Anion ChanneLs Osmoiyre Transport and pH ReGULAtion

Anion channels may be activated during cell proliferation (Nilius and Droogmans, 2001; Shen et al., 2000; Varela et al., 2004) and anion channel blockers may interfere with cell proliferation (Jiang et al., 2004; Pappas and Ritchie, 1998; Phipps et al., 1996; Rouzaire-Dubois et al., 2000; Shen et al., 2000; Wondergem et al., 2001). Moreover, cell proliferation may be impaired in cells lacking functional ClC-3 Cl_ channels (Wang et al., 2002). The signaling of cell proliferation may require transient cell shrinkage at some stage, which may be accomplished by activation of Cl_ channels. As intracellular Cl_ activity is usually above electrochemical equilibrium, activation of Cl_ channels leads to Cl_ exit and thus depolarization. If K+ channels are simultaneously active, the Cl_ exit is paralleled by the exit of K+. The loss of KCl and osmotically obliged water then leads to cell shrinkage (Lang et al., 1998a). In ras oncogene-expressing cells (Ritter et al., 1993), cell shrinkage is required for the initiation of cytosolic Ca2+ oscillations, which are in turn needed for the stimulation of cell proliferation. The initial cell shrinkage is reversed into a later cell swelling, a result ofa shifting cell volume regulatory set point toward greater volumes and a subsequent stimulation of Na+/H+ exchange and/or Na+,K+,2Cl~ cotransport. Activation of Cl_ channels at this later stage may impede cell proliferation.

Activation of Cl_ channels parallels the CD95-induced apoptosis ofJurkat cells (Szabo et al., 1998) and the TNFa- or staurosporine-induced apoptosis of various cell types (Maeno et al., 2000; Okada et al., 2004). Cl_ channels activated during CD95-induced apoptosis are the same as those activated by osmotic cell swelling and participating in regulatory cell volume decrease (Lepple-Wienhues et al., 1998). During both cell swelling (Lepple-Wienhues et al., 1998) and CD95-induced apoptosis (Szabo et al., 1998), the activation of Cl_ channels requires the Src-like kinase Lck56. The kinase is in turn activated by ceramide (Gulbins et al., 1997). In lymphocytes from patients with cystic fibrosis the Cl_ channels cannot be opened by protein kinase A but are activated by cell swelling and Lck56 (Lepple-Wienhues et al., 2001).

Cl_ channel inhibitors may blunt or even disrupt CD95-induced Jurkat cell apoptosis (Szabo et al., 1998), TNFa- or staurosporine-induced apoptosis of various cell types (Maeno et al., 2000; Okada et al., 2004), apoptotic death of cortical neurons (Wei et al., 2004), antimycin A-induced death of proximal renal tubules (Miller and Schnellmann, 1993), GABA-induced enhancement of excitotoxic cell death of rat cerebral neurons (Erdo et al., 1991), cardiomyocyte apoptosis (Takahashi et al., 2005), and eryptosis (Takahashi et al., 2005).

Activation of Cl_ channels leads to cellular loss of KCl and osmotically obliged water and thus to cell shrinkage. Some anion channels further allow exit of organic osmolytes such as taurine (Lang et al., 1998b,e; Moran et al., 2000), an effect contributing to cell shrinkage (Lang As organic osmolytes stabilize cellular proteins (Lang et al., 1998a), their loss could destabilize proteins. Inhibition ofinositol uptake has indeed been shown to induce renal failure, presumably because of apoptotic death of renal tubular cells (Kitamura et al., 1998).

Many Cl~ channels further allow HCO^ exit, leading to cytosolic acidification, a typical feature of cells entering into apoptosis (Lang et al., 2002a; Wenzel and Daniel, 2004). As the DNA-degrading enzyme DNase type II has its pH optimum in the acidic range (for review, see Shrode et al., 1997), acidification is expected to enhance DNA fragmentation. CD95-induced apoptosis is indeed accelerated by the inhibition of Na+/H+ exchange (Lang et al., 2000a).


Cytosolic Ca2+ activity plays a decisive role in the regulation of cell proliferation (Berridge et al., 1998, 2000, 2003; Parekh and Penner, 1997; Santella, 1998; Santella et al., 1998; Whitfield et al., 1995). Growth factors stimulate Ca2+ release through activated Ca2+ channel ICRAC (Qian and Weiss, 1997), which mediates Ca2+ entry, thus triggering and maintaining pulsatile Ca2+ release from intracellular stores yielding oscillations of cytosolic Ca2+ activity. Those oscillations govern a wide variety of cellular functions (Berridge et al., 1998, 2000, 2003; Parekh and Penner, 1997), including depolymerization of actin filaments (Dartsch et al., 1995; Lang et al., 1992, 2000c; Ritter et al., 1997). The depolymerization of actin filaments results in disinhibition of Na+/H+ exchanger and/or Na+,K+, 2Cl~ cotransporter, which both accumulate ions and osmotically obliged water and thus increase cell volume (Lang et al., 1998a). Activation of ICRAC, Ca2+ oscillations, and depolymerization of the actin filament network are prerequisites for the stimulation of cell proliferation (Dartsch et al., 1995; Lang et al., 1992, 2000c; Ritter et al., 1997).

