Structural and stoichiometric determinants of Ca release-activated Ca (CRAC) channel Ca-dependent inactivation

Depletion of intracellular Ca^2 + stores in mammalian cells results in Ca^2 + entry across the plasma membrane mediated primarily by Ca^2 + release-activated Ca^2 + (CRAC) channels. Ca^2 + influx through these channels is required for the maintenance of homeostasis and Ca^2 + signaling in most cell types. One of the main features of native CRAC channels is fast Ca^2 +-dependent inactivation (FCDI), where Ca^2 + entering through the channel binds to a site near its intracellular mouth and causes a conformational change, closing the channel and limiting further Ca^2 + entry. Early studies suggested that FCDI of CRAC channels was mediated by calmodulin. However, since the discovery of STIM1 and Orai1 proteins as the basic molecular components of the CRAC channel, it has become apparent that FCDI is a more complex phenomenon. Data obtained using heterologous overexpression of STIM1 and Orai1 suggest that, in addition to calmodulin, several cytoplasmic domains of STIM1 and Orai1 and the selectivity filter within the channel pore are required for FCDI. The stoichiometry of STIM1 binding to Orai1 also has emerged as an important determinant of FCDI. Consequently, STIM1 protein expression levels have the potential to be an endogenous regulator of CRAC channel Ca^2 + influx. This review discusses the current understanding of the molecular mechanisms governing the FCDI of CRAC channels, including an evaluation of further experiments that may delineate whether STIM1 and/or Orai1 protein expression is endogenously regulated to modulate CRAC channel function, or may be dysregulated in some pathophysiological states.     

Sleep deprivation impairs calcium signaling in mouse splenocytes and leads to a decreased immune response

Background

Sleep is a physiological event that directly influences health by affecting the immune system, in which calcium (Ca^2 +) plays a critical signaling role. We performed live cell measurements of cytosolic Ca^2 + mobilization to understand the changes in Ca^2 + signaling that occur in splenic immune cells after various periods of sleep deprivation (SD).

Methods

Adult male mice were subjected to sleep deprivation by platform technique for different periods (from 12 to 72 h) and Ca^2 + intracellular fluctuations were evaluated in splenocytes by confocal microscopy. We also performed spleen cell evaluation by flow cytometry and analyzed intracellular Ca^2 + mobilization in endoplasmic reticulum and mitochondria. Additionally, Ca^2 + channel gene expression was evaluated

Results

Splenocytes showed a progressive loss of intracellular Ca^2 + maintenance from endoplasmic reticulum (ER) stores. Transient Ca^2 + buffering by the mitochondria was further compromised. These findings were confirmed by changes in mitochondrial integrity and in the performance of the store operated calcium entry (SOCE) and stromal interaction molecule 1 (STIM1) Ca^2 + channels.

Conclusions and general significance

These novel data suggest that SD impairs Ca^2 + signaling, most likely as a result of ER stress, leading to an insufficient Ca^2 + supply for signaling events. Our results support the previously described immunosuppressive effects of sleep loss and provide additional information on the cellular and molecular mechanisms involved in sleep function.     

Regulation of CRAC channels by protein interactions and post-translational modification

Store-operated Ca²⁺ entry (SOCE) is a widespread mechanism to elevate the intracellular Ca²⁺ concentrations and stimulate downstream signaling pathways affecting proliferation, secretion, differentiation and death in different cell types. In immune cells, immune receptor stimulation induces intracellular Ca²⁺ store depletion that subsequently activates Ca²⁺-release-activated-Ca²⁺ (CRAC) channels, a prototype of store-operated Ca²⁺ (SOC) channels. CRAC channel opening leads to activation of diverse downstream signaling pathways affecting proliferation, differentiation, cytokine production and cell death. Recent identification of STIM1 as the endoplasmic reticulum Ca²⁺ sensor and Orai1 as the pore subunit of CRAC channels has provided the much-needed molecular tools to dissect the mechanism of activation and regulation of CRAC channels. In this review, we discuss the recent advances in understanding the associating partners and posttranslational modifications of Orai1 and STIM1 proteins that regulate diverse aspects of CRAC channel function.     

