ATP-regulated Potassium Channels Research Articles

The Role of Ion Channels and Chemokines in Cancer Growth and Metastasis: A Proposed Mode of Action Using Peptides in Cancer Therapy.

Metastasis (Met) largely contributes to the major cause of cancer deaths throughout the world, rather than the growth of the tumor mass itself. The present report brings together several of the pertinent contributors to cancer growth and metastatic processes from an activity standpoint. Such biological activities include the following: (1) cell adherence and detachment; (2) cell-to-cell contact; (3) contact inhibition; (4) the cell interfacing with the extracellular matrix (ECM); (5) tumor cell-to-stroma communication networks; (6) chemotaxis; and (7) cell membrane potential. Moreover, additional biochemical factors that contribute to cancer growth and metastasis have been shown to comprise the following: (a) calcium levels in the extracellular matrix and in intracellular compartments; (b) cation voltage and ATP-regulated potassium channels; (c) selective and non-selective cation channels; and (d) chemokines (cytokines) and their receptors, such as CXCL12 (SDF-1) and its receptor/binding partner, CXCR4. These latter molecular components represent a promising group of an interacting and synchronized set of candidates ideal for peptide therapeutic targeting for cancer growth and metastasis. Such peptides can be obtained from naturally occurring proteins such as alpha-fetoprotein (AFP), an onco-fetal protein and clinical biomarker.

Interaction of ROMK2 channel with lipid kinases DGKE and AGK: Potential channel activation by localized anionic lipid synthesis

In this study, we utilized enzyme-catalyzed proximity labeling with the engineered promiscuous biotin ligase Turbo-ID to identify the proxisome of the ROMK2 channel. This channel resides in various cellular membrane compartments of the cell including the plasma membrane, endoplasmic reticulum and mitochondria. Within mitochondria, ROMK2 has been suggested as a pore-forming subunit of mitochondrial ATP-regulated potassium channel (mitoKATP). We found that ROMK2 proxisome in addition to previously known protein partners included two lipid kinases: acylglycerol kinase (AGK) and diacylglycerol kinase ε (DGKE), which are localized in mitochondria and the endoplasmic reticulum, respectively. Through co-immunoprecipitation, we confirmed that these two kinases are present in complexes with ROMK2 channels. Additionally, we found that the products of AGK and DGKE, lysophosphatidic acid (LPA) and phosphatidic acid (PA), stimulated the activity of ROMK2 channels in artificial lipid bilayers. Our molecular docking studies revealed the presence of acidic lipid binding sites in the ROMK2 channel, similar to those previously identified in Kir2 channels. Based on these findings, we propose a model wherein localized lipid synthesis, mediated by channel-bound lipid kinases, contributes to the regulation of ROMK2 activity within distinct intracellular compartments, such as mitochondria and the endoplasmic reticulum.

Synthesis and SAR of a novel Kir6.2/SUR1 channel opener scaffold identified by HTS

Kir6.2/SUR1 is an ATP-regulated potassium channel that acts as an intracellular metabolic sensor, controlling insulin and appetite-stimulatory neuropeptides secretion. In this Letter, we present the SAR around a novel Kir6.2/SUR1 channel opener scaffold derived from an HTS screening campaign. New series of compounds with tractable SAR trends and favorable potencies are reported.

Pharmacological Characterization of a Recombinant Mitochondrial ROMK2 Potassium Channel Expressed in Bacteria and Reconstituted in Planar Lipid Bilayers

In the inner mitochondrial membrane, several potassium channels that play a role in cell life and death have been identified. One of these channels is the ATP-regulated potassium channel (mitoKATP). The ROMK2 potassium channel is a potential molecular component of the mitoKATP channel. The current study aimed to investigate the pharmacological modulation of the activity of the ROMK2 potassium channel expressed in Escherichia coli bacteria. ROMK2 was solubilized in polymer nanodiscs and incorporated in planar lipid bilayers. The impact of known mitoKATP channel modulators on the activity of the ROMK2 was characterized. We found that the ROMK2 channel was activated by the mitoKATP channel opener diazoxide and blocked by mitoKATP inhibitors such as ATP/Mg2+, 5-hydroxydecanoic acid, and antidiabetic sulfonylurea glibenclamide. These results indicate that the ROMK2 potassium protein may be a pore-forming subunit of mitoKATP and that the impact of channel modulators is not related to the presence of accessory proteins.

