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Biological Research

versión impresa ISSN 0716-9760

Biol. Res. v.35 n.2 Santiago  2002 

Biol Res 35: 209-214, 2002

Ion movements in cell death: from protection to


Centro de Estudios Científicos CECS. Arturo Prat 514, Casilla 1469, Valdivia, Chile


Cell death is preceded by severe disruption of inorganic ion homeostasis. Seconds to minutes after an injury, calcium, protons, sodium, potassium and chloride are exchanged between the cell and its environment. Simultaneously, ions are shifted between membrane compartments inside the cell, whereby mitochondria and endoplasmic reticulum play a crucial role. Depending of the type and severity of injury, two mutually exclusive metastable states can be reached, which predict the final outcome. Cells characterized by large increases in cytosolic [Ca2+], [Na+] and [Mg2+] swell and die by necrosis; alternatively, cells characterized by high [H+] and low [K+], with normal [Na+] and normal to moderate [Ca2+] increases die by apoptosis. The levels of these ions represent central determinants in signaling events leading to cell death. Their movements are explained mechanistically by specific modulation of membrane transport proteins including channels, pumps and carriers.

Key terms: Cell death; membrane channels; apoptosis; necrosis


Steep spatial gradients of inorganic ions are maintained between the cell and its environment. Within the cell itself, gradients can be even bigger, reaching 60 KJ/mol for the difference between the electrochemical potential for Ca2+ between the endoplasmic reticulum and the mitochondria (equivalent to a concentration ratio of 1010:1). The energy accumulated is utilized for general tasks in maintenance of cell homeostasis, with oxidative phosphorylation being an example, and in specialized signaling processes, including muscle contraction, hormone secretion, nerve conduction and synaptic transmission. Maintenance of such gradients requires energy. Thus, it is not surprising that ion gradients slowly dissipate in ischemic tissues. Due to the relative enrichment of non-diffusible anions within a cell and the resulting Gibbs-Donnan effect, a non-energized cell inevitably must gain sodium and water.

The ensuing swelling process leads to cell rupture and release of contents, with collateral damage to neighboring cells and tissue inflammation as a consequence. Because ions and water equilibrate in time frames consistent with the development of anoxic or oxidative death, and some ions, particularly calcium, were known to affect protein activity, early theories advanced in the 1950s proposed that loss of ion homeostasis is central to pathogenic mechanisms of cell death. Today, the ion theory of cell necrosis is well established and advances in our understanding of the molecular mechanisms involved and their relation to irreversibility are rapidly being made (Castro, et al., 2001).

In multicellular organisms, cell death is not equivalent to death of the individual. On the contrary, cell death is an essential part of tissue homeostasis. During every second of an adult human life 100,000 cells are destroyed by the active process of orderly cell disassembly, termed apoptosis. The molecular machinery involved includes receptors, channels, regulators and proteases. Some of these elements, like caspases, are specific to apoptosis while others, like potassium and chloride channels, are co-opted from vital processes. Apoptosis is essential for normal development and central to many pathophysiological developments, as exemplified by AIDS, Cancer and Alzheimer´s disease. These disorders can often be explained by either lack or excess of apoptotic death. In contrast to necrosis, apoptosis is slow, with changes occurring in hours to days, but ion movements are also key events in this type of cell death (see also Razik and Cidlowski, 2002).

This short review outlines recent advances in our mechanistic understanding of cell death, whereby emphasis is laid on a description of ion movements between subcellular compartments, as well as the channels, pumps and transporters that mediate such movements. Recent detailed reviews of these topics can be found in (Barros et al., 2001a; Ermak & Davies, 2002).


Oxidative stress causes intracellular acidification, by several mechanisms including: (i) Energy depletion, with the release of one H+ per ATP molecule hydrolyzed to ADP; (ii) Poly (ADP-ribose) polymerase (PARP) activation by DNA damage releases one H+ per NAD+ used in the reaction; (iii) ATP depletion inhibits proton extrusion through the Na+/H+ exchanger; (iv) Release of H+ from acidic compartments due to lack of ATP-driven pumping; and (v) Increased generation of protons by anaerobic glycolysis or uncoupled-mitochondria. In necrosis, acidosis plays a protective role, as exemplified by massive cell death during alkaline reperfusion, a process referred to as the «pH paradox». Cytoprotection by acidosis, is not explained by changes in Ca2+ or Na+ levels, but may reflect a direct inhibitory effect of low pH on mitochondrial permeability transition (Lemasters et al., 1999). In contrast to necrosis, apoptosis can be facilitated and even triggered by acidosis (Matsuyama et al., 2000). Two mechanisms explaining these effects are the pH dependence of endonucleases (Perez-Sala et al., 1995) and the maintenance of caspase-3 proenzyme dormancy by an intrinsic tripeptide, whose inhibitory effect is ablated at low pH (Roy et al., 2001). On the other hand, translocation of pro-apoptotic Bax to mitochondria is triggered by Na+/H+ exchanger-dependent cytosolic alkalinization (Khaled et al., 1999).


