Evolution of mechanisms of Ca<Superscript>2+</Superscript>-signaling: Role of calcium ions in signal transduction in prokaryotes

The review considers current concepts of mechanisms of Ca²⁺-signaling in cells of prokaryotes. Problems of structural-functional organization of the Ca²⁺-messenger system, mechanisms of membrane transport, and Ca²⁺ homeostasis are discussed. A special attention is paid to analysis of role of calcium ions and Ca²⁺-receptor proteins in regulation of intracellular processes in bacteria.Translated from Zhurnal Evolyutsionnoi Biokhimii i Fiziologii, Vol. 41, No. 1, 2005, pp. 12–17.Original Russian Text Copyright © 2005 by Shemarova, Nesterov.     

Regulation of Ca<Superscript>2+</Superscript> influx in proliferating and differentiating murine myoblasts with participation of L-type Ca<Superscript>2+</Superscript> channels

The basic mechanisms of regulation of Ca²⁺ influx have been studied in murine myoblasts proliferating and differentiating in culture. The presence of L-type Ca²⁺ channels in proliferating myoblasts is shown for the first time. It is also shown that the influx of Ca²⁺ through these channels is regulated by the adrenergic system. The influx of Ca²⁺ after activation of the adrenergic system by addition of adrenaline has been estimated in comparison with the contribution of reticular stocks exhausted by ATP in calcium-free medium. The Ca²⁺ influx in proliferating myoblasts is regulated by β-2 adrenergic receptors whose action is mediated by adenylate cyclase through L-type calcium channels. In differentiating myoblasts, the adrenaline-induced Ca²⁺ influx is substantially lower than in proliferating cells, and maximal influx of Ca²⁺ may be reached only upon exhaustion of reticular stocks.     

Regulation of the RyR channel gating by Ca<Superscript>2+</Superscript> and Mg<Superscript>2+</Superscript>

Ryanodine receptors (RyRs) are the Ca²⁺ release channels in the sarcoplasmic reticulum in striated muscle which play an important role in excitation-contraction coupling and cardiac pacemaking. Single channel recordings have revealed a wealth of information about ligand regulation of RyRs from mammalian skeletal and cardiac muscle (RyR1 and RyR2, respectively). RyR subunit has a Ca²⁺ activation site located in the luminal and cytoplasmic domains of the RyR. These sites synergistically feed into a common gating mechanism for channel activation by luminal and cytoplasmic Ca²⁺. RyRs also possess two inhibitory sites in their cytoplasmic domains with Ca²⁺ affinities of the order of 1 μM and 1 mM. Magnesium competes with Ca²⁺ at these sites to inhibit RyRs and this plays an important role in modulating their Ca²⁺-dependent activity in muscle. This review focuses on how these sites lead to RyR modulation by Ca²⁺ and Mg²⁺ and how these mechanisms control Ca²⁺ release in excitation-contraction coupling and cardiac pacemaking.     

Ca<Superscript>2+</Superscript> channels and Ca<Superscript>2+</Superscript> signals involved in abiotic stress responses in plant cells: recent advances

Calcium (Ca²⁺) signals are essential transducers and regulators in many adaptive and developmental processes in plants. Protective responses of plants to a variety of environmental stress factors are mediated by transient changes of Ca²⁺ concentration in plant cells. Ca²⁺ ions are quickly transported by channel proteins present on the plasma membrane. During responses to external stimuli, various signal molecules are transported directly from extracellular to intracellular compartments via Ca²⁺ channel proteins. Three types of Ca²⁺ channels have been identified in plant cell membranes: voltage-dependent Ca²⁺-permeable channels (VDCCs), which is sorted to depolarization-activated Ca²⁺-permeable channels (DACCs) and hyperpolarization-activated Ca²⁺-permeable channels (HACCs), voltage-independent Ca²⁺-permeable channels (VICCs). They make functions in the abiotic stress such as TPCs, CNGCs, MS channels, annexins which distribute in the organelles, plasma membrane, mitochondria, cytosol, intracelluar membrane. This review summarizes recent advances in our knowledge of many types of Ca²⁺ channels and Ca²⁺ signals involved in abiotic stress resistance and responses in plant cells.     

