Potential Roles of Electrogenic Ion Transport and Plasma Membrane Depolarization in Apoptosis

Apoptosis is characterized by the programmed activation of specific biochemical pathways leading to the organized demise of cells. To date, aspects of the intracellular signaling machinery involved in this phenomenon have been extensively dissected and characterized. However, recent studies have elucidated a novel role for changes in the intracellular milieu of the cells as important modulators of the cell death program. Specially, intracellular ionic homeostasis has been reported to be a determinant in both the activation and progression of the apoptotic cascade. Several apoptotic insults trigger specific changes in ionic gradients across the plasma membrane leading to depolarization of the plasma membrane potential (PMP). These changes lead to ionic imbalance early during apoptosis. Several studies have also suggested the activation and/or modulation of specific ionic transport mechanisms including ion channels, transporters and ATPases, as mediators of altered intracellular ionic homeostasis leading to PMP depolarization during apoptosis. However, the role of PMP depolarization and of the changes in ionic homeostasis during the progression of apoptosis are still unclear. This review summarizes the current knowledge regarding the causes and consequences of PMP depolarization during apoptosis. We also review the potential electrogenic ion transport mechanisms associated with this event, including the net influx/efflux of cations and anions. An understanding of these mechamisms could lead to the generation of new therapeutic approaches for a variety of diseases involving apoptosis.     

Fish red blood cells: characteristics and physiological role of the membrane ion transporters

Several membrane ion transporters playing a role in gas transport and exchanges, cell volume regulation and intracellular acid-base regulation have been identified in fish red blood cells (RBCs). This short review focuses on Na+/K+ATPase and its role in establishing the ionic gradients across the membrane, on the Cl-/HCO3- exchanger and its key role in respiration and possibly in inducing a chloride conductance, on the Na+/H+ exchanger and the recent advances on its molecular mechanisms of activation and regulation, on the different types of K-Cl cotransports, the different hypotheses and suggested models and their role in cell volume regulation. There is no evidence in the literature for ionic channels in fish RBCs. We present original data obtained with the patch-clamp technique that shows for the first time the existence of a DIDS-sensitive chloride anionic conductance measured in whole cell configuration and the presence of a stretch-activated nonselective cationic channel recorded in cell-attached and excised inside-out configuration. The part played by these ionic conductances is discussed in relation with their possible involvement in volume regulation.     

Localization of Protease-Activated Receptors -1 and -2 in Human Mast Cells: Indications for an Amplified Mast Cell Degranulation Cascade

Protease-activated receptors (PARs) belong to a family of G-coupled seven transmembrane receptors that are activated by a proteolytic cleavage of their N-termini. Recent studies suggest the involvement of protease-activated receptors-1 and -2 (PAR-1, PAR-2) activators in mast cell de-granulation in various physiological and pathophysiological processes in inflammatory responses. Although PAR-1 and PAR-2 activating proteases, thrombin and tryptase, have been associated with mast cell activation, PAR-1 and PAR-2 have not been localized within these cells. We describe here the localization of PAR-1 and PAR-2 in mast cells from various normal human tissues using im-munohistochemical and double immunofluorescence techniques. The presence of these receptors on the membrane may explain the actions of accessible extracellular thrombin and tryptase for mast cell activation. In addition to the membrane labeling, these receptors are also localized on the membrane of the intracellular tryptase-positive granules, which may function to sustain further mast cell degranulation upon exocytosis. The localization of these two receptors in mast cells suggests a novel mechanism for controlling mast cell activation through regulation of PARI and PAR-2.     

Dual modulation of a potassium channel by the m1 muscarinic and beta2-adrenergic receptors

Neurotransmitter receptors alter membrane excitability and synaptic efficacy by generating intracellular signals that ultimately change the properties of ion channels. Given their critical role in controlling cell membrane potential, potassium channels are frequently the targets of modulatory signals from many different G protein-coupled receptors. However, due to the heterogeneity of potassium channel expression in vivo, it has been difficult to determine the molecular mechanisms governing the regulation of molecularly defined potassium channels. Through expression studies in Xenopus oocytes and mammalian cells, we found that the m1 muscarinic acetylcholine receptor (mAChR) potently suppresses a cloned delayed rectifier potassium channel, termed RAK, through a pathway involving phospholipase C activation and direct tyrosine phosphorylation of the RAK protein. In contrast, we found that RAK channel activity is strongly enhanced following agonist activation of beta2-adrenergic receptors; this effect requires a single PKA consensus phosphorylation site located near the amino terminus of the channel protein. These results demonstrate that a specific type of potassium channel that is widely expressed in the mammalian brain and heart is subject to both positive and negative regulation by G protein-dependent pathways.     