Calcium ions participate in both necrosis and apoptosis, and several mechanisms of cytosolic calcium overload in stressed cells have been described: (i) Calcium entry through NMDA receptors and voltage-sensitive Ca2+ channels in neuronal excitotoxicity; (ii) Reverse operation of the Na+/Ca2+ exchanger; (iii) Calcium release from intracellular stores; and (iv) Calcium entry through non-selective cation channels. Calcium affects the function of most cell organelles and proteins, so it has been difficult to identify those targets key to its toxicity. Most suggested candidates participate in positive-feedback loops that ensure irreversibility of the process. Examples here are: (i) Mitochondrial Ca2+-overload (Stout et al., 1998); (ii) Ca2+-activated proteases (Wang, 2000); and (iii) Activation of non-selective cation channels (Barros et al., 2001b). Intracellular calcium stores can also determine necrosis, as shown by the requirement of calreticulin, calnexin, as well as IP3- and ryanodine-receptors in necrotic neuronal death in C. elegans (Xu et al., 2001). The absence of a strict correlation between calcium levels and its toxicity suggests «time» and «source» specificity. For instance, blockage of mitochondrial calcium uptake in neurons resulted in diminished glutamate excitotoxicity despite higher levels of cytosolic calcium (Stout et al., 1998). In apoptosis, the role of calcium is cell- and stimulus-dependent. For instance, Fas-dependent apoptosis in lymphocytes does not require calcium, while apoptosis induced in neurons by serum deprivation is calcium-dependent (Lee et al., 1999). One mechanism proposed, involves Ca2+-calcineurin dependent dephosphorylation and activation of the pro-apoptotic protein Bad (Wang et al., 1999). Defects in calcium storage can also trigger apoptosis as shown by the specific activation of caspase 12 (Nakagawa et al., 2000).


The role of magnesium in cell death is not well understood. Despite being present in the mM range only 6% of cytosolic magnesium is free, with most existing in complexes with proteins and ATP (Corkey et al., 1986). In acutely stressed cells, ATP is hydrolyzed and magnesium is released due to its lower affinity for ADP and AMP (Harman et al., 1990). Although Mg+2 ions are known to inhibit the permeability transition pore (Lemasters et al., 1999), their role in pre-necrotic cells has not been determined. However, exogenous administration of magnesium salts have proven protective in several models of ischemia (Maulik et al., 2001). In apoptosis induced by ligation of FAS, the resting level of free Mg+2 increased prior to DNA fragmentation and phosphatidylserine (PS) externalization (Chien et al., 1999). A molecular mechanism for Mg2+-dependent modulation of apoptosis was shown recently in mitochondria where Bax-induced cytochrome C was found to be potentiated by mM concentrations of this divalent ion (Eskes et al., 1998).


Sodium is a major determinant of the necrotic outcome, due both to direct effects on other ions such as calcium and also due to its impact on cell volume. For instance, ATP-depleted hepatocytes swell and lose viability only in presence of sodium (Carini et al., 1999). Inhibition of the Na+/K+ ATPase in energy depleted cells leads to intracellular accumulation of Na+ (Cotran et al., 1999). Activation of both the Na+/H+ exchanger and the Na+/HCO3- co-transporter facilitate hepatic cell death (Carini et al., 1995). The Na+/H+exchanger operating in tandem with the Na+/Ca2+ exchanger facilitates calcium overload and cell death in cardiomyocites (Levitsky et al., 1998). Another mechanism of augmented Na+ influx is suggested by the observation that free-radical donors can rapidly activate non-selective cation channels in several cell types (Barros et al., 2001b). Moreover, pharmacological inhibition of one of these channels abolished cell swelling, cytosolic calcium overload and lysis of liver cells exposed to free-radical donors (Barros et al., 2001b). These channels are ubiquitous, but lack known physiological function in most tissues. They are kept dormant in unstressed cells and become activated by free-radicals and high [Ca2+], and are blocked by ATP. Rapid progress in our understanding of their role in cell death is guaranteed by their recent molecular identification (Launay et al., 2002). Sodium may also play a role in apoptosis, this time being inhibitory, as suggested by the effect of Na+/K+ ATPase inhibition in smooth muscle cells (Orlov et al., 1999).