A critical role of the mechanosensor PIEZO1 in glucose-induced insulin secretion in pancreatic β-cells

Glucose-induced insulin secretion depends on β-cell electrical activity. Inhibition of ATP-regulated potassium (KATP) channels is a key event in this process. However, KATP channel closure alone is not sufficient to induce β-cell electrical activity; activation of a depolarizing membrane current is also required. Here we examine the role of the mechanosensor ion channel PIEZO1 in this process. Yoda1, a specific PIEZO1 agonist, activates a small membrane current and thereby triggers β-cell electrical activity with resultant stimulation of Ca2+-influx and insulin secretion. Conversely, the PIEZO1 antagonist GsMTx4 reduces glucose-induced Ca2+-signaling, electrical activity and insulin secretion. Yet, PIEZO1 expression is elevated in islets from human donors with type-2 diabetes (T2D) and a rodent T2D model (db/db mouse), in which insulin secretion is reduced. This paradox is resolved by our finding that PIEZO1 translocates from the plasmalemma into the nucleus (where it cannot influence the membrane potential of the β-cell) under experimental conditions emulating T2D (high glucose culture). β-cell-specific Piezo1-knockout mice show impaired glucose tolerance in vivo and reduced glucose-induced insulin secretion, β-cell electrical activity and Ca2+ elevation in vitro. These results implicate mechanotransduction and activation of PIEZO1, via intracellular accumulation of glucose metabolites, as an important physiological regulator of insulin secretion.

Influence of Mitochondrial ATP-Sensitive Potassium Channels on Toxic Effect of Amyloid-β 25–35

Amyloid-β (Aβ) is the main component of senile plaques, one of the hallmarks of Alzheimer,s disease. Is been shown that Aβ25–35 decreased neuronal viability while it increased generation of reactive oxygen species (ROS), and albumin (BSA) prevented ROS production and neuronal death in a dose-and time-dependent manner. One of the major sources of ROS is mitochondrion, and is believed that Mitochondrial ATP-regulated potassium channels (mitoKATP) protect synapses and neurons against oxidative and metabolic stress by modulating inner membrane potential and ROS production. Here we investigate the possible participation of MitoKATP channels on toxic effect of Aβ and the protective effect of BSA, by studying the influence of diazoxide (DIAZ) and tolbutamide (TOLB) on the effect of Aβ25–35 in neuronal morphology, cell viability and ROS generation in presence and absence of BSA. DIAZ decreased ROS generation induced by Aβ25–35 in a concentration dependent manner, but increased with the addition of BSA. TOLB increased Aβ25–35 effect on ROS production in a concentration dependent manner, but only in presence of BSA. Neither DIAZ nor TOLB rescued neurons from morphological damage and cell death induced by Aβ25–35. Hence, it could be proposed that MitoKATP channels participate on toxic effects of Aβ25–35, but not in protective effect of BSA, which seems to go through an extraneuronal mechanism.

Structure-Activity Relationships, Pharmacokinetics, and Pharmacodynamics of the Kir6.2/SUR1-Specific Channel Opener VU0071063.

Glucose-stimulated insulin secretion from pancreatic β-cells is controlled by ATP-regulated potassium (KATP) channels composed of Kir6.2 and sulfonylurea receptor 1 (SUR1) subunits. The KATP channel-opener diazoxide is FDA-approved for treating hyperinsulinism and hypoglycemia but suffers from off-target effects on vascular KATP channels and other ion channels. The development of more specific openers would provide critically needed tool compounds for probing the therapeutic potential of Kir6.2/SUR1 activation. Here, we characterize a novel scaffold activator of Kir6.2/SUR1 that our group recently discovered in a high-throughput screen. Optimization efforts with medicinal chemistry identified key structural elements that are essential for VU0071063-dependent opening of Kir6.2/SUR1. VU0071063 has no effects on heterologously expressed Kir6.1/SUR2B channels or ductus arteriole tone, indicating it does not open vascular KATP channels. VU0071063 induces hyperpolarization of β-cell membrane potential and inhibits insulin secretion more potently than diazoxide. VU0071063 exhibits metabolic and pharmacokinetic properties that are favorable for an in vivo probe and is brain penetrant. Administration of VU0071063 inhibits glucose-stimulated insulin secretion and glucose-lowering in mice. Taken together, these studies indicate that VU0071063 is a more potent and specific opener of Kir6.2/SUR1 than diazoxide and should be useful as an in vitro and in vivo tool compound for investigating the therapeutic potential of Kir6.2/SUR1 expressed in the pancreas and brain.

