Am J Physiol Lung Cell Mol Physiol 286: L49-L67, 2004;
doi:10.1152/ajplung.00041.2003
1040-0605/04 $5.00
INVITED REVIEW
Activation of K+ channels: an essential pathway in programmed cell death
Carmelle V. Remillard and
Jason X.-J. Yuan
Division of Pulmonary and Critical Care Medicine, Department of Medicine, School of Medicine, University of California, San Diego, California 92103-8382
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ABSTRACT
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Cell apoptosis and proliferation are two counterparts in sharing the responsibility for maintaining normal tissue homeostasis. In recent years, the process of the programmed cell death has gained much interest because of its influence on malignant cell growth and other pathological states. Apoptosis is characterized by a distinct series of morphological and biochemical changes that result in cell shrinkage, DNA breakdown, and, ultimately, phagocytic death. Diverse external and internal stimuli trigger apoptosis, and enhanced K+ efflux has been shown to be an essential mediator of not only early apoptotic cell shrinkage, but also of downstream caspase activation and DNA fragmentation. The goal of this review is to discuss the role(s) played by K+ transport or flux across the plasma membrane in the regulation of the apoptotic volume decrease and apoptosis. Attention has also been paid to the role of inner mitochondrial membrane ion transport in the regulation of mitochondrial permeability and apoptosis. We provide specific examples of how deregulation of the apoptotic process contributes to pulmonary arterial medial hypertrophy, a major pathological feature in patients with pulmonary arterial hypertension. Finally, we discuss the targeting of K+ channels as a potential therapeutic tool in modulating apoptosis to maintain the balance between cell proliferation and cell death that is essential to the normal development and function of an organism.
apoptosis; ion channels; cell volume regulation; pulmonary artery smooth muscle cells; pulmonary hypertension
CELL DEATH IS CRITICAL for the normal development and function of multicellular organisms. For a tissue to function properly, removal of excess cells or of cells with genetic damage or improper developmental mutations is crucial. Cancer (59), hypertension (151), cardiac disease (36), viral infections (11, 125), and autoimmune (48) and neurodegenerative disorders (189) are all characterized by abnormal cell death regulation. The cellular turnover that results from the balance between cell death and proliferation is important in maintaining tissue homeostasis. Ion channels in both the sarcolemmal and mitochondrial membranes have been implicated in the signal transduction cascades that regulate apoptosis (64). This review focuses on the role played by K+ and K+ transport in the onset and development of cellular changes typical of the apoptotic process, especially in pulmonary vascular smooth muscle cells, and how modulation of K+ efflux and K+ channel function by both pro- and antiapoptotic proteins is a potential therapeutic target for cardiopulmonary diseases.
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APOPTOSIS: AN OVERVIEW
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Apoptosis, or programmed cell death, allows individual cells to die according to a highly controlled series of morphological and biochemical changes (Fig. 1). In the earliest stage of apoptosis, cells undergo shrinkage, the apoptotic cell shrinkage, with little or no change in the structure of intracellular organelles. As will be discussed later, the enhanced activation of ion-selective channels and water-permeable channels (aquaporins) modulates the apoptotic volume decrease (AVD). Nuclear condensation and DNA fragmentation within the nucleus typically occur after apoptotic stimulation and the onset of AVD, as early as 1–4 h after the apoptotic stimulation in the case of human pulmonary artery vascular smooth muscle cells (129), human leukemia HL-60 cells (31), neurons (20), human lymphoid cells (103), and thymocytes (178). The formation of apoptotic bodies (membrane-bound vesicles that pinch off from the dying cell) containing organelles and nuclear fragments constitutes the final step before phagocytosis by resident macrophages and neighboring cells. Apoptotic bodies are then degraded after phagocytosis. Unlike cellular necrosis, apoptosis does not result in an inflammatory response since the intracellular contents are not exposed to the environment prior to phagocytosis, thereby minimizing damage to adjoining healthy cells.
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Fig. 1. Diagram showing the chronological order of morphological and biochemical changes during apoptotic stimulation. The apoptotic volume decrease (AVD) due to K+, Cl-, and H2O efflux occurs before nuclear condensation and DNA fragmentation. Unlike in necrosis, the cellular contents are never released from the dying cells but are ingested during phagocytosis, thereby preventing any inflammatory response by the host tissue.
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Increased proteolytic activity following AVD is a key element in the steps leading to nucleotide fragmentation. The biochemical changes inherent to apoptosis are due to the activation of cytoplasmic proteolytic enzymes, the caspases, by proapoptotic stimuli (41, 68, 161) via one of two pathways (Fig. 2). The extrinsic pathway, or the death receptor pathway, is initiated by the activation of transmembrane death receptors by the binding of proteins such as CD95, tumor necrosis factor-
(TNF-), and Fas ligand. Activation of the death receptors activates the membrane proximal initiator caspase-8 (and/or caspase-10), which then cleaves procaspase-3 to generate the active effector caspase-3. The intrinsic pathway, or the mitochondrial death pathway, requires disruption of the mitochondrial membrane [e.g., by staurosporine (ST), actinomycin D, peroxide, ultraviolet (UV) radiation] and/or the release or translocation of cytochrome c (cyt-c) (81, 183) and other apoptosis-inducing factors from the mitochondrial intermembrane space to the cytoplasm. The precise triggering mechanism for cyt-c release is under investigation. There are suggestions that its release results from 1) physical disruption of the mitochondrial membrane, 2) mitochondrial membrane depolarization, and 3) increased mitochondrial permeability transition (10, 60, 67, 81). Whatever its extrusion mechanism, the released cyt-c binds to apoptotic protease-activating factor 1 (APAF-1) and forms a heptameric APAF-1-cyt-c complex with deoxyadenosine triphosphate/adenosine triphosphate (dATP/ATP), the apoptosome. The apoptosome activates procaspase-9, which in turn activates the downstream effector caspases (caspase-3, -6, -7) in the cytoplasm. Activation of the effector caspases-3/-6/-7 by either death receptor stimulation or mitochondrial disruption leads to chromatin degradation and ultimately to apoptosis.