Mitochondrial Ca<Superscript>2+</Superscript> uptake pathways

Calcium (Ca²⁺) plays diverse roles in all living organisms ranging from bacteria to humans. It is a structural element for bones, an essential mediator of excitation-contraction coupling, and a universal second messenger in the regulation of ion channel, enzyme and gene expression activities. In mitochondria, Ca²⁺ is crucial for the control of energy production and cellular responses to metabolic stress. Ca²⁺ uptake by the mitochondria occurs by the uniporter mechanism. The Mitochondrial Ca2+ Uniporter (MCU) protein has recently been identified as a core component responsible for mitochondrial Ca²⁺ uptake. MCU knockout (MCU KO) studies have identified a number of important roles played by this high capacity uptake pathway. Interestingly, this work has also shown that MCU-mediated Ca²⁺ uptake is not essential for vital cell functions such as muscle contraction, energy metabolism and neurotransmission. Although mitochondrial Ca²⁺ uptake was markedly reduced, MCU KO mitochondria still contained low but detectable levels of Ca²⁺. In view of the fundamental importance of Ca²⁺ for basic cell signalling, this finding suggests the existence of other currently unrecognized pathways for Ca²⁺ entry. We review the experimental evidence for the existence of alternative Ca²⁺ influx mechanisms and propose how these mechanisms may play an integral role in mitochondrial Ca²⁺ signalling.     

Life after the birth of the mitochondrial Na<Superscript>+</Superscript>/Ca<Superscript>2+</Superscript> exchanger,NCLX

Powered by the mitochondrial membrane potential, Ca²⁺ permeates the mitochondria via a Ca²⁺ channel termed Ca²⁺ uniporter and is pumped out by a Na⁺/Ca²⁺ exchanger, both of which are located on the inner mitochondrial membrane. Mitochondrial Ca²⁺ transients are critical for metabolic activity and regulating global Ca²⁺ responses. On the other hand, failure to control mitochondrial Ca²⁺ is a hallmark of ischemic and neurodegenerative diseases. Despite their importance, identifying the uniporter and exchanger remains elusive and their inhibitors are non-specific. This review will focus on the mitochondrial exchanger, initially describing how it was molecularly identified and linked to a novel member of the Na⁺/Ca²⁺ exchanger superfamily termed NCLX. Molecular control of NCLX expression provides a selective tool to determine its physiological role in a variety of cell types. In lymphocytes, NCLX is essential for refilling the endoplasmic reticulum Ca²⁺ stores required for antigendependent signaling. Communication of NCLX with the store-operated channel in astroglia controls Ca²⁺ influx and thereby neuro-transmitter release and cell proliferation. The refilling of the Ca²⁺ stores in the sarcoplasmic reticulum, which is controlled by NCLX, determines the frequency of action potential and Ca²⁺ transients in cardiomyocytes. NCLX is emerging as a hub for integrating glucose-dependent Na⁺ and Ca²⁺ signaling in pancreatic β cells, and the specific molecular control of NCLX expression resolved the controversy regarding its role in neurons and β cells. Future studies on an NCLX knockdown mouse model and identification of human NCLX mutations are expected to determine the role of mitochondrial Ca²⁺ efflux in organ activity and whether NCLX inactivation is linked to ischemic and/or neurodegenerative syndromes. Structure-function analysis and protein analysis will identify the NCLX mode of regulation and its partners in the inner membrane of the mitochondria.     

The mechanism of neuroprotection by positive modulation of Ca<Superscript>2+</Superscript>-activated K<Superscript>+</Superscript> channels of cerebellar neurons in primary culture

Here we show that positive modulators (CyPPA and NS309) of Ca²⁺-activated K⁺ channels of small (SK) and intermediate (IK) conductances in cerebellar neurons decrease glutamate-evoked Ca²⁺ entry into neurons independently on the presence of Mg²⁺ in extracellular media. An analysis of neuronal viability after long-term (240 min) glutamate treatments demonstrated neuroprotective action of CyPPA and NS309. Extracellular Mg²⁺ did not protect neurons from apoptosis during prolonged treatment with glutamate. Activation of SK and IK channels results in local membrane hyperpolarization, which enhances Mg²⁺ block of NMDA receptors and reduces activation of voltage-dependent Ca²⁺ channels, which can explain neuroprotection caused by CyPPA or NS309. The obtained results reveal an important role Ca²⁺-activated K⁺ channels of small and intermediate conductance in the regulation of Ca²⁺ entry into cerebellar neurons via NMDA receptors and voltage-gated Ca²⁺ channels.     

