltage-gated potassium channel could have a different molecular structure with no affinity to agitoxin-2. Western blots of gerbil hippocampal mitochondria and immunohistochemistry on gerbil brain sections confirmed the expression of the Kv1.3 protein in mitochondria. Our findings indicate that gerbil brain mitochondria contain a voltage-gated potassium channel that can influence the function of mitochondria in physiological and pathological conditions and that has properties similar to the surface membrane Kv1.3 channel.     

Mitochondrial ATP-sensitive potassium channels in surgical cardioprotection

ATP-sensitive potassium channels allow for the coupling of membrane potential to cellular metabolic status. Two K(ATP) channel subtypes coexist in the myocardium with one subtype located in the sarcolemma membrane and the other in the inner membrane of the mitochondria. The ATP-sensitive potassium channels can be pharmacologically modulated by a family of structurally diverse agents of varied potency and selectivity, collectively known as potassium channel openers and blockers. Sufficient evidence exists to indicate that the ATP-sensitive potassium channels and in particular the mitochondrial ATP-sensitive potassium channels play an important role both as a trigger and an effector in surgical cardioprotection. In this review, the biochemistry and specificity of the ATP-sensitive potassium channels is examined in relation to surgical cardioprotection.     

Characterization of potassium channels in pancreatic beta cells from ob/ob mice

The patch-clamp technique in the cell-attached mode was used to study the K channels present in the membrane of cultured pancreatic beta cells from ob/ob mice. Three types of K+ channels were regularly observed, with conductances of 64, 20 and 146 pS. The conduction and kinetic properties of the 64 pS channel were similar to those of the ATP-sensitive potassium channel from normal beta cells. Furthermore, glucose blocked the activity of this channel at the same concentrations as that reported for normal cells. The 20 pS and the 146 pS were insensitive to glucose. The latter K+ channel appears to be similar to the large conductance voltage-activated potassium channels described in normal rodent beta cells. Thus, potassium channels in ob/ob pancreatic beta cells in culture are in most respects normal. Other factors may account for the abnormal electrical response to glucose of ob/ob pancreatic islets, such as reversible impairment of their function in vivo or defects not related to potassium permeability.     

Ion channels in human macrophages compared with the U-937 cell line

Summary The human cell line U-937 has been used extensively to model many macrophage functions. We have examined the cell membranes of human monocyte-derived macrophages (HMDM) and U-937 cells to compare membrane properties as expressed by single ion channel currents. The patch-clamp technique was applied to isolated, nonactivated, inside-out patches of cell membranes obtained from HMDM and from the U-937 cell line. Voltage-gated potassium channels of similar conductance but different kinetics are present in both types of cells, and a calcium-activated potassium channel is present only in the HMDM. These differences in ion channel properties suggest fundamentally different behavior between these two cell types at the level of the cell membrane.     

The antidiabetic sulfonylurea glibenclamide is a potent blocker of the ATP-modulated K+ channel in insulin secreting cells

The ATP-sensitive K+ channel of RINm5F insulinoma cells is activated after an intracellular ATP depletion. This activation can be followed by 86Rb+ efflux. Once activated by ATP depletion, the K+ channel can be blocked by the hypoglycemic drug, glibenclamide. The blockade is of a high-affinity type (K0.5 = 0.06 nM). Recording of the activity of ATP-sensitive K+ channels with the patch-clamp technique confirmed that they could be completely blocked with 20 nM glibenclamide.     

An ATP, calcium and voltage sensitive potassium channel in porcine coronary artery smooth muscle cells

There is increasing interest in the roles played by potassium channels of smooth muscle in protecting against ischemic and anoxic insults. Hence, potassium-selective channels were studied in freshly dispersed porcine coronary artery smooth muscle cells using the inside-out variant of the patch-clamp technique. The most abundant potassium channel had a conductance of 148 pS in a 5.4/140 mM K+ gradient, at 0 mV, and was regulated by cytoplasmic ATP (0.05-3.0 mM), cytoplasmic Ca2+ (0.1-10 microM) and voltage. ATP and AMP-PNP (0.5 mM) reduced the probability of channel opening (Po) by 87 and 92%, respectively. This inhibition was partially reversed by the addition of 0.5 mM ADP. ADP on its own (2 mM) reduced Po by 46%. It appears, therefore, that this channel shares properties with both the ATP-sensitive and the calcium-regulated potassium channels, raising the possibility that it plays a central role in the regulation of coronary blood flow.     