Regulation of the mechanosensitive cation channels by ATP and cAMP in leech neurons

Single-channel recordings were used to study the modulation of stretch-activated channels (SACs) by intracellular adenosine nucleotides in identified leech neurons. These channels exhibited two activity modes, spike-like (SL) and multiconductance (MC), displaying different polymodal activation. In the absence of mechanical stimulation, internal perfusion of excised patches with ATP induced robust and reversible activation of the MC but not of the SL mode. The ATP effect on channel activity was dose-dependent within a range of 1 microM-1 mM and was induced at different values of intracellular pH and Ca2+. The non-hydrolyzable ATP analog AMP-PNP, ATP without Mg2+ or ADP also effectively enhanced MC activity. Adenosine mimicked the effect of its nucleotides. At negative membrane potentials, both ATP and adenosine activated the channel. Moreover, ATP but not adenosine induced a flickering block. Addition of cAMP during maximal ATP activation completely and reversibly inhibited the channel, with activation and deactivation times of minutes. However, cAMP alone only induced a weak and rapid channel activation, without inhibitory effects. The expression of these channels in the growth cones of leech neurons, their permeability to Ca2+ and their sensitivity to intracellular cAMP are consistent with a role in the Ca2+ oscillations associated with cell growth.     

Connexin and pannexin as modulators of myocardial injury

Multicellular organisms have developed a variety of mechanisms that allow communication between their cells. Whereas some of these systems, as neurotransmission or hormones, make possible communication between remote areas, direct cell-to-cell communication through specific membrane channels keep in contact neighboring cells. Direct communication between the cytoplasm of adjacent cells is achieved in vertebrates by membrane channels formed by connexins. However, in addition to allowing exchange of ions and small metabolites between the cytoplasms of adjacent cells, connexin channels also communicate the cytosol with the extracellular space, thus enabling a completely different communication system, involving activation of extracellular receptors. Recently, the demonstration of connexin at the inner mitochondrial membrane of cardiomyocytes, probably forming hemichannels, has enlarged the list of actions of connexins. Some of these mechanisms are also shared by a different family of proteins, termed pannexins. Importantly, these systems allow not only communication between healthy cells, but also play an important role during different types of injury. The aim of this review is to discuss the role played by both connexin hemichannels and pannexin channels in cell communication and injury. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.     

Immunologically activated chloride channels involved in degranulation of rat mucosal mast cells.

Crosslinking of type I Fc epsilon receptors (Fc epsilon RI) on the surface of basophils or mast cells initiates a cascade of processes leading to the secretion of inflammatory mediators. We report here a correlation between mediator secretion and the activation of Cl- channels in rat mucosal-type mast cells (line RBL-2H3). Stimulation of RBL cells by either IgE and antigen or by a monoclonal antibody specific for the Fc epsilon RI, resulted in the activation of Cl- ion channels as detected by the patch-clamp technique. Channel activation occurred slowly, within minutes after stimulation. The channel has a slope conductance of 32 pS at potentials between 0 and -100 mV, and an increasing open-state probability with increasing depolarization. Activation of apparently the same Cl- channels could be mimicked without stimulation by isolating inside-out membrane patches in tyrode solution. Parallel inhibition of both Cl- channel activity and mediator secretion, as monitored by serotonin release, was observed by two compounds, the Cl- channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and the anti-allergic drug cromolyn. NPPB inhibited both the antigen-induced Cl- current and the serotonin release, where half-maximal inhibition occurred at similar doses, at 52 microM and 77 microM, respectively. The drug cromolyn, recently found to inhibit immunologically induced mediator secretion from RBL cells upon intracellular application, also blocks Cl- channels (IC50 = 15 microM) when applied to the cytoplasmic side of an inside-out membrane patch. The observed Cl- channel activation upon immunological stimulation and the parallel inhibition of channel current and of serotonin release suggests a functional role for this Cl- channel in mediator secretion from the mast cells studied.     