Trifluoperazine Attenuates Store-Dependent Ca<Superscript>2+</Superscript> Entry in Macrophages

Using Fura-2AM microfluorimetry, we have shown for the first time that preincubation of macrophages with the calsequestrin inhibitor neuroleptic trifluoperazine leads to a significant inhibition of the store-dependent Ca²⁺ entry induced by endoplasmic Ca²⁺-ATPase inhibitors thapsigargin or cyclopiazonic acid in rat peritoneal macrophages. The results suggest calsequestrin involvement in the regulation of the store-dependent Ca²⁺ entry in macrophages.     

Evolution of mechanisms of Ca<Superscript>2+</Superscript>-signaling. Significance of Ca<Superscript>2+</Superscript>-messenger systems during transition of organisms to multicellularity

The review considers Ca²⁺-messenger systems in primitive multicellulars (sponges and hydrozoa organisms). Analysis is performed of Ca²⁺ participation in regulation of early development of the organisms, their mobility, metamorphosis, chemoreception, and some other functions.     

Ca<Superscript>2+</Superscript>-Dependent Phosphorylation of RyR2 Can Uncouple Channel Gating from Direct Cytosolic Ca<Superscript>2+</Superscript> Regulation

Phosphorylation of the cardiac ryanodine receptor (RyR2) is thought to be important not only for normal cardiac excitation-contraction coupling but also in exacerbating abnormalities in Ca²⁺ homeostasis in heart failure. Linking phosphorylation to specific changes in the single-channel function of RyR2 has proved very difficult, yielding much controversy within the field. We therefore investigated the mechanistic changes that take place at the single-channel level after phosphorylating RyR2 and, in particular, the idea that PKA-dependent phosphorylation increases RyR2 sensitivity to cytosolic Ca²⁺. We show that hyperphosphorylation by exogenous PKA increases open probability (P _o) but, crucially, RyR2 becomes uncoupled from the influence of cytosolic Ca²⁺; lowering [Ca²⁺] to subactivating levels no longer closes the channels. Phosphatase (PP1) treatment reverses these gating changes, returning the channels to a Ca²⁺-sensitive mode of gating. We additionally found that cytosolic incubation with Mg²⁺/ATP in the absence of exogenously added kinase could phosphorylate RyR2 in approximately 50% of channels, thereby indicating that an endogenous kinase incorporates into the bilayer together with RyR2. Channels activated by the endogenous kinase exhibited identical changes in gating behavior to those activated by exogenous PKA, including uncoupling from the influence of cytosolic Ca²⁺. We show that the endogenous kinase is both Ca²⁺-dependent and sensitive to inhibitors of PKC. Moreover, the Ca²⁺-dependent, endogenous kinase–induced changes in RyR2 gating do not appear to be related to phosphorylation of serine-2809. Further work is required to investigate the identity and physiological role of this Ca²⁺-dependent endogenous kinase that can uncouple RyR2 gating from direct cytosolic Ca²⁺ regulation.     

Modulation of the reaction cycle of the Na<Superscript>+</Superscript>:Ca<Superscript>2+</Superscript>, K<Superscript>+</Superscript> exchanger

Ca²⁺ concentration in retinal photoreceptor rod outer segment (OS) strongly affects the generator potential kinetics and the receptor light adaptation. The response to intense light stimuli delivered in the dark produce potential changes exceeding 40 mV: since the Ca²⁺ extrusion in the OS is entirely controlled by the Na⁺:Ca²⁺, K⁺ exchanger, it is important to assess how the exchanger ion transport rate is affected by the voltage and, in general, by intracellular factors. It is indeed known that the cardiac Na⁺:Ca²⁺ exchanger is regulated by Mg-ATP via a still unknown metabolic pathway. In the present work, the Na⁺:Ca²⁺, K⁺ exchanger regulation was investigated in isolated OS, recorded in whole-cell configuration, using ionic conditions that activated maximally the exchanger in both forward and reverse mode. In all species examined (amphibia: Rana esculenta and Ambystoma mexicanum; reptilia: Gecko gecko), the forward (reverse) exchange current increased about linearly for negative (positive) voltages and exhibited outward (inward) rectification for positive (negative) voltages. Since hyperpolarisation increases Ca²⁺ extrusion rate, the recovery of the dark level of Ca²⁺ (and, in turn, of the generator potential) after intense light stimuli results accelerated. Mg-ATP increased the size of forward and reverse exchange current by a factor of ∼2.3 and ∼2.6, respectively, without modifying their voltage dependence. This indicates that Mg-ATP regulates the number of active exchanger sites and/or the exchanger turnover number, although via an unknown mechanism. Proceedings of the XVIII Congress of the Italian Society of Pure and Applied Biophysics (SIBPA), Palermo, Sicily, September 2006.     

