Phospholipase C-gamma1 is required for the activation of store-operated Ca2+ channels in liver cells

Repetitive hormone-induced changes in concentration of free cytoplasmic Ca2+ in hepatocytes require Ca2+ entry through receptor-activated Ca2+ channels and SOCs (store-operated Ca2+ channels). SOCs are activated by a decrease in Ca2+ concentration in the intracellular Ca2+ stores, but the molecular components and mechanisms are not well understood. Some studies with other cell types suggest that PLC-gamma (phospholipase C-gamma) is involved in the activation of receptor-activated Ca2+ channels and/or SOCs, independently of PLC-gamma-mediated generation of IP3 (inositol 1,4,5-trisphosphate). The nature of the Ca2+ channels regulated by PLC-gamma has not been defined clearly. The aim of the present study was to determine if PLC-gamma is required for the activation of SOCs in liver cells. Transfection of H4IIE cells derived from rat hepatocytes with siRNA (short interfering RNA) targeted to PLC-gamma1 caused a reduction (by approx. 70%) in the PLC-gamma1 protein expression, with maximal effect at 72-96 h. This was associated with a decrease (by approx. 60%) in the amplitude of the I(SOC) (store-operated Ca2+ current) developed in response to intracellular perfusion with either IP(3) or thapsigargin. Knockdown of STIM1 (stromal interaction molecule type 1) by siRNA also resulted in a significant reduction (approx. 80% at 72 h post-transfection) of the I(SOC) amplitude. Immunoprecipitation of PLC-gamma1 and STIM1, however, suggested that under the experimental conditions these proteins do not interact with each other. It is concluded that the PLC-gamma1 protein, independently of IP3 generation and STIM1, is required to couple endoplasmic reticulum Ca2+ release to the activation of SOCs in the plasma membrane of H4IIE liver cells.     

A store-operated nonselective cation channel in lymphocytes is activated directly by Ca(2+) influx factor and diacylglycerol

Agonist-receptor interactions at the plasma membrane often lead to activation of store-operated channels (SOCs) in the plasma membrane, allowing for sustained Ca(2+) influx. While Ca(2+) influx is important for many biological processes, little is known about the types of SOCs, the nature of the depletion signal, or how the SOCs are activated. We recently showed that in addition to the Ca(2+) release-activated Ca(2+) (CRAC) channel, both Jurkat T cells and human peripheral blood mononuclear cells express novel store-operated nonselective cation channels that we termed Ca(2+) release-activated nonselective cation (CRANC) channels. Here we demonstrate that activation of both CRAC and CRANC channels is accelerated by a soluble Ca(2+) influx factor (CIF). In addition, CRANC channels in inside-out plasma membrane patches are directly activated upon exposure of their cytoplasmic side to highly purified CIF preparations. Furthermore, CRANC channels are also directly activated by diacylglycerol. These results strongly suggest that the Ca(2+) store-depletion signal is a diffusible molecule and that at least some SOCs may have dual activation mechanisms.     

TRPC channels as STIM1-regulated store-operated channels

Receptor-activated Ca(2+) influx is mediated largely by store-operated channels (SOCs). TRPC channels mediate a significant portion of the receptor-activated Ca(2+) influx. However, whether any of the TRPC channels function as a SOC remains controversial. Our understanding of the regulation of TRPC channels and their function as SOCs is being reshaped with the discovery of the role of STIM1 in the regulation of Ca(2+) influx channels. The findings that STIM1 is an ER resident Ca(2+) binding protein that regulates SOCs allow an expanded and molecular definition of SOCs. SOCs can be considered as channels that are regulated by STIM1 and require the clustering of STIM1 in response to depletion of the ER Ca(2+) stores and its translocation towards the plasma membrane. TRPC1 and other TRPC channels fulfill these criteria. STIM1 binds to TRPC1, TRPC2, TRPC4 and TRPC5 but not to TRPC3, TRPC6 and TRPC7, and STIM1 regulates TRPC1 channel activity. Structure-function analysis reveals that the C-terminus of STIM1 contains the binding and gating function of STIM1. The ERM domain of STIM1 binds to TRPC channels and a lysine-rich region participates in the gating of SOCs and TRPC1. Knock-down of STIM1 by siRNA and prevention of its translocation to the plasma membrane inhibit the activity of native SOCs and TRPC1. These findings support the conclusion that TRPC1 is a SOC. Similar studies with other TRPC channels demonstrate their regulation by STIM1 and indicate that all TRPC channels, except TRPC7, function as SOCs.     