KCa and Ca channels: The complex thought

Potassium channels belong to the largest and the most diverse super-families of ion channels. Among them, Ca^2 +-activated K⁺ channels (KCa) comprise many members. Based on their single channel conductance they are divided into three subfamilies: big conductance (BKCa), intermediate conductance (IKCa) and small conductance (SKCa; SK1, SK2 and SK3). Ca^2 + channels are divided into two main families, voltage gated/voltage dependent Ca^2 + channels and non-voltage gated/voltage independent Ca^2 + channels. Based on their electrophysiological and pharmacological properties and on the tissue where there are expressed, voltage gated Ca^2 + channels (Cav) are divided into 5 families: T-type, L-type, N-type, P/Q-type and R-type Ca^2 +. Non-voltage gated Ca^2 + channels comprise the TRP (TRPC, TRPV, TRPM, TRPA, TRPP, TRPML and TRPN) and Orai (Orai1 to Orai3) families and their partners STIM (STIM1 to STIM2). A depolarization is needed to activate voltage-gated Ca^2 + channels while non-voltage gated Ca^2 + channels are activated by Ca^2 + depletion of the endoplasmic reticulum stores (SOCs) or by receptors (ROCs). These two Ca^2 + channel families also control constitutive Ca^2 + entries. For reducing the energy consumption and for the fine regulation of Ca^2 +, KCa and Ca^2 + channels appear associated as complexes in excitable and non-excitable cells. Interestingly, there is now evidence that KCa–Ca^2 + channel complexes are also found in cancer cells and contribute to cancer-associated functions such as cell proliferation, cell migration and the capacity to develop metastases. This article is part of a Special Issue entitled: Calcium signaling in health and disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau.     

TRPC6 participates in the regulation of cytosolic basal calcium concentration in murine resting platelets

Cytosolic-free Ca^2 + plays a crucial role in blood platelet function and is essential for thrombosis and hemostasis. Therefore, cytosolic-free Ca^2 + concentration is tightly regulated in this cell. TRPC6 is expressed in platelets, and an important role for this Ca^2 + channel in Ca^2 + homeostasis has been reported in other cell types. The aim of this work is to study the function of TRPC6 in platelet Ca^2 + homeostasis. The absence of TRPC6 resulted in an 18.73% decreased basal [Ca^2 +]_c in resting platelets as compared to control cells. Further analysis confirmed a similar Ca^2 + accumulation in wild-type and TRPC6-deficient mice; however, passive Ca^2 + leak rates from agonist-sensitive intracellular stores were significantly decreased in TRPC6-deficient platelets. Biotinylation studies indicated the presence of an intracellular TRPC6 population, and subcellular fractionation indicated their presence on endoplasmic reticulum membranes. Moreover, the presence of intracellular calcium release in platelets stimulated with 1-oleoyl-2-acetyl-sn-glycerol further suggested a functional TRPC6 population located on the intracellular membranes surrounding calcium stores. However, coimmunoprecipitation assay confirmed the absence of STIM1–TRPC6 interactions in resting conditions. This findings together with the absence of extracellular Mn^2 + entry in resting wild-type platelets indicate that the plasma membrane TRPC6 fraction does not play a significant role in the maintenance of basal [Ca^2 +]_c in mouse platelets. Our results suggest an active participation of the intracellular TRPC6 fraction as a regulator of basal [Ca^2 +]_c, controlling the passive Ca^2 + leak rate from agonist-sensitive intracellular Ca^2 + stores in resting platelets.     

Cytoskeletal and scaffolding proteins as structural and functional determinants of TRP channels

Transient receptor potential (TRP) channels are six transmembrane-spanning proteins, with variable selectivity for cations, that play a relevant role in intracellular Ca^2 + homeostasis. There is a large body of evidence that shows association of TRP channels with the actin cytoskeleton or even the microtubules and demonstrating the functional importance of this interaction for TRP channel function. Conversely, cation currents through TRP channels have also been found to modulate cytoskeleton rearrangements. The interplay between TRP channels and the cytoskeleton has been demonstrated to be essential for full activation of a variety of cellular functions. Furthermore, TRP channels have been reported to take part of macromolecular complexes including different signal transduction proteins. Scaffolding proteins play a relevant role in the association of TRP proteins with other signaling molecules into specific microdomains. Especially relevant are the roles of the Homer family members for the regulation of TRPC channel gating in mammals and INAD in the modulation of Drosophila TRP channels. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters. Guest Editor: Jean Claude Hervé.     