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Fig. 2. The two major apoptotic pathways involve either membrane death receptor stimulation (extrinsic pathway) and/or mitochondrial disruption (intrinsic pathway). The major proteins involved are shown, as well as the modulatory sites for selected regulatory proteins. AIF, apoptosis-inducing factor; AKT, protein kinase B; APAF-1, apoptotic protease-activating factor 1; ARC, apoptosis repressor with caspase recruitment domain; tBid, truncated Bid; cyt-c, cytochrome c; DIABLO, direct IAP-binding protein with low pI; FADD, Fas-associated death domain protein; c-FLIP, FADD-like ICE (caspase-8) inhibitory protein; IAPs, inhibitors of apoptosis; ROS, reactive oxygen species; Smac, second-mitochondrial-derived activator of caspase;   m, mitochondrial membrane potential.
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Two other mitochondrial proteins, Smac/Diablo (39, 165) and apoptosis-inducing factor (AIF) (77), can be released into the cytoplasm and cause apoptosis via caspase-independent pathways. The initiator caspase-8, initially activated by the extrinsic pathway, can also truncate the cytosolic Bid protein, leading to cyt-c release and then to the activation of the effector caspases-3/-6/-7 via the intrinsic pathway. Mitochondrial proteins released into the cytoplasm, such as Smac/Diablo and Omi/HtrA2 (39, 66, 156, 165, 166), can antagonize the actions of the inhibitors of apoptosis, which directly inhibit caspase activity (35, 175). The death receptor and mitochondrial pathways cross talk with each other in multiple steps to achieve the final goal, activation of the effector caspases.
In summary, apoptosis is a process that plays a critical role in embryonic development and tissue homeostasis. The programmed cell death cascade due to activated death receptors can be divided into at least three functionally distinct stages (60, 104): 1) the initiation or signaling phase in which death-promoting molecules (e.g., TNF- and Fas ligand) bind to death receptors on the cell surface with subsequent recruitment of death domain proteins for activation of caspase-8; 2) the effector phase during which depolarization of mitochondrial membrane potential (m), release of cyt-c from the mitochondrial intermembrane space to the cytoplasm, and/or activation of cytoplasmic caspases take place; and c) the structural alteration and DNA degradation phase in which activated effector caspases lead to the cleavage of the lamin proteins that make up the nuclear lamina (the rigid structure that underlies the nuclear membrane and is involved in chromatin organization) and ICAD [an inhibitor of the caspase-activated deoxyribonuclease (CAD or DFF) responsible for DNA fragmentation], and to the fragmentation and degradation of genomic DNA (161). Indeed, it has been well documented that cells undergoing apoptosis show cell shrinkage, chromatin (nuclear) condensation with subsequent internucleosomal fragmentation of DNA, and membrane redistribution of phospholipids.
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REGULATION OF CELL VOLUME
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Mammalian cellular membranes are highly permeable to water. The movement of water across the membrane occurs via water channels, or aquaporins, which are highly expressed in virtually all cell types (80, 137). Of the eleven known aquaporins, for example, eight have been identified in human pulmonary artery smooth muscle cells (PASMC) to varying degrees (I. Fantozzi and J. X.-J. Yuan, unpublished observations). Animal cell membranes cannot tolerate the hydrostatic pressure gradients produced by the passive transport of water according to its concentration gradient. Therefore, water movement is largely regulated by osmotic gradients across the cell membrane and is rarely a limiting factor in cellular volume changes. In fact, alterations of intra- or extracellular osmolarity typically precede the movement of water and cellular volume changes (93, 94).
Ion transport contributes greatly to the regulation of the transmembrane osmotic gradient (Fig. 3). Most cells achieve and maintain a physiological osmotic balance through the continuous activity of an electrogenic Na+-K+-ATPase pump (3 Na+ out: 2 K+ in), which creates an intracellular environment high in K+ (
140 mM) and low in Na+ (10 mM) (79, 181), as well as various anion and cation cotransporters (108, 121). In most excitable and nonexcitable cells, K+ is the dominant cytoplasmic cation (being 30-fold more concentrated within the cytoplasm than the intercellular space), whereas Na+ and free Ca2+ are more concentrated in the extracellular space (Table 1). Cl- is the major anion in these cells; the cytoplasmic Cl- concentration ([Cl-]cyt) usually ranges from 5 to 15 mM in many excitable cells, although larger variations can be found in smooth muscle cells, neurons, and cardiac muscle depending on species and tissue type (76, 131, 167). [Cl-]cyt in smooth muscle cells, especially vascular smooth muscle cells, however, can be as high as 50 mM (76), suggesting that, in addition to organic anions (such as 
and nitrates), Cl- is a dominant cytoplasmic anion in smooth muscle cells.
Because the cell membrane is permeable to K+ under resting conditions, i.e., the permeability to K+ is much greater than the permeability to other ions, the activity of membrane K+ channels plays a critical role in the regulation of cellular volume. Whole cell K+ current (IK) at any given time is determined by the following equation
where N denotes the total number of functional K+ channels expressed in the plasma membrane; i is the current through a single K+ channel; and Popen is the steady-state open probability of a K+ channel. Therefore, when K+ channels open (i.e., i or Popen rises) and/or the number of functional K+ channels in the plasma membrane increases (i.e., N increases due to upregulation of K+ channel gene expression), the whole cell IK or transmembrane K+ efflux is increased, which would induce or enhance cell volume decrease. In contrast, when K+ channels close (i.e., i, N, or Popen decline), the whole cell IK or transmembrane K+ efflux is decreased, which would inhibit cell volume decrease.