Store-operated Ca(2+) channels and Stromal Interaction Molecule 1 (STIM1) are targets for the actions of bile acids on liver cells

Cholestasis is a significant contributor to liver pathology and can lead to primary sclerosis and liver failure. Cholestatic bile acids induce apoptosis and necrosis in hepatocytes but these effects can be partially alleviated by the pharmacological application of choleretic bile acids. These actions of bile acids on hepatocytes require changes in the release of Ca(2+) from intracellular stores and in Ca(2+) entry. However, the nature of the Ca(2+) entry pathway affected is not known. We show here using whole cell patch clamp experiments with H4-IIE liver cells that taurodeoxycholic acid (TDCA) and other choleretic bile acids reversibly activate an inwardly-rectifying current with characteristics similar to those of store-operated Ca(2+) channels (SOCs), while lithocholic acid (LCA) and other cholestatic bile acids inhibit SOCs. The activation of Ca(2+) entry was observed upon direct addition of the bile acid to the incubation medium, whereas the inhibition of SOCs required a 12 h pre-incubation. In cells loaded with fura-2, choleretic bile acids activated a Gd(3+)-inhibitable Ca(2+) entry, while cholestatic bile acids inhibited the release of Ca(2+) from intracellular stores and Ca(2+) entry induced by 2,5-di-(tert-butyl)-1,4-benzohydro-quinone (DBHQ). TDCA and LCA each caused a reversible redistribution of stromal interaction molecule 1 (STIM1, the endoplasmic reticulum Ca(2+) sensor required for the activation of Ca(2+) release-activated Ca(2+) channels and some other SOCs) to puncta, similar to that induced by thapsigargin. Knockdown of Stim1 using siRNA caused substantial inhibition of Ca(2+)-entry activated by choleretic bile acids. It is concluded that choleretic and cholestatic bile acids activate and inhibit, respectively, the previously well-characterised Ca(2+)-selective hepatocyte SOCs through mechanisms which involve the bile acid-induced redistribution of STIM1.     

ATP and vasopressin activate a single type of store-operated Ca2+ channel, identified by patch-clamp recording, in rat hepatocytes

Hepatocytes are highly polarised epithelial cells that mediate a large number of metabolic pathways, the transcellular movement of numerous ions and metabolites, and the secretion of proteins from both basal and canalicular membrane regions. Hormone-induced changes in the concentration of intracellular Ca2+ play a central role in regulating these functions. Store-operated Ca2+ channels (SOCs) and other Ca2+-permeable channels in the plasma membrane which are activated by hormones are essential for regulating the amount of Ca2+ in the hepatocyte in order to allow these Ca2+ signalling processes to occur. However, the properties of hormone-activated Ca2+ channels in hepatocytes and in other epithelial cells are not well defined. In this study, we have investigated SOCs in cultured rat hepatocytes by patch-clamp recording using IP3 and hormones as activators. We show that IP3 activates a single type of SOC, which, on the basis of its high selectivity for Ca2+ over Na+, inhibition by La3+ and 2-aminoethyl diphenylborate (2-APB), and the time course of fast inactivation, is very similar to CRAC channel in mast cells and lymphocytes. Moreover, a current (ISOC) with properties identical to those of the IP3-activated current can be activated by physiological concentrations of ATP and vasopressin. It is concluded that SOCs with properties similar to those of CRAC channel are present in hepatocytes, highly differentiated primary cells, and these channels can be activated by hormones under conditions close to physiological.     