KATP channels in ductus arteriosus function and pathophysiology: mechanism of action and therapeutic potential

The ductus arteriosus (DA) is an essential fetal vessel connecting the pulmonary artery and aorta. In utero, the DA diverts blood flow away from the developing lungs, shunting it to the systemic circulation where gas exchange occurs within the placenta. After birth, the DA must close to allow adequate perfusion of the newly inflated lungs. Failure of the postnatal DA to constrict results in PDA (patent ductus arteriosus), one of the most common congenital cardiovascular disorders that is associated with significant morbidities and increased infant mortality. PDA disproportionately affects the most critically ill premature neonates, affecting 60–80% of infants weighing <1000g. The only drugs currently available to promote DA closure (indomethacin, ibuprofen, acetaminophen) non-specifically target the prostaglandin pathway, are associated with severe adverse side-effects, and are ineffective in ~30% of patients. Consequently, there is a significant need to identify novel DA-specific regulators of vascular tone in order to develop additional PDA therapeutic options. To this end, we demonstrated that vascular ATP-regulated potassium (KATP) channels, comprised of pore-forming Kir6.1 and regulatory SUR2B subunits, are developmentally regulated and significantly enriched in the mouse and human DA compared to other blood vessels. Of note, gain-of-function mutations in Kir6.1/SUR2B channels underlie Cantu syndrome, an autosomal dominant disorder in which 50% of patients have symptomatic PDA. Using isolated human and mouse DA myography assays and in vivo mouse models, we found that glibenclamide (a non-specific KATP channel inhibitor traditionally used to treat diabetes) induced DA constriction, while having relatively no effect on other vessel types. Moreover, inhibiting KATP channels made DAs more sensitive to ibuprofen-induced constriction, highlighting the potential of KATP channel inhibitors as adjuvant PDA therapies. Importantly, exposure to glibenclamide made human DAs more sensitive to oxygen-induced constriction, a finding of particular importance for cases of PDA in preterm infants who often have substantially lower pO2. Ongoing and future studies are focused on using high-throughput screening assays and medicinal chemistry to identify and optimize vascular-specific (Kir6.1/SUR2B) KATP channel inhibitors with the ultimate goal of developing novel DA-selective PDA therapies that are safer and more effective than those currently available. Support or Funding Information This work was supported by R21HL132805 and AHA15SDG25280015 awarded to ELS, R01DK082884 awarded to JSD, and R35HL140024 awarded to CGN (A) Pressure myography was used to measure changes in lumen diameter in dose response studies of the KATP channel inhibitor, glibenclamide. Vessels were isolated from preterm (day 15 of gestation) and term (day 19 of gestation) mice and mounted on glass pipette tips in microvessel perfusion chambers. (B) DAs are significantly more sensitive to glibenclamide-induced constriction compared to ascending aortas (aAO), descending aortas (dAO), and mesenteric arteries. (A) Neonatal human DA ring suspended in an organ bath for use in wire myography assay. (B) Quantification of results from 7 vessels. Increased tension corresponds to vasoconstriction, while decreased tension corresponds to vasodilation. Pinacidi (KATP channel activator) relaxes the human DA, while glibenclamide (KATP channel inhibitor) used in combination with oxygen makes vessels more sensitive to O2-induced constriction. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Evidence for a mitochondrial ATP-regulated potassium channel in human dermal fibroblasts