The efflux of K+ thereby creates a positive potential outside the cell, which would drag Cl- out of the cell according to its electrochemical gradient, and the membrane hyperpolarization induced by K+ efflux would also activate membrane Cl- channels and further enhance Cl- efflux (192). The resultant accumulation of KCl outside the cell thus shifts the osmotic balance such that water is also extruded from the cell in an attempt to reestablish a normal osmotic gradient. The subsequent cell shrinkage may be functionally important since a doubling of extracellular osmolarity has been shown to trigger apoptosis in lymphocytes (15). Accordingly, the modulation of K+ and Cl- movement as well as K+ and Cl- channel activity is thus crucial in initiating and regulating the apoptotic volume decrease in cells undergoing apoptosis.
Although Na+, K+, and Cl- ions have been implicated in cell shrinkage, Ca2+ ions may also play a role in the regulation of cell volume. After cell swelling, intracellular Ca2+ concentration ([Ca2+]i) increases in some cell types, either due to enhanced sarcolemmal Ca2+ influx or due to Ca2+ release from intracellular stores (93). Although increased [Ca2+]i itself may not have a direct role on the regulation of cell volume, it may affect cytoskeletal elements such as the actin filaments or serve as a signal transduction element to activate other membrane ion channels (e.g., Ca2+-activated K+ and Cl- channels) and transporters. Obviously, both cell swelling and shrinkage result in significant changes in the cytoskeletal architecture. Actin filaments have been found to be depolymerized in swollen cells, possibly due to Ca2+ binding to gelsolin (93). An intact actin filament network is required for the activation of some volume regulatory mechanisms. Disruption due to Ca2+-mediated depolymerization will affect many processes, including 1) Na+ channel activity, 2) insertion of volume regulatory channels into the membrane, 3) regulation of channels by kinases and phospholipids, 4) activation of mechanosensitive anion channels by membrane stretch, and 5) activation of the Na+/H+ exchanger and the Na+/K+/2Cl- cotransporter, resulting in volume deregulation (93).
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MODULATION OF APOPTOTIC STAGES BY K+ FLUX ACROSS THE PLASMA MEMBRANE
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Cell shrinkage is an early hallmark of apoptosis. Apoptotic cell shrinkage occurs in two distinct stages: the initial phase starting before formation of the cyt-c/APAF-1/caspase-9 apoptosome and cell fragmentation and the late phase that is associated with cell fragmentation (123). The early phase of the apoptotic cell shrinkage when cells undergo apoptosis is mainly regulated by the activity of membrane ion channels and transporters (123, 185, 186). The time courses of the effect of apoptosis inducers on morphological changes, cyt-c translocation, caspase activation, and DNA/cell fragmentation have demonstrated that the initial phase of AVD occurs before the release of cyt-c, activation of cytoplasmic caspases, and breakage of cell nuclei. However, a rise in cytoplasmic cyt-c and an increase in active caspases in the cytoplasm have also been demonstrated to contribute to the apoptotic cell shrinkage, mainly the late phase of the volume decrease (73, 129, 143, 169, 180). These results suggest that the early and late phases of cell shrinkage in apoptotic cells may result from different mechanisms. In both stages, membrane ion channels and transporters appear to be involved.
The apoptotic cell shrinkage has been demonstrated to correlate with increased K+ and Cl- efflux and activation of K+ channels (13, 45, 87, 88, 172, 188). Because high cytoplasmic K+ ([K+]cyt) is required to maintain cytoplasmic ion homeostasis and cell volume, any changes of K+ efflux or influx will influence plasma membrane permeability and cell volume. The link between K+ efflux and apoptosis has been further established by experiments using ionophores. Valinomycin, a K+ ionophore that allows K+ efflux based on the K+ electrochemical gradient, can induce apoptosis in many cell types, including neurons (188), thymocytes (4, 29, 30), ascites hepatoma cells (74), and PASMC (87).
K+ uptake (from extracellular fluid to the cytoplasm) is modulated primarily by the ouabain-sensitive Na+-K+-ATPase, or Na+ pump (181). Recent studies have shown that anti-Fas and dexamethasone treatments inactivated an ouabain-sensitive Na+-K+-ATPase pump in lymphocytes (17) and thymocytes (106), significantly decreasing K+ uptake, irreversibly depolarizing the cell membrane, activating voltage-dependent K+ and Cl- channels, and causing apoptosis. What follows is an overall review of the modalities of cytoplasmic K+ efflux and how they regulate apoptosis.
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ACTIVATION OF K+ CHANNELS INDUCES APOPTOTIC CELL SHRINKAGE
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Enhancement of K+ efflux-mediated cell shrinkage is considered to be one of the earliest signs of apoptosis in many cells. The role of K+ channels in apoptosis was proposed by the original work of Yu et al. in 1997 (188) and subsequently supported by other investigators (18, 30, 56, 87, 103, 169). Bortner et al. (18, 169) showed a correlation between the significant decrease in [K+]cyt and the number of shrunken cells when lymphocyte apoptosis was induced by Fas ligand, dexamethasone, ST, and anisomycin (a protein synthesis inhibitor), thereby establishing a link between K+ efflux and AVD. These studies were further reinforced by observations that raising extracellular K+ ([K+]o), which reduces transmembrane K+ concentration gradient and reduces K+ efflux, can inhibit AVD and apoptosis induced by valinomycin, Fas ligand, carbonyl cyanide-p-trifluoromethoxyphenyl hydrazone (FCCP), and ST in neurons (188), PASMC (86, 87), and lymphocytes (18, 56). Inhibition of K+ channel activity with quinine or Ba2+ also prevents cell shrinkage induced by ST or TNF-/cycloheximide (103). In addition, treatment of lymphocytes and cortical neurons with tetrapentylammonium (TPA) inhibits the early stages of apoptosis before caspase activation has occurred (30, 176). These results indicate that an increased K+ efflux via sarcolemmal K+ channels is thus a central mediator of AVD and apoptosis.