In hepatocytes, necrotic Na+ overload is accompanied by net K+ release (Carini et al., 1999), whose effect on necrotic death remains unknown. K+ loss and cell shrinking have been shown to be absolute requirements for apoptosis (Bortner & Cidlowski, 1996; Lang et al., 1998). In several epithelial, lymphoid and neuronal cell lines shrinking, which was termed apoptotic volume decrease (AVD), was located upstream of caspase 3 activation and DNA degradation. In contrast, in Jurkat cells, FAS-induced AVD was blocked by caspase inhibition (Bortner & Cidlowski, 1999). Several K+ channels may be involved in AVD, including the neuronal delayed rectifier (IK) and the NMDA receptor (Yu et al., 1997; 1999). A recent report suggests that the two-pore domain potassium channels used in cell volume regulation (Niemeyer et al., 2001) may also play a role in AVD (Trimarchi et al., 2002). The molecular mechanisms underlying the pro-apoptotic role of potassium loss are not clear. Whereas movement of potassium to the extracellular space is pro-apoptotic, its transport into the mitochondria through diazoxide-sensitive mitoKATP channels is protective. These phenomenon has been studied extensively in heart cells, where channel opening via PKC-dependent mechanisms depolarize the mitochondria, reducing the driving force for mitochondrial calcium uptake and thus explaining the protective role of ischemic preconditioning (Korge et al., 2002; O'Rourke, 2000).


By allowing charge equilibration during cation movements, anion channels are expected to play a permissive role in the kinetics of both swelling and shrinkage, a point that has already been demonstrated for AVD (Maeno et al., 2000; Szabo et al., 1998). In T lymphocytes, FAS-mediated apoptotic chloride efflux occurs through an outwardly rectifying cation channel (ORCC) (Szabo et al., 1998). In addition, chloride movements between organelles may play a role in cell death, as suggested by the pro-apoptotic role of the mitochondrial chloride channel mtCLIC in p53 mediated apoptosis (Fernandez-Salas et al., 2002) and the chloride selectivity of Bax (see below).


The Bcl-2 family deserves special mention in this revision. This large group of cytosolic proteins can either inhibit (e.g. Bcl-2, Bcl-xL) or facilitate (e.g. Bax, Bad, Bid, Bak) apoptotic cell death. As was mentioned above, members of this protein family have been shown to regulate calcium homeostasis by their interaction with membranes of mitochondria and the endoplasmic reticulum. In most cases, the molecular mechanism of action is not known, but the fact that several of these proteins can form ion channels is intriguing (Minn et al., 1997; Schendel et al., 1999; Schlesinger et al., 1997). Anti-apopototic Bcl-2 forms channels which are mildly selective for K+ (PK+/PCl- = 3.9), while Bax forms channels which are mildly selective for Cl- (PK+/PCl- = 0.3)(Schlesinger et al., 1997). It should be noted, however, that all aforementioned biophysical evidence was obtained using artificial membrane bilayers, so the in vivo selectivity is unknown. In addition, it is not clear yet whether the demonstrated modulatory effect of these proteins on apoptosis is related to their ion channel forming capabilities or not.

In summary, data from different cell types suggest that active disruption of ion homeostasis is a major factor determining cell death. In the first minutes of exposure to a lethal stimulus, ions are exchanged between the cell and its environment as well as between intracellular compartments. Some of these movements are pro-necrotic, others are pro-apoptotic and yet others are protective. Most channels, pumps and carriers, the proteins that mediate these movements, are either known or are quickly being identified. Understanding and predicting cell survival or death will require establishing complex models that consider factors like the kinetic parameters of these transporters, ion cross-talk and subcellular ion domains.

This presentation will address the mechanisms involved in ion modulation of necrosis and apoptosis, with an emphasis on the interplay between cytosol, mitochondria and the endoplasmic reticulum. We will show recent data from our laboratory, obtained by confocal microscopy using fluorescent probes, which reveal the temporal sequence of ion changes triggered by oxidants in cultured cells, and how these movements may relate to irreversibility of the process. We will also focus on the role of specific ion channels and how activation/inactivation of these proteins can shift cells between survival, apoptosis and necrosis.


This work was supported by FONDECYT Grant No.1020648. We also thank Fundación Andes for an Equipment Grant to CECS. CECS is a Millennium Science Institute.


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To whom correspondence should be addressed: Felipe Barros. CECS. Arturo Prat 514, Casilla 1469, Valdivia, Chile. Phone: + 56 63 234500. Fax: + 56 63 234515.e-mail:

Received: May 30, 2002. In revised form: June 24, 2002. Accepted: July 11, 2002

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