Extracellular protease trypsin activates amiloride-insensitive sodium channels in human leukemia cells

Sodium influx is tightly regulated in the cells of blood origin. Amiloride-insensitive sodium channels were identified as one of the main sodium-transporting pathways in leukemia cells. To date, all known regulatory pathways of these channels are coupled with intracellular actin cytoskeleton dynamics. Here, to search for physiological mechanisms controlling epithelial Na⁺ channel (ENaC)-like channels, we utilized leukemia K562 cells as a unique model to examine single channel behavior in a whole-cell patch-clamp experiments. We have shown for the first time that extracellular serine protease trypsin directly activates sodium channels in plasma membrane of K562 cells. The whole-cell single current recordings clearly demonstrate no inhibition of trypsin-activated channels by amiloride or benzamil. Involvement of proteolytic cleavage in channel opening was confirmed in experiments with soybean trypsin inhibitor. More importantly, stabilization of F-actin with intracellular phalloidin did not prevent trypsin-induced channel activation indicating no implication of cytoskeleton rearrangements in stimulatory effect of extracellular protease. Our data reveals a novel mechanism modulating amiloride-insensitive ENaC-like channel activity and integral sodium permeability in leukemia cells.     

Ionic mechanisms of histamine-induced responses in guinea pig intracardiac neurons

Histamine, released from mast cells, can modulate the activity of intrinsic neurons in the guinea pig cardiac plexus. The present study examined the ionic mechanisms underlying the histamine-induced responses in these cells. Histamine evokes a small membrane depolarization and an increase in neuronal excitability. Using intracellular voltage recording from individual intracardiac neurons, we were able to demonstrate that removal of extracellular sodium reduced the membrane depolarization, whereas inhibition of K+ channels by 1 mM Ba2+, 2 mM Cs+, or 5 mM tetraethylammonium had no effect. The depolarization was also not inhibited by either 10 microM Gd3+ or a reduced Cl- solution. The histamine-induced increase in excitability was unaffected by K+ channel inhibitors; however, it was reduced by either blockage of voltage-gated Ca2+ channels with 200 microM Cd2+ or replacement of extracellular Ca2+ with Mg2+. Conversely, alterations in intracellular calcium with thapsigargin or caffeine did not inhibit the histamine-induced effects. However, in cells treated with both thapsigargin and caffeine to deplete internal calcium stores, the histamine-induced increase in excitability was decreased. Treatment with the phospholipase C inhibitor U73122 also prevented both the depolarization and the increase in excitability. From these data, we conclude that histamine, via activation of H1 receptors, activates phospholipase C, which results in 1) the opening of a nonspecific cation channel, such as a transient receptor potential channel 4 or 5; and 2) in combination with either the influx of Ca2+ through voltage-gated channels or the release of internal calcium stores leads to an increase in excitability.     

Direct ion channel gating: A new function for intracellular messengers

1. There is widespread belief that intracellular messengers [e.g., Ca2+, cyclic AMP, cyclic GMP, inositol-1,4,5-triphosphate (IP3)] assert their actions primarily through activation of protein kinases. 2. In studies of excitable cells protein kinase activation has been shown to alter membrane ionic conductance, presumably through phosphorylation of ion channels (see Levitan, 1985). However, recent reports from several laboratories indicate that intracellular messengers can also affect membrane ionic conductances directly without invoking protein kinase activation. 3. In this article we examine those examples of direct activation of ionic conductances by intracellular messengers which are supported by single-channel studies of isolated membrane patches. The list of cell types displaying this kind of response is growing and includes cells of neuronal as well as nonneuronal origin.     

Pore-forming colicins: synthesis, extracellular release, mode of action, immunity

Colicins are bacterial toxins encoded by plasmids which also confer immunity to producing cells. In a first stage, colicins are synthesized in the cytoplasm of colicinogenic cells. Subsequently they are released into the extracellular medium following the action of a small protein synthesized coordinately with the colicins. This protein is a lipoprotein and causes a non-specific increase in the envelope permeability, in particular, through the activation of an outer membrane phospholipase. After release into the medium, colicins kill sensitive cells in 3 defined steps: adsorption onto a specific receptor at the surface of the bacterium, translocation across the outer membrane and action. A specific domain of the colicin molecule is responsible for each of these steps. The most common colicins are those which kill by depolarizing the cytoplasmic membrane with the formation of voltage-dependent ionic channels. Immunity is conferred to producing cells by a membrane protein which interacts with the colicin and prevents formation or functioning of these ionic channels formed by its C-terminal domain.     