Mitochondrial ATP-regulated potassium channels are present in the inner membrane of the mitochondria of various cells. In the present study, we show for the first time mitochondrial ATP-regulated potassium channels in human dermal fibroblast cells. Using the patch-clamp technique on the inner mitochondrial membrane of fibroblasts, we detected a potassium channel with a mean conductance equal to 100 pS in symmetric 150 mM KCl. The activity of this channel was inhibited by a complex of ATP/Mg2+ and activated by potassium channel openers such as diazoxide or BMS 191095. Channel activity was inhibited by antidiabetic sulfonylurea glibenclamide and 5-hydroxydecanoic acid. The influence of substances modulating ATP-regulated potassium channel activity on oxygen consumption and membrane potential of isolated fibroblast mitochondria was also studied. Additionally, the potassium channel opener diazoxide lowered the amount of superoxide formed in isolated fibroblast mitochondria. Using reverse transcriptase-PCR, we found an mRNA transcript for the KCNJ1(ROMK) channel. The presence of ROMK protein was observed in the inner mitochondrial membrane fraction. Moreover, colocalization of the ROMK protein and a mitochondrial marker in the mitochondria of fibroblast cells was shown by immunofluorescence. In summary, the ATP-regulated mitochondrial potassium channel in a dermal fibroblast cell line have been identified.

OxLDL antibody inhibits MCP-1 release in monocytes/macrophages by regulating Ca2+ /K+ channel flow.

oxLDL peptide vaccine and its antibody adoptive transferring have shown a significantly preventive or therapeutic effect in atherosclerotic animal model. The molecular mechanism behind this is obscure. Here, we report that oxLDL induces MCP‐1 release in monocytes/macrophages through their TLR‐4 (Toll‐like receptor 4) and ERK MAPK pathway and is calcium/potassium channel‐dependent. Using blocking antibodies against CD36, TLR‐4, SR‐AI and LOX‐1, only TLR‐4 antibody was found to have an inhibitory effect and ERK MAPK‐specific inhibitor (PD98059) was found to have a dramatic inhibitory effect compared to inhibitors of other MAPK group members (p38 and JNK MAPKs) on oxLDL‐induced MCP‐1 release. The release of cytokines and chemokines needs influx of extracellular calcium and imbalance of efflux of potassium. Nifedipine, a voltage‐dependent calcium channel (VDCC) inhibitor, and glyburide, an ATP‐regulated potassium channel (K+ ATP) inhibitor, inhibit oxLDL‐induced MCP‐1 release. Potassium efflux and influx counterbalance maintains the negative potential of macrophages to open calcium channels, and our results suggest that oxLDL actually induces the closing of potassium influx channel – inward rectifier channel (Kir) and ensuing the opening of calcium channel. ERK MAPK inhibitor PD98059 inhibits oxLDL‐induced Ca2+/Kir channel alterations. The interfering of oxLDL‐induced MCP‐1 release by its monoclonal antibody is through its FcγRIIB (CD32). Using blocking antibodies against FcγRI (CD64), FcγRIIB (CD32) and FcγRIII (CD16), only CD32 blocking antibody was found to reverse the inhibitory effect of oxLDL antibody on oxLDL‐induced MCP‐1 release. Interestingly, oxLDL antibody specifically inhibits oxLDL‐induced ERK MAPK activation and ensuing Ca2+/Kir channel alterations, and MCP‐1 release. Thus, we found a molecular mechanism of oxLDL antibody on inhibition of oxLDL‐induced ERK MAPK pathway and consequent MCP‐1 release.

What do we not know about mitochondrial potassium channels?

In this review, we summarize our knowledge about mitochondrial potassium channels, with a special focus on unanswered questions in this field. The following potassium channels have been well described in the inner mitochondrial membrane: ATP-regulated potassium channel, Ca2+-activated potassium channel, the voltage-gated Kv1.3 potassium channel, and the two-pore domain TASK-3 potassium channel. The primary functional roles of these channels include regulation of mitochondrial respiration and the alteration of membrane potential. Additionally, they modulate the mitochondrial matrix volume and the synthesis of reactive oxygen species by mitochondria. Mitochondrial potassium channels are believed to contribute to cytoprotection and cell death. In this paper, we discuss fundamental issues concerning mitochondrial potassium channels: their molecular identity, channel pharmacology and functional properties. Attention will be given to the current problems present in our understanding of the nature of mitochondrial potassium channels. This article is part of a Special Issue entitled ‘EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2–6, 2016’, edited by Prof. Paolo Bernardi.