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

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

颈动脉注射腺苷对去缓冲神经大鼠延髓腹外侧头端区神经元放电的影响

在28只切断双侧缓冲神经的Sprague－Dawley大鼠,应用细胞外记录方法,观察了72个自发放电单位中颈动脉注射腺苷对延髓腹外侧头端（RVLM）区神经元自发放电活动的影响。所得结果如下：（1）颈动脉注射腺苷（25μg/kg）,31个单位的放电频率由23．5±3．0下降至（16．5±2．6）spikes／s（P＜0．001）,血压和心率无明显变化（P＞0．05）;（2）在24个单位中,应用非选择性腺苷受体拮抗剂8-苯茶碱（8-phenyltheophylline,15μg/kg）和选择性腺苷A1受体持抗剂8-环戊-1,3-二丙基黄嘌呤（8－cyclopentyl-1,3－dipropylxanthine,50μg/kg）均可完全阻断腺苷的抑制效应;（3）在应用ATP敏感性钾通道阻断剂格列苯脲（500μg/kg）的12个单位中,腺苷的上述效应亦被消除。以上结果提示,腺苷对RVLM区神经元自发放电有抑制作用,而此作用与A1受体介导的ATP敏感性钾通道开放有关。     

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.     

PKA-mediated phosphorylation of the human K(ATP) channel: separate roles of Kir6.2 and SUR1 subunit phosphorylation.

ATP-sensitive potassium (K(ATP)) channels play important roles in many cellular functions such as hormone secretion and excitability of muscles and neurons. Classical ATP-sensitive potassium (K(ATP)) channels are heteromultimeric membrane proteins comprising the pore-forming Kir6.2 subunits and the sulfonylurea receptor subunits (SUR1 or SUR2). The molecular mechanism by which hormones and neurotransmitters modulate K(ATP) channels via protein kinase A (PKA) is poorly understood. We mutated the PKA consensus sequences of the human SUR1 and Kir6.2 subunits and tested their phosphorylation capacities in Xenopus oocyte homogenates and in intact cells. We identified the sites responsible for PKA phosphorylation in the C-terminus of Kir6.2 (S372) and SUR1 (S1571). Kir6.2 can be phosphorylated at its PKA phosphorylation site in intact cells after G-protein (Gs)-coupled receptor or direct PKA stimulation. While the phosphorylation of Kir6.2 increases channel activity, the phosphorylation of SUR1 contributes to the basal channel properties by decreasing burst duration, interburst interval and open probability, and also increasing the number of functional channels at the cell surface. Moreover, the effect of PKA could be mimicked by introducing negative charges in the PKA phosphorylation sites. These data demonstrate direct phosphorylation by PKA of the K(ATP) channel, and may explain the mechanism by which Gs-coupled receptors stimulate channel activity. Importantly, they also describe a model of heteromultimeric ion channels in which there are functionally distinct roles of the phosphorylation of the different subunits.     

Single potassium channels of human glioma cells.

Single potassium channels in the membrane of human malignant glioma cells U-118MG were studied using the technique of patch clamp in cell-attached and inside-out configurations. Three types of potassium channels were found which differed from each other under conditions close to physiological in their conductance and gating characteristics. The lowest-conductance channel (20 pS near the reversal potential) showed a mild outward rectification up to 45 pS at positive voltages and spontaneous modes of high and low activity. At extreme values of potentials its activity was generally low. The intermediate conductance channel had an S-shaped I-V curve, giving a conductance of 63 pS at reversal, and a low and voltage independent opening probability. The high-conductance (215 pS) channel was found to be activated by both membrane potential and Ca2+ ions and blocked by internal sodium at high voltages. The current-voltage curves of all three channel types displayed saturation.     