CD95 receptor triggering is paralleled by inhibition of ICRAC in Jurkat T lymphocytes (Dangel et al., 2005; Lepple-Wienhues et al., 1999). Inhibition of ICRAC prevents activation and proliferation of lymphocytes but does not necessarily lead to apoptotic cell death. At a later stage, CD95 stimulation may, in some cells, lead to a sustained increase of cytosolic Ca2+ activity, which has been shown to trigger apoptosis in a variety of nucleated cells

(Berridge et al., 2000; Green and Reed, 1998; Liu et al., 2005; Parekh and Penner, 1997; Parekh and Putney, 2005; Spassova etal., 2004). Furthermore, Ca2+-permeable cation channels trigger apoptosis-like suicidal death of erythrocytes (eryptosis) (Brand et al., 2003; Lang et al., 2002b, 2003b). Accordingly, eryptosis is elicited by exposure to the Ca2+ ionophore iono-mycin (Berg etal., 2001; Bratosin etal., 2001; Daugas etal., 2001; Lang et al., 2002b, 2003b) and blunted in the nominal absence of Ca2+ (Lang et al., 2003b). The Ca2+-permeable erythrocyte cation channels are activated by osmotic shock (Huber et al., 2001), oxidative stress (Duranton et al., 2002), energy depletion (Lang et al., 2003b), and infection with the malaria pathogen Plasmodium falciparum (Duranton et al., 2003; Lang et al., 2003b, 2004a). Energy depletion is presumably effective through impairment of GSH replenishment, thus weakening the antioxidative defense ofthe erythrocytes (Bilmen et al., 2001; Mavelli et al., 1984). The erythrocyte cation channels are inhibited by Cl_ and are activated by replacement of Cl_ with gluconate (Duranton et al., 2002; Huber et al., 2001). Similar or identical cation channels are activated by incubation of human erythrocytes in low ionic strength (Bernhardt et al., 1991; Jones and Knauf, 1985; LaCelle and Rothsteto, 1966) or by depolarization (Bennekou, 1993; Christophersen andBennekou, 1991; Kaestner etal., 1999).

Increased cytosolic Ca2+ concentrations somehow trigger the scrambling of the erythrocyte cell membrane (Zhou et al., 2002) with breakdown of phosphatidylserine asymmetry and phosphatidylserine exposure at the cell surface (Lang et al., 2003b). The cation channels are activated by prostaglandin E2, which is released upon osmotic shock (Lang et al., 2005a). The cation channel blockers amiloride (Lang et al., 2003b) and ethylisopropylamiloride (Lang et al., 2003c) blunt the phosphatidylserine exposure following osmotic shock.

Cell volume-sensitive cation channels are similarly expressed in nucleated cells, such as airway epithelia cells (Chan et al., 1992), vascular smooth muscle, colon carcinoma and neuroblastoma cells (Koch and Korbmacher, 1999), cortical collecting duct cells (Volk et al., 1995), hepatocytes (Wehner et al., 1995, 2000), mast cells (Cabado et al., 1994), and macrophages (Gamper etal., 2000). Cation channels activated by Cl_ removal are expressed in salivary and lung epithelial cells (Dinudom et al., 1995; Marunaka et al., 1994; Tohda et al., 1994). Whether or not those channels participate in the stimulation of apoptosis remains elusive.


Several K+ channels participate in the regulation of cell proliferation (Patel and Lazdunski, 2004; Wang, 2004). Growth factors activate K+ channels (Enomoto et al., 1986; Faehling et al., 2001; Lang et al., 1991;

Liu et al., 2001; O'Lague et al., 1985; Sanders et al., 1996; Wiecha et al., 1998), and enhanced K+ channel activity is observed in tumor cells (DeCoursey et al., 1984; Mauro et al., 1997; Nilius and Wohlrab, 1992; Pappas and Ritchie, 1998; Pappone and Ortiz-Miranda, 1993; Patel and Lazdunski, 2004; Skryma et al., 1997; Strobl et al., 1995; Wang, 2004; Zhou et al., 2003). In ras oncogene-expressing cells, repetitive activation of Ca2+-sensitive K+ channels by oscillating cytosolic Ca2+ activity leads to oscillations of cell membrane potential (Lang et al., 1991). Several K+ channel inhibitors disrupt cell proliferation (for review, see Wang 2004). K+ channel activation is apparently important for the early G1 phase of the cell cycle (Wang et al., 1998; Wonderlin and Strobl, 1996). The maintenance of cell membrane potential by K+ channels provides the electrical driving force for Ca2+ entry through ICRAC (Parekh and Penner, 1997), which is required for stimulation of cell proliferation.