Orai1-NFAT signalling pathway triggered by T cell receptor stimulation

T cell receptor (TCR) stimulation plays a crucial role in development, homeostasis, proliferation, cell death, cytokine production, and differentiation of T cells. Thus, in depth understanding of TCR signalling is crucial for development of therapy targeting inflammatory diseases, improvement of vaccination efficiency, and cancer therapy utilizing T cell-based strategies. TCR activation turns on various signalling pathways, one of the important one being the Ca²⁺-calcineurin-nuclear factor of activated T cells (NFAT) signalling pathway. Stimulation of TCRs triggers depletion of intracellular Ca²⁺ store and in turn, initiates store-operated Ca²⁺ entry (SOCE), one of the major mechanisms to raise the intracellular Ca²⁺ concentrations in T cells. Ca²⁺-release-activated-Ca²⁺ (CRAC) channels are a prototype of store-operated Ca²⁺ (SOC) channels in immune cells that are very well characterized. Recent identification of STIM1 as the endoplasmic reticulum (ER) Ca²⁺ sensor and Orai1 as the pore subunit has dramatically advanced the understanding of CRAC channels and provides a molecular tool to investigate the physiological outcomes of Ca²⁺ signalling during immune responses. In this review, we focus on our current understanding of CRAC channel activation, regulation, and downstream calcineurin-NFAT signaling pathway.     

Frequency decoding of calcium oscillations

Background

Calcium (Ca^2 +) oscillations are ubiquitous signals present in all cells that provide efficient means to transmit intracellular biological information. Either spontaneously or upon receptor ligand binding, the otherwise stable cytosolic Ca^2 + concentration starts to oscillate. The resulting specific oscillatory pattern is interpreted by intracellular downstream effectors that subsequently activate different cellular processes. This signal transduction can occur through frequency modulation (FM) or amplitude modulation (AM), much similar to a radio signal. The decoding of the oscillatory signal is typically performed by enzymes with multiple Ca^2 + binding residues that diversely can regulate its total phosphorylation, thereby activating cellular program. To date, NFAT, NF-κB, CaMKII, MAPK and calpain have been reported to have frequency decoding properties.

Scope of review

The basic principles and recent discoveries reporting frequency decoding of FM Ca^2 + oscillations are reviewed here.

Major conclusions

A limited number of cellular frequency decoding molecules of Ca^2 + oscillations have yet been reported. Interestingly, their responsiveness to Ca^2 + oscillatory frequencies shows little overlap, suggesting their specific roles in cells.

General significance

Frequency modulation of Ca^2 + oscillations provides an efficient means to differentiate biological responses in the cell, both in health and in disease. Thus, it is crucial to identify and characterize all cellular frequency decoding molecules to understand how cells control important cell programs.     

Aequorin-based genetic approaches to visualize Ca signaling in developing animal systems

Background

In recent years, as our understanding of the various roles played by Ca^2 + signaling in development and differentiation has expanded, the challenge of imaging Ca^2 + dynamics within living cells, tissues, and whole animal systems has been extended to include specific signaling activity in organelles and non-membrane bound sub-cellular domains.

Scope of review

In this review we outline how recent advances in genetics and molecular biology have contributed to improving and developing current bioluminescence-based Ca^2 + imaging techniques. Reporters can now be targeted to specific cell types, or indeed organelles or domains within a particular cell.

Major conclusions

These advances have contributed to our current understanding of the specificity and heterogeneity of developmental Ca^2 + signaling. The improvement in the spatial resolution that results from specifically targeting a Ca^2 + reporter has helped to reveal how a ubiquitous signaling messenger like Ca^2 + can regulate coincidental but different signaling events within an individual cell; a Ca^2 + signaling paradox that until now has been hard to explain.

General significance

Techniques used to target specific reporters via genetic means will have applications beyond those of the Ca^2 + signaling field, and these will, therefore, make a significant contribution in extending our understanding of the signaling networks that regulate animal development. This article is part of a Special Issue entitled Biochemical, biophysical and genetic approaches to intracellular calcium signalling.     