Pulses of Cell Ca<Superscript>2+</Superscript> and the Dynamics of Tight Junction Opening and Closing

A mathematical modeling of tight junction (TJ) dynamics was elaborated in a previous study (Kassab, F., Marques, R.P., Lacaz-Vieira, F. 2002. Modeling tight junction dynamics and oscillations. J. Gen. Physiol. 120:237–247) to better understand the dynamics of TJ opening and closing, as well as oscillations of TJ permeability that are observed in response to changes of extracellular Ca²⁺ levels. In this model, TJs were assumed to be specifically controlled by the Ca²⁺ concentration levels at the extracellular Ca²⁺ binding sites of zonula adhaerens. Despite the fact that the model predicts all aspects of TJ dynamics, we cannot rule out the likelihood that changes of intracellular Ca²⁺ concentration (Ca²⁺ _cell), which might result from changes \ of extracellular Ca²⁺ concentration (Ca²⁺ _extl), contribute to the observed results. In order to address this aspect of TJ regulation, fast Ca²⁺-switch experiments were performed in which changes of Ca²⁺ _cell were induced using the Ca²⁺ ionophore A23187 or thapsigargin, a specific inhibitor of the sarco-endoplasmic reticulum Ca²⁺-ATPase. The results indicate that the ionophore or thapsigargin per se do not affect basal tissue electrical conductance (G), showing that the sealing of TJs is not affected by a rise in Ca²⁺ _cell. When TJs were kept in a dynamic state, as partially open structures or in oscillation, conditions in which the junctions are very sensitive to disturbances that affect their regulation, a rise of Ca²⁺ _cell never led to a decline of G, indicating that a rise of Ca²⁺ _cell does not trigger per se TJ closure. On the contrary, always the first response to a rise of Ca²⁺ _cell is an increase of G that, in most cases, is a transient response. Despite these observations we cannot assure that a rise of Ca²⁺ _cell is without effect on the TJs, since an increase of Ca²⁺ _cell not only causes a transient increase of G but, in addition, during oscillations a rise of Ca²⁺ _cell induced by the Ca²⁺ ionophore transiently halted the oscillatory pattern of TJs. The main conclusion of this study is that TJ closure that is observed when basolateral Ca²⁺ concentration (Ca²⁺ _bl) is increased after TJs were opened by Ca²⁺ _bl removal cannot be ascribed to a rise of Ca²⁺ _cell and might be a consequence of Ca²⁺ binding to extracellular Ca²⁺ sites.     

Plant Ca<Superscript>2+</Superscript>-ATPases

Plant calcium pumps, similarly to animal Ca²⁺ pumps, belong to the superfamily of P-type ATPase comprising also the plasma membrane H⁺-ATPase of fungi and plants, Na⁺/K⁺ ATPase of animals and H⁺/K⁺ ATPase of mammalian gastric mucosa. According to their sensitivity to calmodulin the plant Ca²⁺-ATPases have been divided into two subgroups: type IIA (homologues of animal SERCA) and type IIB (homologues of animal PMCA). Regardless of the similarities in a protein sequence, the plant Ca²⁺ pumps differ from those in animals in their cellular localization, structure and sensitivity to inhibitors. Genomic investigations revealed multiplicity of plant Ca²⁺-ATPases; they are present not only in the plasma membranes and ER but also in membranes of most of the cell compartments, such as vacuole, plastids, nucleus or Golgi apparatus. Studies using yeast mutants made possible the functional and biochemical characterization of individual plant Ca²⁺-ATMPases. Plant calcium pumps play an essential role in signal transduction pathways, they are responsible for the regulation of [Ca²⁺] in both cytoplasm and endomembrane compartments. These Ca²⁺-ATPases appear to be involved in plant adaptation to stress conditions, like salinity, chilling or anoxia.     