Modification of potassium channel kinetics by histidine-specific reagents

We have examined the actions of histidine-specific reagents on potassium channels in squid giant axons. External application of 20-500 microM diethylpyrocarbonate (DEP) slowed the opening of potassium channels with little or no effect on closing rates. Sodium channels were not affected by these low external concentrations of DEP. Internal application of up to 2 mM DEP had no effect on potassium channel kinetics. Steady-state potassium channel currents were reduced in an apparently voltage-dependent manner by external treatment with this reagent. The shape of the instantaneous current-voltage relation was not altered. The voltage-dependent probability of channel opening was shifted toward more positive membrane potentials, thus accounting for the apparent voltage-dependent reduction of steady-state current. Histidine-specific photo-oxidation catalyzed by rose bengal produced alterations in potassium channel properties similar to those observed with DEP. The rate of action of DEP was consistent with a single kinetic class of histidine residues. In contrast to the effects on ionic currents, potassium channel gating currents were not modified by treatment with DEP. These results suggest the existence of a histidyl group (or groups) on the external surface of potassium channels important for a weakly voltage-dependent conformational transition. These effects can be reproduced by a simple kinetic model of potassium channels.     

Potassium channels in the mitochondria of unicellular eukaryotes and plants

The functional characterisation of potassium channels found in the mitochondria of plants and unicellular eukaryotes is critically discussed herein, with a focus on the ATP-sensitive potassium channel and the large-conductance Ca²⁺-activated potassium channel (mitoBK_Ca channel). The physiological functions of these channels are not completely understood. We discuss the functional connections and roles of potassium channels, uncoupling protein and alternative oxidase, three energy-dissipating systems that exist in the mitochondrial respiratory chain of plants and some unicellular eukaryotes, which include preventing the production of reactive oxygen species.     

Involvement of K(+) channels in the inhibitory effects of adenosine on anoxia-induced [Ca(2+) ](i) increase in cultured rat hippocampal CA1 neurons.

Changes in intracellular free Ca(2+) concentration ([Ca(2+)](i)) in cultured hippocampal CA1 neurons isolated from newborn rats were measured by a confocal laser scanning microscope, using the Ca(2+) indicator Fluo-3. The results showed that exogenous adenosine (100 microM) significantly attenuated the increase of neuronal [Ca(2+)](i) induced by acute anoxia. This effect of adenosine could be suppressed by the adenosine A1 receptor antagonist 8-cyclopentyltheophylline. Moreover, potassium channel blockers, aminopyridine, and glipizide could also block the inhibitory role of adenosine, but tetraethylammonium had no effect. These results suggest that adenosine may activate 4-AP or ATP-sensitive potassium channels via an A1-receptor-mediated mechanism and consequently inhibit anoxia-induced [Ca(2+)](i) elevation in hippocampal neurons.     

颈动脉内注射腺苷对去缓冲神经大鼠最后区神经元放电的影响

在45只切断双侧缓冲神经的SpragueDawley大鼠,应用细胞外记录方法,观察了颈动脉内注射腺苷对76个最后区(AP)神经元自发放电活动的影响。所得结果如下:(1)在记录到的42个自发放电单位中,颈动脉内注射腺苷(25μg/kg)引起其中29个单位的放电频率由626±075下降至474±076spikes/s(P<001),6个单位放电频率由413±077增加至472±083spikes/s(P<005),另外7个单位放电频率无明显变化,而血压和心率在实验中无变化;(2)在应用非选择性腺苷受体拮抗剂8苯茶碱(8phenyltheophylline,15μg/kg)的10个单位,腺苷对放电的抑制效应可被完全阻断;(3)应用选择性腺苷A1受体拮抗剂8环戊1,3二丙基黄嘌呤(8cyclopentyl1,3dipropylxanthine,50μg/kg)亦可有效地阻断腺苷对12个单位的抑制效应;(4)应用ATP敏感性钾通道阻断剂格列苯脲(500μg/kg)的12个单位,腺苷的上述效应也被消除。以上结果提示,腺苷对AP区神经元自发放电有抑制作用,而此作用与A1受体介导的ATP敏感性钾通道开放有关。     

Sulfonylureas correct trafficking defects of ATP-sensitive potassium channels caused by mutations in the sulfonylurea receptor