Regulation of the mechanosensitive cation channels by ATP and cAMP in leech neurons

Single-channel recordings were used to study the modulation of stretch-activated channels (SACs) by intracellular adenosine nucleotides in identified leech neurons. These channels exhibited two activity modes, spike-like (SL) and multiconductance (MC), displaying different polymodal activation. In the absence of mechanical stimulation, internal perfusion of excised patches with ATP induced robust and reversible activation of the MC but not of the SL mode. The ATP effect on channel activity was dose-dependent within a range of 1 μM-1 mM and was induced at different values of intracellular pH and Ca²⁺. The non-hydrolyzable ATP analog AMP-PNP, ATP without Mg²⁺ or ADP also effectively enhanced MC activity. Adenosine mimicked the effect of its nucleotides. At negative membrane potentials, both ATP and adenosine activated the channel. Moreover, ATP but not adenosine induced a flickering block. Addition of cAMP during maximal ATP activation completely and reversibly inhibited the channel, with activation and deactivation times of minutes. However, cAMP alone only induced a weak and rapid channel activation, without inhibitory effects. The expression of these channels in the growth cones of leech neurons, their permeability to Ca²⁺ and their sensitivity to intracellular cAMP are consistent with a role in the Ca²⁺ oscillations associated with cell growth.     

Electrical correlates of secretion in endocrine and exocrine cells

Many types of secretory cells including neurons and cells of endocrine and exocrine glands show changes in electrical potential and resistance when secretion is stimulated. These electrical correlates result from the movement of ions across the cell membrane through specific ion-selective channels. In neurons and certain endocrine cells (such as pancreatic beta cells and certain cells of the anterior pituitary), these channels are voltage dependent and open transiently upon depolarization leading to action potentials. Thus some endocrine cells are electrically excitable, a property previously held to occur only in nerve and muscle. In other nonexcitable endocrine and exocrine cells (such as the pancreas and parotid), ion channels are responsive to either occupancy of specific membrane receptors or changes in intracellular metabolites and second messengers. Ion fluxes through these latter channels also lead to changes in the electrical potential and resistance, but these changes are generally more sustained and action potentials are not seen. The entry of Ca2+ through both voltage-dependent and voltage-independent ion channels plays a major role in the activation of secretion via exocytosis.     

Stable activation of single Ca2+ release-activated Ca2+ channels in divalent cation-free solutions

The regulation of store-operated, calcium-selective channels in the plasma membrane of rat basophilic leukemia cells (RBL-2H3 m1), an immortalized mucosal mast cell line, was studied at the single-channel level with the patch clamp technique by removing divalent cations from both sides of the membrane. The activity of the single channels in excised patches could be modulated by Ca(2+), Mg(2+), and pH. The maximal activation of these channels by divalent cation-free conditions occurred independently of depletion of intracellular Ca(2+) stores, whether in excised patches or in whole cell mode. Yet, a number of points of evidence establish these single-channel openings as amplified store-operated channel events. Specifically, (i) the single channels are exquisitely sensitive to inhibition by intracellular Ca(2+), and (ii) both the store-operated current and the single-channel openings are completely blocked by the capacitative calcium entry blocker, 2-aminoethoxydiphenyl borane. In addition, in Jurkat T cells single-channel openings with lower open probability have been observed in the whole cell mode with intracellular Mg(2+) present (Kerschbaum, H. H., and Cahalan, M. D. (1999) Science 283, 836-839), and in RBL-2H3 m1 cells a current with similar properties is activated by store depletion.     

The use of the potential-sensitive fluorescent probe bisoxonol in mast cells.

The regulation of the plasma membrane potential of rat peritoneal mast cells at the resting state and during activation was investigated using bisoxonol as a potential-sensitive fluorescent dye. Fluorescence microphotography showed that this negatively charged probe was not only present in the plasma membrane, but was also distributed in the cytoplasm. The intracellular localization of bisoxonol was confirmed by conducting experiments which showed that bisoxonol fluorescence was not enhanced in ATP-permeabilized mast cells. Rotenone (10(-7) M) and oligomycin (10(-6) M) did not change the fluorescence of bisoxonol showing, therefore, mitochondrial depolarization was not recorded with bisoxonol and suggesting that bisoxonol may represent a useful probe to study plasma membrane potential changes in the absence of exocytosis. We showed that, in non-stimulated mast cells, the blockade of the sodium pump enhanced the fluorescence of bisoxonol as did gramicidin a non selective ionophore used to fully depolarize the cells. High concentration of potassium (30 mM) as well as different ionic channel blockers did not significantly change the fluorescence intensity of bisoxonol, suggesting that ionic channel permeabilities were not involved in maintaining the resting plasma membrane potential of mast cells. Mast cells stimulated by compound 48/80 completely lost the fluorescence, shown by fluorescence microphotography, suggesting that exocytotic phenomena might induce a dye redistribution which is not only due to changes in the plasma membrane potential. In mast cells pretreated with pertussis toxin, which blocks mast cell-exocytosis, compound 48/80 induced a delayed (2 min) decrease of bisoxonol fluorescence which was shown to be dependent on the activity of the sodium pump. Considering that bisoxonol is a useful potential-sensitive probe in exocytosis-deprived mast cells, our results suggest that the sodium pump is mainly involved in the changes of plasma membrane potential of mast cells.     