GABA transmission via ATP-dependent K+channels regulates α-synuclein secretion in mouse striatum

α-Synuclein is readily released in human and mouse brain parenchyma, even though the normal function of the secreted protein has not been yet elucidated. Under pathological conditions, such as in Parkinson's disease, pathologically relevant species of α-synuclein have been shown to propagate between neurons in a prion-like manner, although the mechanism by which α-synuclein transfer induces degeneration remains to be identified. Due to this evidence extracellular α-synuclein is now considered a critical target to hinder disease progression in Parkinson's disease. Given the importance of extracellular α-synuclein levels, we have now investigated the molecular pathway of α-synuclein secretion in mouse brain. To this end, we have identified a novel synaptic network that regulates α-synuclein release in mouse striatum. In this brain area, the majority of α-synuclein is localized in corticostriatal glutamatergic terminals. Absence of α-synuclein from the lumen of brain-isolated synaptic vesicles suggested that they are unlikely to mediate its release. To dissect the mechanism of α-synuclein release, we have used reverse microdialysis to locally administer reagents that locally target specific cellular pathways. Using this approach, we show that α-synuclein secretion in vivo is a calcium-regulated process that depends on the activation of sulfonylurea receptor 1-sensitive ATP-regulated potassium channels. Sulfonylurea receptor 1 is distributed in the cytoplasm of GABAergic neurons from where the ATP-dependent channel regulates GABA release. Using a combination of specific agonists and antagonists, we were able to show that, in the striatum, modulation of GABA release through the sulfonylurea receptor 1-regulated ATP-dependent potassium channels located on GABAergic neurons controls α-synuclein release from the glutamatergic terminals through activation of the presynaptic GABAB receptors. Considering that sulfonylurea receptors can be selectively targeted, our study highlights the potential use of the key molecules in the α-synuclein secretory pathway to aid the discovery of novel therapeutic interventions for Parkinson's disease.

Effectors of large-conductance calcium-activated potassium channel modulate glutamate excitotoxicity in organotypic hippocampal slice cultures

Mitochondria have been suggested as a potential target for cytoprotective strategies. It has been shown that increased K+ uptake mediate by mitochondrial ATP-regulated potassium channels (mitoKATP channel) or large-conductance Ca2+-activated potassium channels (mitoBKCa channel) may provide protection in different models of cell death. Since recent findings demonstrated the presence of BKCa channels in neuronal mitochondria, the goal of the present study was to test the potential neuroprotective effects of BKCa channel modulators. Using organotypic hippocampal slice cultures exposed to glutamate, we demonstrated that preincubation of the slices with the BKCa channel opener NS1619 resulted in decreased neuronal cell death measured as reduced uptake of propidium iodide. This neuroprotective effect was reversed by preincubation with the BKCa channel inhibitors paxilline and Iberiotoxin (IbTx). Moreover, mitochondrial respiration measurements revealed that NS1619 induced an IbTx-sensitive increase in state 2 respiration of isolated brain mitochondria. In addition, electrophysiological patch-clamp studies confirmed the presence of BKCa channels in mitoplasts isolated from embryonic hippocampal cells. Taken together, our results confirm presence of BKCa channel in rat hippocampal neurons mitochondria and suggest putative role for mitoBKCa in neuroprotection.

Activation of Mitochondrial Uncoupling Protein 4 and ATP-Sensitive Potassium Channel Cumulatively Decreases Superoxide Production in Insect Mitochondria.

It has been evidenced that mitochondrial uncoupling protein 4 (UCP4) and ATP-regulated potassium channel (mKATP channel) of insect Gromphadorhina coqereliana mitochondria decrease superoxide anion production. We elucidated whether the two energy-dissipating systems work together on a modulation of superoxide level in cockroach mitochondria. Our data show that the simultaneous activation of UCP4 by palmitic acid and mKATP channel by pinacidil revealed a cumulative effect on weakening mitochondrial superoxide formation. The inhibition of UCP4 by GTP (and/or ATP) and mKATP channel by ATP elevated superoxide production. These results suggest a functional cooperation of both energy-dissipating systems in protection against oxidative stress in insects.