Okada and Maeno (123) classified the apoptotic cell shrinkage into two stages: the early volume decrease that occurs before cyt-c release and caspase activation and the late volume decrease that occurs concurrently with DNA fragmentation and nuclear breakage. In PASMC, our recent results suggest that, when cells are treated with ST, voltage-gated K+ current (IK(V)) increases within 30 min immediately followed by a reduction of cell size/volume (Fig. 4) (129). The ST-mediated nuclear breakage and condensation, determined by 4',6'-diamidino-2-phenylindole (DAPI) staining (and terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) assay, occurs after the ST-mediated increase in IK(V) and cell volume decrease (Fig. 4) (129). Furthermore, cytoplasmic application of recombinant cyt-c enhances 4-aminopyridine (4-AP)-sensitive voltage-gated K+ (KV) currents in these cells (Fig. 5) independently of caspase-9 activation (129), suggesting that cyt-c-mediated opening of K+ channels precedes caspase-9 activation. These observations indicate that activation of K+ channels is involved in both the early and late volume decrease. In cells challenged by apoptosis inducers (e.g., ST, TNF-, UV light, dexamethasone) or death triggers, K+ channels may be activated by an unknown mechanism to initiate the early volume decrease and further activated by cyt-c to maintain the early stage of cell shrinkage. The cyt-c-mediated KV channel activation may also play an important role in initiating the late volume decrease associated with cell fragmentation.
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Fig. 4. Whole cell voltage-gated K+ current [IK(V)] increase precedes staurosporine (ST)-induced AVD in rat pulmonary artery smooth muscle cells (rPASMC). A: averaged IK(V), elicited by 300-ms test potentials ranging from -60 to +80 mV (-70 mV holding potential) in control (untreated, Cont) cells and following ST (0.02 µM) treatment for 0.5 (middle) and 2.5 (right) h. B: phase-contrast images of cell before ST (0.02 µM) and after 2.5- and 3.5-h ST treatment (middle and right). C: time courses of ST-induced enhancement of IK(V) (red circles), cell volume (AVD, blue triangles), and nuclear breakage (green bars) show that AVD and IK(V) enhancement occur within a similar short time frame, whereas nuclear breakage starts 4–6 h after ST treatment. D: inverse correlation of the amplitude of IK(V) and cell size in PASMC treated with ST (r2 = 0.91), indicating that increased currents lead to cell shrinkage. [Modified from Platoshyn et al. (129).]
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Fig. 5. Cytoplasmic application of cyt-c enhances IK(V) in PASMC. A: representative currents were elicited by 300-ms test potentials from -60 to +80 mV from a holding potential of -70 mV. Recordings were obtained immediately (0 min) or 20 min following whole cell access during which recombinant cyt-c (5 µM, dissolved in the pipette solution) was allowed to dialyze into the cell. B: time course of cyt-c-induced IK(V) increase after membrane rupture. [Modified from Platoshyn et al. (129).]
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ACTIVATION OF K+ CHANNELS INDUCES APOPTOSIS
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In addition to K+-permeable channels, K+ efflux is also controlled by many different mechanisms in excitable cells, such as an electroneutral K+-Cl- symporter and a K+-H+ and 
exchanger system (121). Enhanced K+ efflux through K+ channels, however, is a major pathway for K+ loss. Five classes of K+ channels have been identified in excitable cells: KV channels, Ca2+-activated K+ (KCa) channels, ATP-sensitive K+ (KATP) channels, inwardly rectifying K+ (KIR) channels, and tandem pore K+ (KT, two-pore six-domain) channels (97, 105, 115). The enhanced activity of four of these channels has been implicated in apoptosis induced by the stimulation of either the mitochondrial or death receptor apoptotic pathways, as is discussed below.
ST, a potent apoptosis inducer in almost all cell types, enhances the activity of a 4-AP-sensitive KV channel in human and rat PASMC (Fig. 6A) (37, 85) and of a tetraethylammonium (TEA)-sensitive K+ channel in mouse neocortical neurons (188). Activation of the 4-AP-sensitive KV channels by the nitric oxide (NO) donor S-nitroso-N-acetyl-penicillamine (SNAP) also causes apoptosis in PASMC (Fig. 6B). In rat and human PASMC treated with ST, the maximum enhancement of KV currents occurs within 6 h, whereas apoptosis is maximal after 24 h of treatment (Fig. 4C), suggesting that KV channel activation occurs rapidly following the challenge of apoptotic inducers or death triggers and likely precedes caspase activation and DNA degradation (43, 86, 129). A similar 4-AP-sensitive K+ current is activated by UV radiation in myeloblastic leukemia cells (173). In rat fetal neurons, the sulfhydryl-oxidizing agent 2,2'-dithiodipyridine activates a TEA-sensitive K+ current with similar kinetics to that produced by the 4-AP-sensitive KV channels (109), whereas neuronal apoptosis is associated with a significant increase in KV currents (188). K+ currents sensitive to TPA, a TEA analog, were also detected in thymocytes and cortical neurons treated with dexamethasone and ST, respectively (30, 176). In thymocytes, TPA prevented all characteristics of dexamethasone-induced apoptosis, including m dissipation, cytosolic K+ efflux, chromatin condensation, and caspase and endonuclease activation (30). These results using TPA as a K+ channel inhibitor should be interpreted with caution, as the compound has been shown to have multiple nonspecific effects on voltage-dependent Ca2+ and Na+ channels' activity, as well as on K+ channel activity, in cortical neurons (176).