Microdomains of high calcium are not required for exocytosis in RBL-2H3 mucosal mast cells

We have previously shown that store-associated microdomains of high Ca(2+) are not essential for exocytosis in RBL-2H3 mucosal mast cells. We have now examined whether Ca(2+) microdomains near the plasma membrane are required, by comparing the secretory responses seen when Ca(2+) influx was elicited by two very different mechanisms. In the first, antigen was used to activate the Ca(2+) release-activated Ca(2+) (CRAC) current (I(CRAC)) through CRAC channels. In the second, a Ca(2+) ionophore was used to transport Ca(2+) randomly across the plasma membrane. Since store depletion by Ca(2+) ionophore will also activate I(CRAC), different means of inhibiting I(CRAC) before ionophore addition were used. Ca(2+) responses and secretion in individual cells were compared using simultaneous indo-1 microfluorometry and constant potential amperometry. Secretion still takes place when the increase in intracellular Ca(2+) occurs diffusely via the Ca(2+) ionophore, and at an average intracellular Ca(2)+ concentration that is no greater than that observed when Ca(2+) entry via CRAC channels triggers secretion. Our results suggest that microdomains of high Ca(2+) near the plasma membrane, or associated with mitochondria or Ca(2+) stores, are not required for secretion. Therefore, we conclude that modest global increases in intracellular Ca(2+) are sufficient for exocytosis in these nonexcitable cells.     

Activation and desensitization of the caffeine-sensitive cation channels and calcium stores have no persistent effect on the electrophysiological properties of leech P neurones

In leech P neurones caffeine activates unselective ion channels in the plasma membrane and induces intracellular Ca2+ release (Schoppe, J., Hochstrate, P., Schlue, W.-R., 1997. Caffeine mediates cation influx and intracellular Ca2+ release in leech P neurones. Cell Calcium 22, 385-397). These effects are prominent only upon the first caffeine exposure, while subsequent applications are largely ineffective; i.e. both plasma membrane channels and intracellular Ca2+ release mechanism desensitize irreversibly. In order to examine whether this desensitization is paralleled by irreversible changes in the electrophysiological parameters of the cells, we investigated the action of caffeine on changes in membrane potential and the cytosolic free Ca2+ concentration, which were induced by varying the ionic composition of the extracellular fluid or by application of 5-hydroxytryptamine. Neither the resting values nor any of the experimentally induced shifts in membrane potential or cytosolic Ca2+ concentration were affected by caffeine, which suggests strongly that activation and/or desensitization of the caffeine-sensitive ion channels and Ca2+ stores have no long-lasting effect on the relevant electrochemical gradients, membrane conductances, or transport mechanisms.     

Arachidonic acid, ARC channels, and Orai proteins

A critical role for arachidonic acid in the regulation of calcium entry during agonist activation of calcium signals has become increasingly apparent in numerous studies over the past 10 years or so. In particular, low concentrations of this fatty acid, generated as a result of physiologically relevant activation of appropriate receptors, induces the activation of a unique, highly calcium-selective conductance now known as the ARC channel. Activation of this channel is specifically dependent on arachidonic acid acting at the intracellular surface of the membrane, and is entirely independent of any depletion of internal calcium stores. Importantly, a specific role of this channel in modulating the frequency of oscillatory calcium signals in various cell types has been described. Recent studies, subsequent to the discovery of STIM1 and the Orai proteins and their role in the store-operated CRAC channels, have revealed that these same proteins are also integral components of the ARC channels and their activation. However, unlike the CRAC channels, activation of the ARC channels depends on the pool of STIM1 that is constitutively resident in the plasma membrane, and the pore of these channels is comprised of both Orai1 and Orai3 subunits. The clear implication is that CRAC channels and ARC channels are closely related, but have evolved to play unique roles in the modulation of calcium signals—largely as a result of their entirely distinct modes of activation. Given this, although the precise details of how arachidonic acid acts to activate the channels remain unclear, it seems likely that the specific molecular features of these channels that distinguish them from the CRAC channels – namely Orai3 and/or plasma membrane STIM1 – will be involved.