Identification of the ATP Regulated Potassium Channel in Mitochondria of Fibroblast Cells

The transport of potassium ions into the mitochondrial matrix can trigger the protection of the injured cardiac and neuronal tissues. A growing body of evidence suggests that ischemic preconditioning, short episodes of ischemia that increase tissue tolerance to lethal insults, could be mimicked by the administration of openers of mitochondrial potassium channels. Hence, the identification of ion channels present in the inner mitochondrial membrane of fibroblasts is important in distinguishing possible protective mechanisms in these cells.In our research, inner membrane mitochondrial ion channels of the human fibroblast cell line were investigated using a patch-clamp and biochemical techniques. The single channel activity of mitochondrial potassium channels were investigated using a patch-clamp technique. In the inner mitochondrial membrane of fibroblast we detected ATP-regulated potassium channel (mitoKATP channel) with mean conductance equal to 100 ± 3 pS. The activity of this channel was inhibited by complex of ATP/Mg2+ and activated by the potassium channel opener BMS191095 and diazoxide. The influence of substances modulating ATP-regulated potassium channel activity on the bioenergetics, oxygen consumption and membrane potential, of isolated human dermal fibroblast mitochondria was also studied.Our findings indicate presence of the ATP-regulated potassium channels in the inner mitochondrial membranes of human fibroblast.This study was supported by a grant MERIS PBS1/B8/1/2012 from the National Centre of Research and Development.

ATP-regulated potassium channels and voltage-gated calcium channels in pancreatic alpha and beta cells: similar functions but reciprocal effects on secretion.

Closure of ATP-regulated K(+) channels (K(ATP) channels) plays a central role in glucose-stimulated insulin secretion in beta cells. K(ATP) channels are also highly expressed in glucagon-producing alpha cells, where their function remains unresolved. Under hypoglycaemic conditions, K(ATP) channels are open in alpha cells but their activity is low and only ~1% of that in beta cells. Like beta cells, alpha cells respond to hyperglycaemia with K(ATP) channel closure, membrane depolarisation and stimulation of action potential firing. Yet, hyperglycaemia reciprocally regulates glucagon (inhibition) and insulin secretion (stimulation). Here we discuss how this conundrum can be resolved and how reduced K(ATP) channel activity, via membrane depolarisation, paradoxically reduces alpha cell Ca(2+) entry and glucagon exocytosis. Finally, we consider whether the glucagon secretory defects associated with diabetes can be attributed to impaired K(ATP) channel regulation and discuss the potential for remedial pharmacological intervention using sulfonylureas.

Direct Activation ofβ-Cell KATPChannels with a Novel Xanthine Derivative

ATP-regulated potassium (KATP) channel complexes of inward rectifier potassium channel (Kir) 6.2 and sulfonylurea receptor (SUR) 1 critically regulate pancreatic islet β-cell membrane potential, calcium influx, and insulin secretion, and consequently, represent important drug targets for metabolic disorders of glucose homeostasis. The KATP channel opener diazoxide is used clinically to treat intractable hypoglycemia caused by excessive insulin secretion, but its use is limited by off-target effects due to lack of potency and selectivity. Some progress has been made in developing improved Kir6.2/SUR1 agonists from existing chemical scaffolds and compound screening, but there are surprisingly few distinct chemotypes that are specific for SUR1-containing KATP channels. Here we report the serendipitous discovery in a high-throughput screen of a novel activator of Kir6.2/SUR1: VU0071063 [7-(4-(tert-butyl)benzyl)-1,3-dimethyl-1H-purine-2,6(3H,7H)-dione]. The xanthine derivative rapidly and dose-dependently activates Kir6.2/SUR1 with a half-effective concentration (EC50) of approximately 7 μM, is more efficacious than diazoxide at low micromolar concentrations, directly activates the channel in excised membrane patches, and is selective for SUR1- over SUR2A-containing Kir6.1 or Kir6.2 channels, as well as Kir2.1, Kir2.2, Kir2.3, Kir3.1/3.2, and voltage-gated potassium channel 2.1. Finally, we show that VU0071063 activates native Kir6.2/SUR1 channels, thereby inhibiting glucose-stimulated calcium entry in isolated mouse pancreatic β cells. VU0071063 represents a novel tool/compound for investigating β-cell physiology, KATP channel gating, and a new chemical scaffold for developing improved activators with medicinal chemistry.