Assessment of the role of the inositol 1,4,5-trisphosphate receptor in the activation of transient receptor potential channels and store-operated Ca2+ entry channels

The mechanism for coupling between Ca(2+) stores and store-operated channels (SOCs) is an important but unresolved question. Although SOCs have not been molecularly identified, transient receptor potential (TRP) channels share a number of operational parameters with SOCs. The question of whether activation of SOCs and TRP channels is mediated by the inositol 1,4,5-trisphosphate receptor (InsP(3)R) was examined using the permeant InsP(3)R antagonist, 2-aminoethoxydiphenyl borate (2-APB) in both mammalian and invertebrate systems. In HEK293 cells stably transfected with human TRPC3 channels, the actions of 2-APB to block carbachol-induced InsP(3)R-mediated store release and carbachol-induced Sr(2+) entry through TRPC3 channels were both reversed at high agonist levels, suggesting InsP(3)Rs mediate TRPC3 activation. However, electroretinogram recordings of the light-induced current in Drosophila revealed that the TRP channel-mediated responses in wild-type as well as trp and trpl mutant flies were all inhibited by 2-APB. This action of 2-APB is likely InsP(3)R-independent since InsP(3)Rs are dispensable for the light response. We used triple InsP(3)R knockout DT40 chicken B-cells to further assess the role of InsP(3)Rs in SOC activation. (45)Ca(2+) flux analysis revealed that although DT40 wild-type cells retained normal InsP(3)Rs mediating 2-APB-sensitive Ca(2+) release, the DT40InsP(3)R-k/o cells were devoid of functional InsP(3)Rs. Using intact cells, all parameters of Ca(2+) store function and SOC activation were identical in DT40wt and DT40InsP(3)R-k/o cells. Moreover, in both cell lines SOC activation was completely blocked by 2-APB, and the kinetics of action of 2-APB on SOCs (time dependence and IC(50)) were identical. The results indicate that (a) the action of 2-APB on Ca(2+) entry is not mediated by the InsP(3)R and (b) the effects of 2-APB provide evidence for an important similarity in the function of invertebrate TRP channels, mammalian TRP channels, and mammalian store-operated channels.     

Effect of 2,5-anhydro-D-mannitol on membrane potential in rat hepatocyte couplets and hepatocyte monolayer cultures

The fructose analogue 2,5-anhydro-D-mannitol (2,5-AM), which depletes liver cells of ATP, has been shown to alter liver cell membrane potential (V(m)) in situ and in superfused liver slices. To study this effect of 2,5-AM on hepatocytes in more detail, patch-clamp experiments in the current-clamp mode were performed using two established models, rat hepatocyte couplets and confluent rat hepatocytes in primary culture. 2,5-AM, which has previously been shown to hyperpolarize hepatocytes in superfused liver slices and in vivo, failed to alter V(m) of hepatocyte couplets. Increasing intracellular Ca(2+) by addition of thapsigargin or ionomycin also did not evoke a change of V(m). This is most likely due to a lack of Ca(2+)-dependent K(+) channels in rat hepatocyte couplets. In contrast, 2,5-AM depolarized the cells in confluent hepatocyte monolayers. This depolarization was mimicked after inhibition of Na(+)/K(+) ATPase by ouabain. Ouabain was also able to block 2, 5-AM's effect on monolayer V(m). Thus, 2,5-AM affects the membrane potential of isolated and cultured hepatocytes in a way not comparable with cells integrated in the liver.     

Upregulated TRP and enhanced capacitative Ca(2+) entry in human pulmonary artery myocytes during proliferation

A rise in cytosolic Ca(2+) concentration ([Ca(2+)](cyt)) due to Ca(2+) release from intracellular Ca(2+) stores and Ca(2+) influx through plasmalemmal Ca(2+) channels plays a critical role in mitogen-mediated cell growth. Depletion of intracellular Ca(2+) stores triggers capacitative Ca(2+) entry (CCE), a mechanism involved in maintaining Ca(2+) influx and refilling intracellular Ca(2+) stores. Transient receptor potential (TRP) genes have been demonstrated to encode the store-operated Ca(2+) channels that are activated by Ca(2+) store depletion. In this study, we examined whether CCE, activity of store-operated Ca(2+) channels, and human TRP1 (hTRP1) expression are essential in human pulmonary arterial smooth muscle cell (PASMC) proliferation. Chelation of extracellular Ca(2+) and depletion of intracellularly stored Ca(2+) inhibited PASMC growth in media containing serum and growth factors. Resting [Ca(2+)](cyt) as well as the increases in [Ca(2+)](cyt) due to Ca(2+) release and CCE were all significantly greater in proliferating PASMC than in growth-arrested cells. Consistently, whole cell inward currents activated by depletion of intracellular Ca(2+) stores and the mRNA level of hTRP1 were much greater in proliferating PASMC than in growth-arrested cells. These results suggest that elevated [Ca(2+)](cyt) and intracellularly stored [Ca(2+)] play an important role in pulmonary vascular smooth muscle cell growth. CCE, potentially via hTRP1-encoded Ca(2+)-permeable channels, may be an important mechanism required to maintain the elevated [Ca(2+)](cyt) and stored [Ca(2+)] in human PASMC during proliferation.     

