Molecular interplay between ion channels and the
regulation of apoptosis

MONA A RAZIK AND JOHN A CIDLOWSKI*

The Laboratory of Signal Transduction. Molecular Endocrinology Group
National Institute of Environmental Health Sciences. National Institutes of Health. 111 Alexander Drive, Research Triangle Park, NC 27709. Phone (919) 541-1564. Fax (919) 541-1367

ABSTRACT

Apoptosis is the programmed and deliberate destruction of specific cells. This process occurs during normal development and maintains cellular homeostasis. Disruption or malfunction of apoptosis is implicated in diseases like cancer, AIDS as well as neurodegenerative disorders. The movement of monovalent ions appears to set the stage for the induction of the self-destruction machinery by creating an intracellular environment that favors activation and coordinated execution of the apoptotic program. Understanding the components and steps involved in this intricate process can further our insight to diseases and reveal new approaches for therapeutic treatment.

GENERAL ASPECTS OF CELL DEATH AND APOPTOSIS

Cell homeostasis is achieved through an orchestrated balance between continuous cycles of cell proliferation and apoptosis. Apoptotic stimuli are, in general terms, either intracellular or extracellular by nature. Additionally, cues for activating the death process can either be of environmental or developmental origin. Some of the known apoptotic stimuli include the family of tumor necrosis factor ligands such as the Fas Ligand (Nagata and Goldstein, 1995), glucocorticoids, UV irradiation, toxins, cytotoxic drugs that interfere with the cell cycle, serum and/or growth factor deprivation as well as pathogens. Once the decision to die is reached, signals to a common death program are activated and executed, resulting in the elimination of toxic, pathogen invaded or auto-aggressive immune cells. The proteins necessary for apoptosis are usually present in the cellular cytosol, as evidenced by studies showing that cells lacking nuclei undergo apoptosis when treated with the protein kinase inhibitor staurosporine (Steller, 1995).

Morphological characteristics of apoptosis include cell shrinkage, protein degradation, chromatin condensation, and DNA degradation to yield the characteristic 180 to 200 base pair ladder. Additionally, phosphatidylserine accumulates in the exterior leaflet of the plasma membrane. Also, loss of monovalent ions, a decrease in the mitochondrial membrane potential, the release of cytochrome c, and eventually blebbing into apoptotic bodies are observed. Ultimately, these events result in engulfment of the dying cell by neighboring macrophages. Thus, apoptotic cells are rapidly removed from an organism, thereby avoiding activation of an inflammatory response. This mode of cell death contrasts very strikingly to necrosis, a pathological mode of cell death resulting from trauma, where cells swell due to increase in intracellular Mg²⁺, Na⁺ and Ca²⁺, rupture and trigger inflammation (Barros et al., 2002; Simon et al., 2002).

Regulation of cell volume

The plasma membrane of eukaryotic cells is permeable to water and most non-polar molecules, but less permeable to ions. Thus, to regulate the osmotic pressure of cells, which have an abundance of negatively charged organic molecules confined to their intracellular compartments, cells utilize channels and transporters to maintain ionic balance. As mentioned, different ways exist for ions to move across the plasma membrane, including electro diffusion, electro neutral exchange, co-transport and electrogenic exchange. Cells respond to changes in the osmolarity of their environment via regulatory volume increase (RVI) or regulatory volume decrease (RVD) mechanisms. For example, a cell placed in a hypertonic environment initially shrinks. This activates an RVI mechanism and as a consequence the cell quickly reverts back to normal volume. The RVI response is initiated by an influx of Na⁺ and Cl^- ions, subsequent replacement of Na⁺ by K⁺ via the Na⁺/K⁺-ATPase, resulting in a net increase in intracellular K⁺ and Cl^- and the movement of water into the cell. Alternatively, if a cell is placed in a hypotonic environment, the inverse process takes place with initial cell swelling followed by activation of the RVD response and return of the cell to its normal volume. RVD is achieved by expelling mainly K⁺ and Cl^- ions followed by the associated movement of water out of the cell (Eggermont et al., 2001). In addition to the involvement of the Na⁺/K⁺-ATPase during the RVD response, other ionic transport mechanisms may be involved, including the Na⁺/K⁺/2Cl^- co transporter, the Na⁺/H⁺ and the Cl^-/HCO₃ exchangers, as well as the individual ion channels. The activation of any given ionic transport mechanism is cell-type specific. In cells that fail to regulate their volume, a change in the membrane potential arises due to a deficit in charge across the plasma membrane.

Ionic Control of Apoptosis

A common feature of cells undergoing apoptosis is cell shrinkage, irrespective of the apoptotic stimuli (Kerr et al., 1972). Cell shrinkage has been shown to result from a dramatic efflux of intracellular Na⁺ and K⁺ ions (Bortner et al., 1997). Additionally, the intracellular ion concentration of a dying cell was shown to be an important factor in induction of apoptosis. In normal lymphocytes the concentration of K⁺ is 150mM; however, in striking contrast, K⁺ concentrations in shrunken cells are around 35mM. This decrease in K⁺ concentration has been shown to favor activation of proteases, mainly caspases, and apoptotic nucleases, which lead to cell death (Hughes Jr. et al., 1997; Cain et al., 2001). Loss of intracellular K⁺ was shown to be restricted to the shrunken cell population. Furthermore, in Jurkat T-cells, stimulated with anti-Fas antibodies sustained plasma membrane depolarization, attributable to increased intracellular Na⁺ but reduced K⁺ uptake, is observed prior to cell shrinkage (Bortner et al., 2001). Glucocorticoid induced apoptosis in rat thymocytes also results in the depolarization of the plasma membrane and when the Na⁺/K⁺-ATPase was inhibited by addition of ouabain, cells became more sensitive to apoptotic stimuli (Mann et al., 2001). Thus, inhibition of the Na⁺/K⁺- ATPase expedited plasma membrane depolarization and enhanced sensitivity to apoptotic stimuli.