New Mitochondrial Potassium Channels

Mitochondrial potassium channels play an important role in cytoprotection. The following potassium channels have been described in the inner mitochondrial membrane: the ATP-regulated potassium channel, the large conductance calcium activated potassium channel, the voltage-gated potassium channel and the twin-pore domain TASK-3 potassium channel. Potassium channels in the inner mitochondrial membrane are modulated by inhibitors and activators (potassium channel openers) previously described for plasma membrane potassium channels. The majority of mitochondrial potassium channel modulators exhibit a broad spectrum of off-target effects. These include uncoupling properties, inhibition of the respiratory chain and effects on cellular calcium homeostasis. Therefore, the rational application of channel inhibitors or activators is crucial to understanding the cellular consequences of mitochondrial channel inhibition or activation. In this paper, new observations on mitochondrial potassium channel will be discussed: 1). their molecular identity, 2). their interaction with potassium channel openers and inhibitors and 3). their functional role.

Reinterpreting the Action of ATP Analogs on KATP Channels

Neuroendocrine-type K(ATP) channels, (SUR1/Kir6.2)4, couple the transmembrane flux of K(+), and thus membrane potential, with cellular metabolism in various cell types including insulin-secreting β-cells. Mutant channels with reduced activity are a cause of congenital hyperinsulinism, whereas hyperactive channels are a cause of neonatal diabetes. A current regulatory model proposes that ATP hydrolysis is required to switch SUR1 into post-hydrolytic conformations able to antagonize the inhibitory action of nucleotide binding at the Kir6.2 pore, thus coupling enzymatic and channel activities. Alterations in SUR1 ATPase activity are proposed to contribute to neonatal diabetes and type 2 diabetes risk. The regulatory model is partly based on the reduced ability of ATP analogs such as adenosine 5'-(β,γ-imino)triphosphate (AMP-PNP) and adenosine 5'-O-(thiotriphosphate) (ATPγS) to stimulate channel activity, presumably by reducing hydrolysis. This study uses a substitution at the catalytic glutamate, SUR(1E1507Q), with a significantly increased affinity for ATP, to probe the action of these ATP analogs on conformational switching. ATPγS, a slowly hydrolyzable analog, switches SUR1 conformations, albeit with reduced affinity. Nonhydrolyzable AMP-PNP and adenosine 5'-(β,γ-methylenetriphosphate) (AMP-PCP) alone fail to switch SUR1, but do reverse ATP-induced switching. AMP-PCP displaces 8-azido-[(32)P]ATP from the noncanonical NBD1 of SUR1. This is consistent with structural data on an asymmetric bacterial ABC protein that shows that AMP-PNP binds selectively to the noncanonical NBD to prevent conformational switching. The results imply that MgAMP-PNP and MgAMP-PCP (AMP-PxP) fail to activate K(ATP) channels because they do not support NBD dimerization and conformational switching, rather than by limiting enzymatic activity.

Effects of KATP channel openers diazoxide and pinacidil in coronary-perfused atria and ventricles from failing and non-failing human hearts

This study compared the effects of ATP-regulated potassium channel (KATP) openers, diazoxide and pinacidil, on diseased and normal human atria and ventricles. We optically mapped the endocardium of coronary-perfused right (n=11) or left (n=2) posterior atrial–ventricular free wall preparations from human hearts with congestive heart failure (CHF, n=8) and non-failing human hearts without (NF, n=3) or with (INF, n=2) infarction. We also analyzed the mRNA expression of the KATP targets Kir6.1, Kir6.2, SUR1, and SUR2 in the left atria and ventricles of NF (n=8) and CHF (n=4) hearts. In both CHF and INF hearts, diazoxide significantly decreased action potential durations (APDs) in atria (by −21±3% and −27±13%, p<0.01) and ventricles (by −28±7% and −28±4%, p<0.01). Diazoxide did not change APD (0±5%) in NF atria. Pinacidil significantly decreased APDs in both atria (−46 to −80%, p<0.01) and ventricles (−65 to −93%, p<0.01) in all hearts studied. The effect of pinacidil on APD was significantly higher than that of diazoxide in both atria and ventricles of all groups (p<0.05). During pinacidil perfusion, burst pacing induced flutter/fibrillation in all atrial and ventricular preparations with dominant frequencies of 14.4±6.1Hz and 17.5±5.1Hz, respectively. Glibenclamide (10μM) terminated these arrhythmias and restored APDs to control values. Relative mRNA expression levels of KATP targets were correlated to functional observations. Remodeling in response to CHF and/or previous infarct potentiated diazoxide-induced APD shortening. The activation of atrial and ventricular KATP channels enhances arrhythmogenicity, suggesting that such activation may contribute to reentrant arrhythmias in ischemic hearts.