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Fig. 6. Apoptotic inducers increase K+ efflux via sarcolemmal K+ channels in PASMC. ST (0.1 µM, A) and S-nitroso-N-acetyl penicillamine (SNAP, 0.1 mM; B) treatments activate 4-aminopyridine (4-AP)-sensitive KV currents [whole cell IK(V) recordings in A and B] in human PASMC (hPASMC) and rPASMC, respectively. The development of apoptotic nuclei induced by ST and SNAP, characterized by nuclear shrinkage and condensation (quantified using DAPI staining and TUNEL assay), is inhibited by the application of 4-AP, a relatively selective KV channel antagonist (bar graphs). TEA, tetraethylammonium. [Modified from Krick et al. (86, 88).] ***P < 0.001 vs. ST (A) or SNAP (B).
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In addition to KV channels, activation of KCa channels has also been implicated in AVD and apoptosis. In vascular smooth muscle cells, for example, FCCP, which dissipates the proton gradient across the inner mitochondrial membrane (IM) and disrupts the m, causes an increase in cytoplasmic free Ca2+ concentration and enhances K+ efflux via iberiotoxinand TEA-sensitive KCa channels (Fig. 7A) (38, 87). Activation of KCa channels by the NO donor SNAP (Fig. 7B) and by diydroepiandrosterone (DHEA) also induces apoptosis in human PASMC (86, 88). TNF-, a death receptor agonist, activates Ca2+-dependent and protein kinase C (PKC)-activated KCa channels, increases K+ currents and efflux, and induces apoptosis in rat liver HTC cells (120). Furthermore, hydrogen peroxide (H2O2)-mediated apoptosis (134) is associated with activation of TREK KT channels (162), whereas cromakalim induces neuronal apoptosis by activating KATP channels (188). Conductance of the human ether-a-go-go channels markedly promotes H2O2-induced apoptosis in various tumor cell lines (170). Nevertheless, inhibition of K+ channels by Ba2+ and quinine attenuates apoptosis and increases viability of ST- or TNF-/cycloheximide-treated cells in human lymphoid (U-937) and epithelial (HeLa) cells, hybrid neuroblastoma/glioma (NG108-5) cells, rat pheochromoytoma (PC12) cells (103), and liver HTC cells (120).
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Fig. 7. Proapoptotic agents stimulate K+ efflux via iberiotoxin (IBTX)-sensitive Ca2+-activated (KCa) channels in PASMC. Unitary KCa currents (IK(CA)) were recorded in hPASMC and rPASMC stimulated with carbonyl cyanide-p-trifluoromethoxyphenyl hydrazone (FCCP, 5 µM; A) or SNAP (0.1 mM, B) and following drug washout. Single channel conductance of the evoked currents was 250 pS, typical for KCa channels in vascular smooth muscle. The application of 100 nM IBTX, a selective inhibitor of these large-conductance KCa channels, attenuated the apoptosis induced by FCCP and SNAP (histograms) in rPASMC and hPASMC. [Modified from Krick et al. (87, 88).] ***P < 0.001 vs. FCCP (A) or SNAP (B).
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There is evidence that the proapoptotic stimulation of K+ channels may be mediated by auxiliary modulatory proteins and kinases. KChAP (K+ channel-associated protein/protein inhibitor of activated STAT) is a K+ channel modulatory protein belonging to the protein inhibitor of the STAT family, members of which are known to interact with transcription factors such as the proapoptotic p53 protein (90, 179). KChAP induces apoptosis in prostate cancer cell lines by increasing K+ efflux and causing cell shrinkage; KChAP is also increased by ST treatment (179). On the basis of the latter study, it is believed that KChAP increases p53 levels and stimulates phosphorylation of p53 residue serine 15, leading to elevation of p21 levels and apoptosis (38, 179). Although PKC is involved in numerous cell functions, its role in modulating apoptosis is unclear. There is evidence that the enhanced K+ efflux produced by Fas and TNF- can be blocked by PKC stimulation (56, 120), i.e., PKC inhibition promotes cell shrinkage. Tyrosine kinase-mediated phosphorylation appears to play a more important role in modulating cell survival than PKC. In cortical neurons (187) and lymphocytes (157), tyrosine kinase inhibition (by herbimycin A, lavendustin A, or genistein) attenuates Fas- and ceramide-induced apoptosis and upregulation of N-type and delayed-rectifier K+ channels. A more recent study showed that inhibition of tyrosine phosphorylation also suppresses the activity of the Na+-K+-ATPase pump in cortical neurons, leading to apoptosis (177). Although kinases may modulate K+ channel activity, kinase stimulation also may be dependent on K+ channel activation. For example, in myeloblastic leukemia cells, UV-stimulated K+ currents subsequently activate the JNK/SAPK signaling pathway to cause apoptosis (177). These observations indicate that phosphorylation of apoptotic proteins and of membrane channels plays an important role in regulating cell survival and, in particular, in enhancing the proapoptotic role of K+ channels.
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MODULATION OF CYT-C RELEASE AND CASPASE ACTIVATION BY K+ EFFLUX
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The release of cyt-c from the mitochondrial intermembrane space is pivotal to apoptosis, since formation of the cyt-c/APAF-1/caspase-9 apoptosome triggers the activation of the effector caspases-3/-6/-7. ST (86, 103), TNF-/cycloheximide (103), NO (88), Fas ligand (56), etoposide (183), and UV irradiation (52, 90) all cause cyt-c release into the cytosol. Mitochondrial membrane depolarization induced by FCCP and NO also causes cyt-c release (10, 67). In many of these cases (except Fas ligand), caspase inhibitors do not prevent the release of cyt-c, indicating that cyt-c release occurs before activation of caspase-3 (19, 60, 78, 81, 129, 164, 183).