Strong alkalinization of Chara cell surface in the area of cell wall incision as an early event in mechanoperception

Mechanical wounding of cell walls occurring in plants under the impact of pathogens or herbivores can be mimicked by cell wall incision with a glass micropipette. Measurements of pH at the surface of Chara corallina internodes following microperforation of cell wall revealed a rapid (10–30 s) localized alkalinization of the apoplast after a lag period of 10–20 s. The pH increase induced by incision could be as large as 3 pH units and relaxed slowly, with a halftime up to 20 min. The axial pH profile around the incision zone was bell-shaped and localized to a small area, extending over a distance of about 100 μm. The pH response was suppressed by lowering cell turgor upon the replacement of artificial pond water (APW) with APW containing 50 mM sorbitol. Stretching of the plasma membrane during its impression into the cell wall defect is likely to activate the Ca^2 + channels, as evidenced from sensitivity of the incision-induced alkalinization to the external calcium concentration and to the addition of Ca^2 +-channel blockers, such as La^3 +, Gd^3 +, and Zn^2 +. The maximal pH values attained at the incision site (~ 10.0) were close to pH in light-dependent alkaline zones of Chara cells. The involvement of cytoskeleton in the origin of alkaline patch was documented by observations that the incision-induced pH transients were suppressed by the inhibitors of microtubules (oryzalin and taxol) and, to a lesser extent, by the actin inhibitor (cytochalasin B). The results indicate that the localized increase in apoplastic pH is an early event in mechanoperception and depends on light, cytoskeleton, and intracellular calcium.     

Inositol 1,4,5-trisphosphate receptor-isoform diversity in cell death and survival

Cell-death and -survival decisions are critically controlled by intracellular Ca^2 + homeostasis and dynamics at the level of the endoplasmic reticulum (ER). Inositol 1,4,5-trisphosphate (IP₃) receptors (IP₃Rs) play a pivotal role in these processes by mediating Ca^2 + flux from the ER into the cytosol and mitochondria. Hence, it is clear that many pro-survival and pro-death signaling pathways and proteins affect Ca^2 + signaling by directly targeting IP₃R channels, which can happen in an IP₃R-isoform-dependent manner. In this review, we will focus on how the different IP₃R isoforms (IP₃R1, IP₃R2 and IP₃R3) control cell death and survival. First, we will present an overview of the isoform-specific regulation of IP₃Rs by cellular factors like IP₃, Ca^2 +, Ca^2 +-binding proteins, adenosine triphosphate (ATP), thiol modification, phosphorylation and interacting proteins, and of IP₃R-isoform specific expression patterns. Second, we will discuss the role of the ER as a Ca^2 + store in cell death and survival and how IP₃Rs and pro-survival/pro-death proteins can modulate the basal ER Ca^2 + leak. Third, we will review the regulation of the Ca^2 +-flux properties of the IP₃R isoforms by the ER-resident and by the cytoplasmic proteins involved in cell death and survival as well as by redox regulation. Hence, we aim to highlight the specific roles of the various IP₃R isoforms in cell-death and -survival signaling. This article is part of a Special Issue entitled: Calcium signaling in health and disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau.     

Modeling local and global intracellular calcium responses mediated by diffusely distributed inositol 1,4,5-trisphosphate receptors

Considerable insight into intracellular Ca²⁺ responses has been obtained through the development of whole cell models that are based on molecular mechanisms, e.g., single channel kinetics of the inositol 1,4,5-trisphosphate (IP₃) receptor Ca²⁺ channel. However, a limitation of most whole cell models to date is the assumption that IP₃ receptor Ca²⁺ channels (IP₃Rs) are globally coupled by a “continuously stirred” bulk cytosolic [Ca²⁺], when in fact open IP₃Rs experience elevated “domain” Ca²⁺ concentrations. Here we present a 2N+2-compartment whole cell model of local and global Ca²⁺ responses mediated by N=100,000 diffusely distributed IP₃Rs, each represented by a four-state Markov chain. Two of these compartments correspond to bulk cytosolic and luminal Ca²⁺ concentrations, and the remaining 2N compartments represent time-dependent cytosolic and luminal Ca²⁺ domains associated with each IP₃R. Using this Monte Carlo model as a starting point, we present an alternative formulation that solves a system of advection-reaction equations for the probability density of cytosolic and luminal domain [Ca²⁺] jointly distributed with IP₃R state. When these equations are coupled to ordinary differential equations for the bulk cytosolic and luminal [Ca²⁺], a realistic but minimal model of whole cell Ca²⁺ dynamics is produced that accounts for the influence of local Ca²⁺ signaling on channel gating and global Ca²⁺ responses. The probability density approach is benchmarked and validated by comparison to Monte Carlo simulations, and the two methods are shown to agree when the number of Ca²⁺ channels is large (i.e., physiologically realistic). Using the probability density approach, we show that the time scale of Ca²⁺ domain formation and collapse (both cytosolic and luminal) may influence global Ca²⁺ oscillations, and we derive two reduced models of global Ca²⁺ dynamics that account for the influence of local Ca²⁺ signaling on global Ca²⁺ dynamics when there is a separation of time scales between the stochastic gating of IP₃Rs and the dynamics of domain Ca²⁺.     