The role of K+ channels in apoptosis is less obvious. In some cells, inhibition of K+ channels participates in the stimulation of apoptosis (Bankers-Fulbright et al., 1998; Chin et al., 1997; Han et al., 2004; Miki et al., 1997; Pal et al., 2004; Patel and Lazdunski, 2004), and activation ofK+ channels inhibits apoptosis (Jakob and Krieglstein, 1997; Lauritzen et al., 1997). Along those lines, extensive neuronal cell death is observed in mice carrying a mutation of G-coupled inward rectifier K+ channels (Weaver mice) (Harrison and Roffler-Tarlov, 1998; Migheli et al., 1995, 1997; Murtomaki et al., 1995; Oo et al., 1996).

However, in other cells apoptosis is stimulated by activation of K+ channels (Wei et al., 2004; Yu et al., 1997) and inhibited by increase of extracellular K+ concentration (Colom et al., 1998; Lang et al., 2003e; Prehn etal., 1997) orK+ channel blockade (Gantner etal., 1995; Lang etal., 2003e). Cellular loss of K+ apparently favors apoptosis in a wide variety of cells (Beauvais et al., 1995; Benson et al., 1996; Bortner and Cidlowski, 1999, 2004; Bortner et al., 1997; Gomez-Angelats et al., 2000; Hughes and Cidlowski, 1999; Hughes et al., 1997; Maeno et al., 2000; Montague et al., 1999; Perez et al., 2000; Yurinskaya et al., 2005a,b). Moreover, activation of K+ channels hyperpolarizes the cell membrane, thus increasing the electrical driving force for Cl~ exit. Depending on Cl~ channel activity, K+ channel activity leads to cellular loss of KCl with osmotically obliged water and hence to apoptotic cell shrinkage (Lang et al., 1998a).

In Jurkat lymphocytes, CD95 activation is followed within a few minutes by inhibition of Kv1.3 K+ channels (Szabo et al., 1996, 1997, 2004), the cell volume regulatory K+ channel of those cells (Deutsch and Chen, 1993). CD95 triggering leads to tyrosine phosphorylation of the Kv1.3 channel protein (Gulbins et al., 1997; Szabo et al., 1996). Accordingly, CD95-induced inhibition of Kv1.3 requires Lck56 (Gulbins et al., 1997; Szabo et al., 1996). The inhibitory effect of CD95 triggering is mimicked by the sphingomyelinase product ceramide, which similarly induces apoptosis

(Gulbins et al., 1997). In other cells, Kv1.3 is similarly regulated by tyrosine phosphorylation (Holmes et al., 1996). Moreover, Kv1.3 is upregulated by the serum and glucocorticoid-inducible kinase (Lang et al., 2003a), which similarly inhibits apoptosis (Aoyama et al., 2005). Following CD95 activation, the early inhibition of Kv1.3 is followed by late activation of Kv1.3 (Storey et al., 2003). Early inhibition ofKv1.3 channels in CD95-activated cells may serve to prevent premature cell shrinkage which otherwise may interfere with signaling of apoptosis (Lang et al., 1998a). The late activation of Kv1.3 channels during the execution phase of apoptosis supports apoptotic cell shrinkage (Storey et al., 2003).

In suicidal erythrocytes, Ca2+-sensitive K+ channels (GARDOS channels) are activated by increased cytosolic Ca2+ activity (Brugnara et al., 1993; Del Carlo et al., 2002; Dunn, 1998; Gardos, 1958; Grygorczyk and Schwarz, 1983; Leinders et al., 1992; Pellegrino and Pellegrini, 1998; Shindo et al., 2000). Activation of GARDOS channels hyperpolarizes the cell membrane and, because of high erythrocyte Cl_ permeability, leads to parallel exit ofK+ and Cl~. The cellular loss ofKCl and osmotically obliged water leads to cell shrinkage, which in turn favors phosphatidylserine exposure (Lang et al., 2003d). An increase of extracellular K+ or a pharmacological inhibition of GARDOS channels blunts cell shrinkage and has a moderate inhibitory effect on phosphatidylserine scrambling following exposure to the Ca2+ ionophore ionomycin (Lang et al., 2003d). Erythrocyte shrinkage stimulates formation of the platelet-activating factor, which in turn activates a sphingomyelinase (Lang et al., 2005b). The ceramide generated by the sphingomyelinase then sensitizes the cell for the scrambling effect of Ca2+ (Lang et al., 2004b, 2005b).

The same or similar channels could participate in the stimulation of both cell proliferation and apoptosis. The effect of channel activation depends on further properties of the cell. For instance, it may depend on the activity of other channels. Activation of K+ channels without parallel activity of electrogenic anion transporters or Cl~ channels, for instance, hyperpolarizes the cell membrane but does not shrink the cell (Lang et al., 1998a). Moreover, activation ofK+ channels may increase Ca2+ entry and cytosolic Ca2+ activity only in the presence of active Ca2+ channels.

The effect may further depend on the temporal pattern of channel activation. The oscillating K+ channel activity typical of proliferating cells (Lang et al., 1991; Pandiella et al., 1989) has different effects as sustained K+ channel activation typical of apoptotic cells (Lang et al., 2003d). Oscillations of

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