Down-Modulation of Ca<Superscript>2+</Superscript> Channels by Endogenously Released ATP and Opioids: from the Isolated Chromaffin Cell to the Slice of Adrenal Medullae

Modifications in Ca²⁺ influx may lead to profound changes in the cell activity associated with Ca²⁺-dependent processes, from muscle contraction and neurotransmitter release to calcium-mediated cell death. Therefore, calcium entry into the cell requires fine regulation. In this context, understanding of the modulation of voltage-dependent Ca²⁺ channels seems to be critical. The modulatory process results in the enhancement or decrement of calcium influx that may regulate the local and global cytosolic Ca²⁺ concentrations. Here, we summarize the well-established data on this matter described in isolated chromaffin cells by our laboratory and others, and the new results we have obtained in a more physiological preparation: freshly isolated slices of mouse adrenal medullae.     

Nicotine-induced Ca<Superscript>2+</Superscript>-myristoyl Switch of Neuronal Ca<Superscript>2+</Superscript> Sensor VILIP-1 in Hippocampal Neurons: A Possible Crosstalk Mechanism for Nicotinic Receptors

Visinin-like protein (VILIP-1) belongs to the neuronal Ca²⁺ sensor family of EF-hand Ca²⁺-binding proteins that regulate a variety of Ca²⁺-dependent signal transduction processes in neurons. It is an interaction partner of α4β2 nicotinic acetylcholine receptor (nAChR) and increases surface expression level and agonist sensitivity of the receptor in oocytes. Nicotine stimulation of nicotinic receptors has been reported to lead to an increase in intracellular Ca²⁺ concentration by Ca²⁺-permeable nAChRs, which in turn might lead to activation of VILIP-1, by a mechanism described as the Ca²⁺-myristoyl switch. It has been postulated that this will lead to co-localization of the proteins at cell membranes, where VILIP-1 can influence functional activity of α4-containing nAChRs. In order to test this hypothesis we have investigated whether a nicotine-induced and reversible Ca²⁺-myristoyl switch of VILIP-1 exists in primary hippocampal neurons and whether pharmacological agents, such as antagonist specific for distinct nAChRs, can interfere with the Ca²⁺-dependent membrane localization of VILIP-1. Here we report, that only α7- but not α4-containing nAChRs are able to elicit a Ca²⁺-dependent and reversible membrane-translocation of VILIP-1 in interneurons as revealed by employing the specific receptor antagonists dihydro-beta-erythroidine and methylallylaconitine. The nAChRs are associated with processes of synaptic plasticity in hippocampal neurons and they have been implicated in the pathology of CNS disorders, including Alzheimer’s disease and schizophrenia. VILIP-1 might provide a novel functional crosstalk between α4- and α7-containing nAChRs.     

Sigma-1 Receptor Antagonist Haloperidol Attenuates Store-Dependent Ca<Superscript>2+</Superscript> Entry in Macrophages

Using Fura-2AM microfluorimetry, we have shown for the first time that preincubation of macrophages with sigma-1 receptor antagonist haloperidol leads to a significant inhibition of the store-dependent Ca²⁺ entry induced by endoplasmic Ca²⁺-ATPase inhibitors thapsigargin or cyclopiazonic acid in rat peritoneal macrophages. The results suggest the involvement of the sigma-1 receptor in the regulation of storedependent Ca²⁺ entry in macrophages.     

Anoxia-induced elevation of cytosolic Ca<Superscript>2+</Superscript> concentration depends on different Ca<Superscript>2+</Superscript> sources in rice and wheat protoplasts