Both cyt-c release and caspase-3 activation are readily attenuated by inhibition of sarcolemmal K+ channels by quinine and Ba2+ (103), suggesting that activation of K+ channels occurs before cyt-c release and caspase activation in apoptotic cells. In addition, a decrease in [K+]cyt enhances caspase activation and limits cyt-c release in lymphocytes (16). Physiological [K+]cyt also inhibits formation of the APAF-1/cyt-c/caspase-9 apoptosome (21), while increasing [K+]o (which reduces the driving force for K+ efflux) inhibits death receptor-mediated apoptosis before cyt-c release and caspase-8 activation can occur (21, 160). Therefore, maintenance of physiological and high [K+]cyt not only inhibits AVD but also suppresses cyt-c release from the mitochondria and inhibits cytoplasmic caspase activation, the deciding factors in cell death.
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MODULATION OF CASPASE ACTIVITY AND DNA FRAGMENTATION BY CYTOPLASMIC K+
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The final phase of apoptosis involves degradation of the nucleus and its contents. Internucleosomal DNA fragmentation is typically visualized as DNA laddering, i.e., DNA fragments that migrate as multiples of 200 bp during agarose gel electrophoresis correspond to strands of DNA cleaved in internucleosomal sites (112). Cytoplasmic K+ in physiological concentration (140 mM) inhibits chromatin condensation and DNA fragmentation, likely through suppression of caspase and endonuclease activities (29). The suppression of endonucleases and caspases is mimicked by sarcolemmal K+ channel inhibition (30) during apoptotic stimulation by dexamethasone (a glucocorticoid receptor agonist) and etoposide (a topoisomerase inhibitor and genetoxic agent). Similar effects are also observed with quinine and Ba2+ in ST- or TNF-/cycloheximide-treated U-937, HeLa, PC12, and NG108-15 cells (103).
Although K+ efflux may also occur via K+-Cl- cotransporters and the combined K+-H+ exchange/ exchange system, most efflux occurs via K+ channels. Decreased [K+]cyt, due to enhanced K+ efflux through opened sarcolemmal K+ channels, also enhances endonuclease activity. This suggests that the [K+]cyt, transmembrane K+ gradient, and function and expression of sarcolemmal K+ channels all contribute to regulating the early (e.g., by modulating AVD) and late (e.g., by modulating caspase activity) stages of apoptosis (16, 112). Indeed, in a cell-free system (isolated nuclei), a decrease in [K+] from 140 to 80 mM caused a 1.6-fold increase in apoptosis induced by the apoptosis-inducing factor (29). In lymphocytes, an 8-h treatment with ST decreased [K+]cyt from 140 to 50 mM, whereas a decrease in [K+] in assay buffer from 150 to 80 mM caused a 2.4-fold increase in DNA degradation in isolated nuclei (72). Similar experiments performed in rat PASMC show that increasing [K+] in the assay buffer enhanced caspase-3 activity (Fig. 8A) (105). The NO-induced apoptosis (Fig. 6B) and increase in caspase-3 (Fig. 8B) were both attenuated by K+ channel inhibition by 4-AP, TEA, and high [K+]o. These results provide evidence that a high [K+]cyt is required to suppress the activation of apoptotic processes (e.g., activation of caspases and endonucleases), whereas K+ efflux relieves the inhibition on cytoplasmic caspases and nucleases, thereby enhancing apoptosis.
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Fig. 8. Inverse correlation between [K+] and caspase-3 (Casp-3) activity. A: increasing [K+] in the assay buffer from 0 to 25, 75, 100, and 150 mM causes a marked decrease in caspase-3 activity [means ± SE, determined as the optical density (OD) at 405 nm of the caspase-3-cleaved product]. [Reproduced with permission from Elsevier from Mandegar et al. (105).] B: SNAP (0.1 mM) treatment significantly increases caspase-3 concentration (means ± SE) in PASMC. The SNAP-induced enhancement is attenuated by inhibiting K+ channel activity with increased extracellular K+ concentration ([K+]o) or TEA (1 mM). **P < 0.01 vs. solid bar. NOR, SNAP in physicological solution; 40 K, 40 mM [K+].
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CL- EFFLUX ALSO AFFECTS APOPTOSIS
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As was discussed earlier, Cl- efflux is tightly coupled to K+ efflux, especially in cells undergoing apoptosis. It is therefore not surprising that numerous apoptosis inducers can trigger Cl- channel activity. ST-induced AVD and apoptosis are significantly reduced by Cl- channel inhibitors such as 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB), 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), phloretin, and 4-acetamido-4'-isothiocyanostilbene in HeLa, U-937, PC12, and NG108-15 cells (103). Apoptosis induced by TNF- treatment of rat liver HTC cells is reversed by NPPB and N-phenylanthranilic acid (DPC) (120). However, at least in HeLa and U-937 cells, application of anthracene-9-carboxylate and furosemide does not prevent ST-induced apoptosis, thereby eliminating cAMP-activated cystic fibrosis transmembrane regulator (CFTR) channels and Na+-K+-2Cl- and Na+-Cl- symporters as possible Cl- extrusion pathways (103), although the role of CFTR in apoptosis is under debate (58, 110). Fas ligand/CD95 binding-mediated apoptosis is partially inhibited by indanyloxyacetic acid, DPC, and DIDS in lymphocytes (158). Many of these Cl- channel antagonists also attenuate cyt-c release, caspase-3 activation, and DNA fragmentation in the same cells (103, 136). The Cl- channels involved in apoptosis may possibly be members of the Ca2+-activated or volume-sensitive channel families identified in mammalian cells based on their pharmacological properties (95, 152).
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MODULATION OF K+ CHANNEL ACTIVITY BY ANTIAPOPTOTIC PROTEINS
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The important role played by [K+]cyt and K+ channel activity in AVD and apoptosis is further enhanced by the fact that the antiapoptotic proteins Bcl-2, an antiapoptotic member of the Bcl-2 family, and ARC (apoptosis repressor with caspase recruitment domain), an antiapoptotic protein in cardiac and skeletal myocytes, modulate sarcolemmal K+ channel function (43, 44).