ORAI-mediated calcium influx in T cell proliferation, apoptosis and tolerance

Ca²⁺ homeostasis controls a diversity of cellular processes including proliferation and apoptosis. A very important aspect of Ca²⁺ signaling is how different Ca²⁺ signals are translated into specific cell functions. In T cells, Ca²⁺ signals are induced following the recognition of antigen by the T cell receptor and depend mainly on Ca²⁺ influx through store-operated CRAC channels, which are mediated by ORAI proteins following their activation by STIM proteins. The complete absence of Ca²⁺ influx caused by mutations in Stim1 and Orai1 leads to severe immunodeficiency. Here we summarize how Ca²⁺ signals are tuned to regulate important T cell functions as proliferation, apoptosis and tolerance, the latter one being a special state of immune cells in which they can no longer respond properly to an otherwise activating stimulus. Perturbations of Ca²⁺ signaling may be linked to immune suppressive diseases and autoimmune diseases.     

The dual face of connexin-based astroglial Ca communication: A key player in brain physiology and a prime target in pathology

For decades, studies have been focusing on the neuronal abnormalities that accompany neurodegenerative disorders. Yet, glial cells are emerging as important players in numerous neurological diseases. Astrocytes, the main type of glia in the central nervous system , form extensive networks that physically and functionally connect neuronal synapses with cerebral blood vessels. Normal brain functioning strictly depends on highly specialized cellular cross-talk between these different partners to which Ca^2 +, as a signaling ion, largely contributes. Altered intracellular Ca^2 + levels are associated with neurodegenerative disorders and play a crucial role in the glial responses to injury. Intracellular Ca^2 + increases in single astrocytes can be propagated toward neighboring cells as intercellular Ca^2 + waves, thereby recruiting a larger group of cells. Intercellular Ca²⁺ wave propagation depends on two, parallel, connexin (Cx) channel-based mechanisms: i) the diffusion of inositol 1,4,5-trisphosphate through gap junction channels that directly connect the cytoplasm of neighboring cells, and ii) the release of paracrine messengers such as glutamate and ATP through hemichannels (‘half of a gap junction channel’). This review gives an overview of the current knowledge on Cx-mediated Ca^2 + communication among astrocytes as well as between astrocytes and other brain cell types in physiology and pathology, with a focus on the processes of neurodegeneration and reactive gliosis. Research on Cx-mediated astroglial Ca^2 + communication may ultimately shed light on the development of targeted therapies for neurodegenerative disorders in which astrocytes participate. This article is part of a Special Issue entitled: Calcium signaling in health and disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau.     

Coupled gating modifies the regulation of cardiac ryanodine receptors by luminal Ca

Cardiac ryanodine receptors (RYR2s) infrequently exhibit coupled gating that is manifested by synchronous opening and closing. To better characterize this phenomenon, we investigated the regulation of coupled RYR2 channels by luminal Ca^2 + focusing on effects that are likely mediated by the true luminal activation mechanism. By reconstituting an ion channel into a planar lipid bilayer and using substantially lower concentration of luminal Ba^2 + (8 mM, the virtual absence of Ca^2 +) and luminal Ca^2 + (8 mM), we show that response of coupled RYR2 channels to caffeine at a diastolic cytosolic Ca^2 + (90 nM) was affected by luminal Ca^2 + in a similar manner as for the single RYR2 channel except the gating behavior. Whereas, the single RYR2 channel responded to luminal Ca^2 + by prolongation in open and closed times, coupled RYR2 channels seemed to be resistant in this respect. In summary, we conclude that the class of Ca^2 + sites located on the luminal face of coupled RYR2 channels that is responsible for the channel potentiation by luminal Ca^2 + is functional and not structurally hindered by the channel coupling. Thus, the idea about non-functional luminal Ca^2 + sites as a source of the apparent gating resistance of coupled RYR2 channels to luminal Ca^2 + appears to be ruled out.     