The anoxia-dependent elevation of cytosolic Ca²⁺ concentration, [Ca²⁺]_cyt, was investigated in plants differing in tolerance to hypoxia. The [Ca²⁺]_cyt was measured by fluorescence microscopy in single protoplasts loaded with the calcium-fluoroprobe Fura 2-AM. Imposition of anoxia led to a fast (within 3 min) significant elevation of [Ca²⁺]_cyt in rice leaf protoplasts. A tenfold drop in the external Ca²⁺ concentration (to 0.1 mM) resulted in considerable decrease of the [Ca²⁺]_cyt shift. Rice root protoplasts reacted upon anoxia with higher amplitude. Addition of plasma membrane (verapamil, La³⁺ and EGTA) and intracellular membrane Ca²⁺-channel antagonists (Li⁺, ruthenium red and cyclosporine A) reduced the anoxic Ca²⁺-accumulation in rice. Wheat protoplasts responded to anoxia by smaller changes of [Ca²⁺]_cyt. In wheat leaf protoplasts, the amplitude of the Ca²⁺-shift little depended on the external level of Ca²⁺. Wheat root protoplasts were characterized by a small shift of [Ca²⁺]_cyt under anoxia. Plasmalemma Ca²⁺-channel blockers had little effect on the elevation of cytosolic Ca²⁺ in wheat protoplasts. Intact rice seedlings absorbed Ca²⁺ from the external medium under anoxic treatment. On the contrary, wheat seedlings were characterized by leakage of Ca²⁺. Verapamil abolished the Ca²⁺ influx in rice roots and Ca²⁺ efflux from wheat roots. Anoxia-induced [Ca²⁺]_cyt elevation was high particularly in rice, a hypoxia-tolerant species. In conclusion, both external and internal Ca²⁺ stores are important for anoxic [Ca²⁺]_cyt elevation in rice, whereas the hypoxia-intolerant wheat does not require external sources for [Ca²⁺]_cyt rise. Leaf and root protoplasts similarly responded to anoxia, independent of their organ origin.     

Regulation of Plant Plasma Membrane H<Superscript>+</Superscript>- and Ca<Superscript>2+</Superscript>-ATPases by Terminal Domains

In the last few years, major progress has been made to elucidate the structure, function, and regulation of P-type plasma membrane H⁺-and Ca²⁺-ATPases. Even though a number of regulatory proteins have been identified, many pieces are still lacking in order to understand the complete regulatory mechanisms of these pumps. In plant plasma membrane H⁺- and Ca²⁺-ATPases, autoinhibitory domains are situated in the C- and N-terminal domains, respectively. A model for a common mechanism of autoinhibition is discussed.     

Ryanodine-induced vasoconstriction of the gerbil spiral modiolar artery depends on the Ca<Superscript>2+</Superscript> sensitivity but not on Ca<Superscript>2+</Superscript> sparks or BK channels

Background

In many vascular smooth muscle cells (SMCs), ryanodine receptor-mediated Ca²⁺ sparks activate large-conductance Ca²⁺-activated K⁺ (BK) channels leading to lowered SMC [Ca²⁺]_i and vasodilation. Here we investigated whether Ca²⁺ sparks regulate SMC global [Ca²⁺]_i and diameter in the spiral modiolar artery (SMA) by activating BK channels.

Methods

SMAs were isolated from adult female gerbils, loaded with the Ca²⁺-sensitive flourescent dye fluo-4 and pressurized using a concentric double-pipette system. Ca²⁺ signals and vascular diameter changes were recorded using a laser-scanning confocal imaging system. Effects of various pharmacological agents on Ca²⁺ signals and vascular diameter were analyzed.

Results

Ca²⁺ sparks and waves were observed in pressurized SMAs. Inhibition of Ca²⁺ sparks with ryanodine increased global Ca²⁺ and constricted SMA at 40 cmH₂O but inhibition of Ca²⁺ sparks with tetracaine or inhibition of BK channels with iberiotoxin at 40 cmH₂O did not produce a similar effect. The ryanodine-induced vasoconstriction observed at 40 cmH₂O was abolished at 60 cmH₂O, consistent with a greater Ca²⁺-sensitivity of constriction at 40 cmH₂O than at 60 cmH₂O. When the Ca²⁺-sensitivity of the SMA was increased by prior application of 1 nM endothelin-1, ryanodine induced a robust vasoconstriction at 60 cmH₂O.

Conclusions

The results suggest that Ca²⁺ sparks, while present, do not regulate vascular diameter in the SMA by activating BK channels and that the regulation of vascular diameter in the SMA is determined by the Ca²⁺-sensitivity of constriction.

     

The role of mitochondrial palmitate/Ca<Superscript>2+</Superscript>-activated pore in palmitate-induced apoptosis

The evidence of possible involvement of the mitochondrial cyclosporin A-insensitive palmitate/Ca²⁺-activated pore in palmitate-induced apoptosis is presented. It has been established that the opening of the palmitate/Ca²⁺-activated pore results in the high-amplitude swelling of mitochondria and the release of the apoptosis-inducing factor from organelles. These processes are accompanied by a short-term slight decrease of membrane potential, which recovers in 1 min. The possible role of the palmitate/Ca²⁺-activated pore in the induction of palmitate-induced apoptosis is discussed.