Bcl-2 is a large family of proteins with contrasting effects on apoptosis. Although structurally similar, some members of the family (Bax, Bak, Bad, Bid, Bim, PUMA, Noxa, Blk, Bik/Nbk, Hrk/DP5, Bok/Mbl, Bcl-xS) promote apoptosis, whereas others (Bcl-2, Bcl-xL, Bcl-2, A1, Mcl-1, Boo) inhibit apoptosis (1, 154). Proapoptotic Bcl-2 proteins are cytoplasmic and activate only with apoptotic stimulation. Some (like Bak, Bax, and truncated Bid) can translocate and insert themselves into the mitochondrial membrane upon apoptotic stimulation, thereby enhancing cyt-c release and causing apoptosis (62, 98, 101). The antiapoptotic protein Bcl-2 is mainly located in the endoplasmic reticulum (ER) membrane, the nuclear envelope, and the outer mitochondrial membrane (OM). The designation of Bcl-2 and Bcl-xL as antiapoptotic proteins has been blurred by recent evidence that cleavage by caspase-3 converts them into proapoptotic proteins similar to Bax (22, 23). Furthermore, mitochondrial Bcl-2 can cause apoptosis, whereas ER Bcl-2 protects against apoptosis induced by Bax overexpression (174). Therefore, the physical origin or location of Bcl-2 may be an important regulator in apoptosis.
Bcl-2 genes are regulated by cytokines and other death-survival signals at different levels. For example, antiapoptotic genes are induced transcriptionally by certain cytokines, whereas antiapoptotic Bax genes are induced as part of the p53-mediated damage response (3, 14). In addition, Bcl-2 can protect against apoptosis induced by 
- and UV-irradiation, cytokine withdrawal, glucocorticoid treatment, and ST (25), but not against apoptosis induced by ligand binding to CD95 death receptors in lymphocytes (153).
Bcl-2 inhibits apoptosis primarily by blocking cyt-c release into the cytoplasm (81, 183), although it can also protect against apoptosis via 1) inhibition of some proapoptotic proteins (see Fig. 2) (62, 98, 101, 155), 2) restoration of the high ATP-to-ADP ratio in the cytosol by facilitating mitochondrial ATP/ADP exchange (163), 3) direct antioxidant effects (24, 69), 4) regulation of Ca2+ content in the mitochondria and sarcoplasmic reticulum (65, 91, 197), and 5) maintenance of a negative m via enhanced proton efflux or formation of mitochondrial cation channels (5, 142, 145). In addition to these more well-characterized effects, the antiapoptotic Bcl-2 protein has been shown to prevent apoptosis by, at least in part, acting on sarcolemmal K+ channels in vascular smooth muscle cells. In rat PASMC, overexpression of the human bcl-2 gene using an adenoviral vector 1) markedly increases the protein expression of Bcl-2 (Fig. 9Aa), 2) decreases current density of the 4-AP-sensitive KV channels (Fig. 9, Ab–d), 3) downregulates mRNA expression of KV1.1, KV1.5, and KV2.1 channels as well as their representative whole cell KV currents (Fig. 9B), and 4) inhibits ST-mediated apoptosis (Fig. 9C) (43). These results suggest that inhibition of KV channel activity may serve as an additional mechanism involved in the Bcl-2-mediated antiapoptotic effect in vascular smooth muscle cells. The precise mechanisms by which Bcl-2 downregulates mRNA expression of KV channels and inhibits KV channel activity are unknown.
The recruitment and activation of caspases is central to the regulation of apoptosis. One of the protein-protein interaction motifs involved in death receptor-mediated apoptosis involves a caspase recruitment domain (CARD). ARC is a cardiac and skeletal muscle CARD-containing protein that binds to the initiator caspases-2/-8 and significantly attenuates death receptor-induced apoptosis (83, 161). Multiple mechanisms are involved in the antiapoptotic effect of ARC on cardiomyocytes: 1) inhibition of caspase activation (83); 2) blockade of hypoxia/ischemia-induced cyt-c release (42); and 3) prevention of H2O2-mediated loss of membrane integrity and disruption of the m (116).
In addition to the inhibitory effects on cyt-c release (42) and mitochondrial disruption (116), overexpression of ARC in cardiomyocytes 1) blocks sarcolemmal KV channels (Fig. 10A), which possibly contributes to inhibition of the apoptotic cell shrinkage, and 2) inhibits ST-mediated activation of K+ channels (Fig. 10B) and apoptosis (Fig. 10C) (44). The precise mechanism(s) by which ARC blocks KV channels remains unclear. The ARC-mediated inhibition of ST-induced increase in IK(V) may be partially due to its inhibiting cyt-c release (42), because cytoplasmic dialysis of cyt-c increases IK(V) in vascular smooth muscle cells (Fig. 5) (129).
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Fig. 10. Overexpression of ARC decreases KV channel activity in embryonic rat heart H9c2 cells. A: Western blot analysis (top) showing the ARC protein levels in Neo cells (empty vector) and cells stably transfected with the human ARC-5 gene. Representative currents (bottom), elicited by test potentials between -60 and +80 mV (holding potential -70 mV), in a Neo cell and an ARC-5-transfected cell. B: averaged currents at +80 mV (holding potential, -70 mV) in Neo cells and ARC-5-transfected cells before (Cont) and after (ST) treatment with ST (0.02 µM). C: summarized data showing the percentage of cells undergoing apoptosis in Neo and ARC-5-transfected cells before (Cont) and after (ST) exposure to ST. ***P < 0.001 vs. Neo. [From Ekhterae et al. (44).]