MD simulations of the central pore of ryanodine receptors and sequence comparison with 2B protein from coxsackie virus

The regulation of intracellular Ca^2 + triggers a multitude of vital processes in biological cells. Ca^2 + permeable ryanodine receptors (RyRs) are the biggest known ion channels and play a key role in the regulation of intracellular calcium concentrations, particularly in muscle cells. In this study, we construct a computational model of the pore region of the skeletal RyR and perform molecular dynamics (MD) simulations. The dynamics and distribution of Ca^2 + around the luminal pore entry of the RyR suggest that Ca^2 + ions are channeled to the pore entry due to the arrangement of (acidic) amino acids at the extramembrane surface of the protein. This efficient mechanism of Ca^2 + supply is thought to be part of the mechanism of Ca^2 + conductance of RyRs. Viral myocarditis is predominantly caused by coxsackie viruses that induce the expression of the protein 2B which is known to affect intracellular Ca^2 + homeostasis in infected cells. From our sequence comparison, it is hypothesized, that modulation of RyR could be due to replacement of its transmembrane domains (TMDs) by those domains of the viral channel forming protein 2B of coxsackie virus. This article is part of a Special Issue entitled: Viral Membrane Proteins — Channels for Cellular Networking.     

Ion Channel Phenotype of Melanoma Cell Lines

Melanoma cells are transformed melanocytes of neural crest origin. K⁺ channel blockers have been reported to inhibit melanoma cell proliferation. We used whole-cell recording to characterize ion channels in four different human melanoma cell lines (C8161, C832C, C8146, and SK28). Protocols were used to identify voltage-gated (K_V), Ca²⁺-activated (K_Ca), and inwardly rectifying (K_IR) K⁺ channels; swelling-sensitive Cl⁻ channels (Cl_swell); voltage-gated Ca²⁺ channels (Ca_V) and Ca²⁺ channels activated by depletion of intracellular Ca²⁺ stores (CRAC); and voltage-gated Na⁺ channels (Na_V). The presence of Ca²⁺ channels activated by intracellular store depletion was further tested using thapsigargin to elicit a rise in [Ca²⁺] _i . The expression of K⁺ channels varied widely between different cell lines and was also influenced by culture conditions. K_IR channels were found in all cell lines, but with varying abundance. Whole-cell conductance levels for K_IR differed between C8161 (100 pS/pF) and SK28 (360 pS/pF). K_Ca channels in C8161 cells were blocked by 10 nm apamin, but were unaffected by charybdotoxin (CTX). K_Ca channels in C8146 and SK28 cells were sensitive to CTX (K _d = 4 nm), but were unaffected by apamin. K_V channels, found only in C8146 cells, activated at ∼−20 mV and showed use dependence. All melanoma lines tested expressed CRAC channels and a novel Cl_swell channel. Cl_swell current developed at 30 pS/sec when the cells were bathed in 80% Ringer solution, and was strongly outwardly rectifying (4:1 in symmetrical Cl⁻). We conclude that different melanoma cell lines express a diversity of ion channel types. Received: 2 April 1996/Revised: 22 August 1996     

Extracellular calcium depletion transiently elevates oxygen consumption in neurosecretory PC12 cells through activation of mitochondrial Na/Ca exchange

Fluctuating extracellular Ca²⁺ regulates many aspects of neuronal (patho)physiology including cell metabolism and respiration. Using fluorescence-based intracellular oxygen sensing technique, we demonstrate that depletion of extracellular Ca²⁺ from 1.8 to ≤ 0.6 mM by chelation with EGTA induces a marked spike in O₂ consumption in differentiated PC12 cells. This respiratory response is associated with the reduction in cytosolic and mitochondrial Ca²⁺, minor depolarization on the mitochondrial membrane, moderate depolarization of plasma membrane, and no changes in NAD(P)H and ATP. The response is linked to the influx of extracellular Na⁺ and the subsequent activation of mitochondrial Na⁺/Ca²⁺ and Na⁺/H⁺ exchange. The mitochondrial Na⁺/Ca²⁺ exchanger (_mNCX) activated by Na⁺ influx reduces Ca²⁺ and increases Na⁺ levels in the mitochondrial matrix. The excess of Na⁺ activates the mitochondrial Na⁺/H⁺ exchanger (NHE) increasing the outward pumping of protons, electron transport and O₂ consumption. Reduction in extracellular Na⁺ and inhibition of Na⁺ influx through the receptor operated calcium channels and plasmalemmal NHE reduce the respiratory response. Inhibition of the _mNCX, L-type voltage gated Ca²⁺ channels or the release of Ca²⁺ from the endoplasmic reticulum also reduces the respiratory spike, indicating that unimpaired intercompartmental Ca²⁺ exchange is critical for response development.