Response to a particular stimulus is cell specific, and depends on the specific context. Hence, glucocorticoids induce apoptosis in lymphocytes but have a protective, antiapoptotic effect on serum deprived hepatoma cells (Evans-Storm and Cidlowski, 2000). Furthermore, in Fas ligand treated Jurkat T-cells increased intracellular calcium levels were observed. While such calcium transients were not an essential component of the signal transduction pathway leading to cell death, they were found to be necessary for activation of the apoptotic nucleases involved in chromatin degradation, and in this manner important for late stages of DNA catabolism (Scoltock et al., 2000). Together this evidence indicates that apoptosis is regulated by signal and cell type specific mechanisms, but common early events including plasma membrane depolarization and the efflux of ions, K⁺ being the most important, create an inductive environment for execution of the death process.

The Fas signal transduction pathway

Fas ligand is expressed mostly on the surface of T-cells, while Fas, which belongs to the tumor necrosis factor (TNF) family of receptors, is expressed by a variety of cell types. When the Fas receptor binds Fas ligand, cross-linking of the Fas receptor takes place and apoptosis is induced in the target cell. This signal transduction pathway has some steps that are specific to Fas, while others, involving the mitochondria, are commonly observed with other death stimuli. Fas has been extensively studied as a model system to elucidate the apoptotic cascade of events involved in both induction and protection of cells from apoptosis (Nagata and Goldstien,1995). The Fas receptor is a 45kDa transmembrane protein comprised of an extracellular ligand binding domain and a cytoplasmic effector domain. The cytoplasmic domain, of approximately 70 amino acid residues, is necessary and sufficient for transmitting the apoptotic signal and is referred to as the death domain. Upon ligand binding, receptor molecules aggregate and form signaling complexes that recruit, via adaptor proteins (Fas associated death domain or FADD), procaspase 8. Fas receptor bound to FADD and associated with caspase 8 form the DISC (Death -inducing signaling complex) which initiates the caspase cascade, through cleavage and activation of downstream caspases (Fig.1). Caspase 8 is activated by interactions with proteins in its proximity, and can be inhibited by the decoy c-FLIP (Flice Inhibitory Protein). When genetically manipulated caspase 8 deficient Jurkat T-cells were activated via Fas, initiation of apoptosis was blocked and no loss of K⁺ or cell shrinkage occurred (Vu et al., 2001). Furthermore the processing and activation of caspase 8, caspase 3 and Bid are regulated and can be blocked by protein kinase C activation (Gomez-Angelats and Cidlowski, 2001). A rapid increase in intracellular Na⁺ and inhibition of K⁺ uptake initially takes place prior to cell shrinkage (Bortner et al., 2001). Shrinkage resulting from the efflux of ions and particularly the loss of K⁺ create a favorable environment

for caspase activation (Bortner et al., 1997). Further amplification of the caspase cascade takes place at the level of the mitochondria. Caspase 8 cleaves Bid, a pro-apoptotic member of the Bcl-2 family, and processed Bid activates the mitochondria pathway. Interestingly Bid deficient mice are resistant to Fas induced apoptosis, hence underscoring the importance of Bid in signal transduction events downstream of Fas (Yin, 1999). Once mitochondrial function is perturbed, a number of pro-apoptotic proteins are released, including cytochrome c. In the cytoplasm, cytochrome c binds to the adaptor molecule Apaf-1 at a 2:1 ratio in the presence of dATP or ATP to form a complex (Zou, 1999). The latter recruits procaspase 9 to form the apoptosome. Caspase 9 is activated and in turn activates downstream caspases. Formation of the apoptosome has been shown to be sensitive to the intracellular K⁺ concentration whereby apoptosome assembly is prevented at physiological K⁺ levels (Cain et al., 2001). Inhibitors of apoptosis (IAP) family members prevent activation of several caspases, including caspase 9, and thus interfere with apoptosome formation (Roy et al., 1997). Additionally, smac/DIABLO, when released from the mitochondria, promotes caspase activation by binding to IAPs and preventing their inhibitory function (Verhagen et al., 2000; Du et al., 2000). Regardless of the apoptotic stimuli, cytochrome c released from the mitochondria is a common step that leads to formation of the apoptosome. Using Jurkat T-cells expressing dominant negative caspase 9 and UV irradiation as the death stimuli, it was shown that activation of caspase 9 is a critical step for progression of apoptosis (Vu et al., 2001).

In conclusion, apoptosis is essential in maintaining cellular homeostasis, and is orchestrated by the coordinated promotion of ion fluxes and subsequent activation of caspases. Changes in intracellular ionic concentrations, and K⁺ in particular, are essential for induction and progression of apoptosis. The molecular mechanisms connecting ion fluxes to the apoptotic machinery are still unknown, and these aspects remain to be investigated for different kinds of cell death. We have begun to elucidate ionic signals controlling death and survival of cells. The goal is to be able to manipulate these events by controlling ion fluxes, and thus to treat therapeutically diseases caused by insufficient apoptosis or unrestrained proliferation.

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*To whom correspondence should be addressed. e-mail: cidlowski@niehs.nih.gov

Received: June 06, 2002. In revised form: June 26, 2002. Accepted: July 14, 2002