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The inhibitory effects of the antiapoptotic proteins Bcl-2 and ARC on plasmalemmal K+ channels in smooth muscle cells and cardiomyocytes further support the theory that activation of K+ channels is a critical step for cells to undergo apoptosis, whereas inhibition of K+ channels attenuates apoptosis, which would facilitate cell proliferation and cause tissue remodeling.
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ROLE OF MITOCHONDRIAL ION FLUX IN APOPTOSIS
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Changes in mitochondrial membrane permeability (MMP) determine the ultimate fate of cells irrespective of the nature of the proapoptotic stimuli. Therefore, the mitochondria play a central role in modulating apoptosis by 1) integrating different signal transduction cascades to a common pathway (47) initiated by MMP alterations and 2) releasing soluble proteins (i.e., cyt-c, Smac/Diablo, AIF, procaspases) from the mitochondrial intermembrane space into the cytosol.
The two well-defined compartments (i.e., intermembrane space and matrix) within the mitochondria regulate its activity. Under physiological conditions, the folded IM (Fig. 11) is almost impermeable, allowing the respiratory chain within the matrix (the region surrounded by the IM) to generate an electrochemical gradient that regulates the highly negative (-150 to -200 mV) m via the production and translocation of H+. Disruption of MMP may result from defective ATP/ADP exchange between the matrix and cytosol mediated by the adenine nucleotide translocase (ANT) on the IM and voltage-dependent anion channels (VDAC) on the mitochondrial OM. Persistent membrane impermeability to ATP/ADP exchange ultimately results in loss of OM integrity, rendering it permeable to soluble proteins. IM permeabilization (visualized by cytofluorometry) results from disruption of m.
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Fig. 11. Cross-sectional view of the mitochondria and the inner and outer membrane channels contribute to the regulation of apoptosis. IM, inner mitochondrial membrane, OM, outer mitochondrial membrane, VDAC, voltage-dependent anion channel; ANT, adenine nucleotide translocase; mtKCa, mitochondrial KCa channel, mtKATP, mitochondrial KATP channel, mtCLIC, mitochondrial intracellular Cl- channel.
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The numerous channels and exchangers that populate the mitochondrial IM and OM play significant roles in the modulation of m and apoptosis. What follows is a brief discussion of selected mitochondrial ion channels and how ion permeability within the mitochondria also contributes to cyt-c release and apoptosis. A focused review of mitochondrial K+ channels will follow in a separate section. For more in-depth information, readers should refer to recent publications dealing with mitochondrial cation transport, m regulation, and control of cellular function (12, 47, 89, 118, 124, 175).
The VDAC, or mitochondrial porin, is a large-diameter OM channel serving as a voltage-dependent permeability pathway for large uncharged molecules (<5 kDa) such as NADH and metabolites. Together with the ANT on the IM, the VDAC forms the so-called mitochondrial permeability transition pore, a nonselective channel that, when opened, allows for the equilibration of ions within the matrix and intermembrane space, thereby dissipating the H+ gradient and disrupting or depolarizing m (60, 89, 147). VDAC activity is required for apoptotic m loss or depolarization to occur (146). Alone, the VDAC is weakly anion selective (i.e., permeable to Cl-, DIDS sensitive) (146).
Bax channels are formed on the OM when soluble Bax proteins translocate from the cytosol upon apoptotic stimulation. Bax channels exhibit cation (K+, Na+) selectivity and, like VDAC, do not allow cyt-c release from the mitochondrial intermembrane space to the cytosol (146). However, both Bax and Bak proteins can interact with OM VDAC to form a large pore. In consequence, disruption of VDAC integrity allows cyt-c to pass into the cytosol at a rate of 10 molecules·s-1·channel-1 (68, 146).
A mitochondrial intracellular Cl- channel (mtCLIC) has been identified on the mitochondrial IM (46); no other mitochondrial IM Cl- channels have been identified to date. Expression of mtCLIC is regulated by p53 and TNF-, two potent proapoptotic agents, suggesting that it may be a common downstream effector for these two apoptotic stimulants. It has been suggested that changes in mitochondrial IM permeability to Cl- via mtCLIC and changes in m lead to activation of the mitochondrial permeability transition pore and apoptosis.
Bcl-2 channels on the mitochondrial OM are mostly closed at neutral pH. At more acidic pH (pH 5.4), Bcl-2 forms cation channels (142). The antiapoptotic Bcl-2 and Bcl-xL channels increase cell survival by causing m hyperpolarization, leading to 1) decreased cyt-c release, 2) increased mitochondrial uptake of cationic fluorescent dyes (e.g., rhodamine-123), 3) increased Ca2+ uptake, 4) increased resistance to disruption of m, 5) enhanced H+ efflux in the presence of m-depolarizing stimuli (without influencing K+ e
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ABSTRACT
APOPTOSIS: AN OVERVIEW
exchangers. Cell shrinkage (bottom) is characterized by increased cytoplasmic H2O removal (via aquaporins) due to K+ and Cl- efflux via K+ and Cl- channels, K+/Cl- cotransporters, and coupled 
exchangers.
) and bcl-2-transfected (
) cells (c). Current density of IK(V) at +80 mV is signifi-cantly decreased in cells infected with bcl-2 (d). B: single cell RT-PCR amplified products (a) for human Bcl-2 (267 bp) and rat Kv1.1 (298 bp), Kv1.5 (196 bp), Kv2.1 (269 bp), and 
-actin (267 bp) as well as the corresponding whole cell IK (b) in a control cell (-bcl-2) and a bcl-2-infected cell (+bcl-2). -RT, RT performed in the absence of reverse transcriptase. M, 1 kb plus DNA ladder marker. C: ST (0.02 µM)-induced apoptosis is inhibited in cells infected with bcl-2. ***P < 0.001 vs. -bcl-2. [From Ekhterae et al. (