The pancreatic ATP-sensitive potassium (K(ATP)) channel, a complex of four sulfonylurea receptor 1 (SUR1) and four potassium channel Kir6.2 subunits, regulates insulin secretion by linking metabolic changes to beta-cell membrane potential. Sulfonylureas inhibit K(ATP) channel activities by binding to SUR1 and are widely used to treat type II diabetes. We report here that sulfonylureas also function as chemical chaperones to rescue K(ATP) channel trafficking defects caused by two SUR1 mutations, A116P and V187D, identified in patients with congenital hyperinsulinism. Sulfonylureas markedly increased cell surface expression of the A116P and V187D mutants by stabilizing the mutant SUR1 proteins and promoting their maturation. By contrast, diazoxide, a potassium channel opener that also binds SUR1, had no effect on surface expression of either mutant. Importantly, both mutant channels rescued to the cell surface have normal ATP, MgADP, and diazoxide sensitivities, demonstrating that SUR1 harboring either the A116P or the V187D mutation is capable of associating with Kir6.2 to form functional K(ATP) channels. Thus, sulfonylureas may be used to treat congenital hyperinsulinism caused by certain K(ATP) channel trafficking mutations.     

Inhibitors of ATP-sensitive potassium channels stimulate intestinal cholecystokinin secretion

Recently, a role for adenosine 5′-triphosphate(ATP)-sensitive potassium channels in the regulation of cholecystokinin (CCK) secretion has been described in STC-1 cells, an intestinal CCK-secreting cell line. To examine whether a similiar mechanism might participate in the regulation of hormone secretion from native CCK cells, the effects of two established inhibitors of ATP-sensitive potassium channels (e.g. glucose, disopyramide) were examined on CCK release from dispersed murine intestinal cells. Both glucose and disopyramide were found to stimulate CCK secretion. Furthermore, CCK release induced by glucose was inhibited by the calcium channel blocker diltiazem. It is concluded that, ATP-sensitive potassium channels may play a role in the regulation of intestinal CCK secretion.     

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.     

A novel sulfonylurea receptor family member expressed in the embryonic Drosophila dorsal vessel and tracheal system.

Sulfonylurea receptors (SURx) are required subunits of the ATP-sensitive potassium channel. SURx alone is electrophysiologically inert. However, when SURx is combined with an inward rectifier Kir6.2 subunit, ATP-sensitive potassium channel activity is generated. We report the identification, characterization, and localization of Dsur, a novel Drosophila gene that is highly related to the vertebrate SUR family. The Dsur coding sequence contains structural features characteristic of the ABC transporter family and, in addition, harbors 1.7 kilobases of a distinctive sequence that does not share homology with any known gene. When Dsur alone is expressed in Xenopus oocytes glibenclamide-sensitive potassium channel activity occurs. During Drosophila embryogenesis, the Dsur gene is specifically expressed in the developing tracheal system and dorsal vessel. Studies of the Drosophila genome support that only a single Dsur gene is present. Our data reveal conservation of glibenclamide-sensitive potassium channels in Drosophila and suggest that Dsur may play an important role during Drosophila embryogenesis. The lack of gene duplication in the Drosophila system provides a unique opportunity for functional studies of SUR using a genetic approach.     

Putative Structural and Functional Coupling of the Mitochondrial BKCa Channel to the Respiratory Chain

Potassium channels have been found in the inner mitochondrial membranes of various cells. These channels regulate the mitochondrial membrane potential, the matrix volume and respiration. The activation of these channels is cytoprotective. In our study, the single-channel activity of a large-conductance Ca²⁺-regulated potassium channel (mitoBK_Ca channel) was measured by patch-clamping mitoplasts isolated from the human astrocytoma (glioblastoma) U-87 MG cell line. A potassium-selective current was recorded with a mean conductance of 290 pS in symmetrical 150 mM KCl solution. The channel was activated by Ca²⁺ at micromolar concentrations and by the potassium channel opener NS1619. The channel was inhibited by paxilline and iberiotoxin, known inhibitors of BK_Ca channels. Western blot analysis, immuno-gold electron microscopy, high-resolution immunofluorescence assays and polymerase chain reaction demonstrated the presence of the BK_Ca channel β4 subunit in the inner mitochondrial membrane of the human astrocytoma cells. We showed that substrates of the respiratory chain, such as NADH, succinate, and glutamate/malate, decrease the activity of the channel at positive voltages. This effect was abolished by rotenone, antimycin and cyanide, inhibitors of the respiratory chain. The putative interaction of the β4 subunit of mitoBK_Ca with cytochrome c oxidase was demonstrated using blue native electrophoresis. Our findings indicate possible structural and functional coupling of the mitoBK_Ca channel with the mitochondrial respiratory chain in human astrocytoma U-87 MG cells.