Store-operated Ca2+ channels in prostate cancer epithelial cells: function,regulation, and role in carcinogenesis

Ca2+ homeostasis mechanisms, in which the Ca2+ entry pathways play a key role, are critically involved in both normal function and cancerous transformation of prostate epithelial cells. Here, using the lymph node carcinoma of the prostate (LNCaP) cell line as a major experimental model, we characterize prostate-specific store-operated Ca2+ channels (SOCs)--a primary Ca2+ entry pathway for non-excitable cells--for the first time. We show that prostate-specific SOCs share major store-dependent, kinetic, permeation, inwardly rectifying, and pharmacological (including dual, potentiation/inhibition concentration-dependent sensitivity to 2-APB) properties with "classical" Ca2+ release-activated Ca2+ channels (CRAC), but have a higher single channel conductance (3.2 and 12pS in Ca2+- and Na+-permeable modes, respectively). They are subject to feedback inhibition via Ca2+-dependent PKC, CaMK-II and CaM regulatory pathways and are functionally dependent on caveolae integrity. Caveolae also provide a scaffold for spatial co-localization of SOCs with volume-regulated anion channels (VRAC) and their Ca2+-mediated interaction. The TRPC1 and TRPV6 members of the transient receptor potential (TRP) channel family are the most likely molecular candidates for the formation of prostate-specific endogenous SOCs. Differentiation of LNCaP cells to an androgen-insensitive, apoptotic-resistant neuroendocrine phenotype downregulates SOC current. We conclude that prostate-specific SOCs are important determinants in the transition to androgen-independent prostate cancer.     

Voltage-independent calcium influx in smooth muscle

In smooth muscle cells, agonists such as neurotransmitters or hormones can induce an increase in [Ca(2+)](i) via a release of intracellular stored calcium or/and an influx of extracellular calcium. The calcium entry pathway operates through a variety of plasmalemmal calcium channels which involve voltage-dependent and voltage-independent calcium channels. Voltage-independent calcium channels include (1) receptor-operated channels (ROCs) activated by agonist-receptor interaction and, in the majority of cases, the downstream signal transduction proteins, (2) store-operated channels (SOCs) activated by the emptying of intracellular Ca(2+) store (mainly the sarcoplasmic reticulum), (3) mechanosensitive or stretch-activated channels (SACs) activated by membrane stretch. Generally, voltage-independent calcium channels are calcium permeable non-selective cation channels with electrophysiological differences, complex regulatory mechanisms and pharmacology. Although the molecular identity of voltage-independent calcium channels is not yet fully elucidated, there are growing evidences that these channels correspond to a new family of membrane proteins encoded by mammalian homologues of specific transient receptor potential (TRP) genes. Several types of TRP proteins are ubiquitously expressed in smooth muscle cells and variations in the expression depend on tissue and species. More recently, other proteins such as Orai1 and STIM1 proteins have been also proposed as participating in the molecular identity of voltage-independent calcium channels. These channels control phenomena such as smooth muscle cells proliferation and/or contraction.     

The mammalian TRPC cation channels

Transient Receptor Potential-Canonical (TRPC) channels are mammalian homologs of Transient Receptor Potential (TRP), a Ca(2+)-permeable channel involved in the phospholipase C-regulated photoreceptor activation mechanism in Drosophila. The seven mammalian TRPCs constitute a family of channels which have been proposed to function as store-operated as well as second messenger-operated channels in a variety of cell types. TRPC channels, together with other more distantly related channel families, make up the larger TRP channel superfamily. This review summarizes recent findings on the structure, regulation and function of the apparently ubiquitous TRPC cation channels.     

The nature and mechanism of activation of the hepatocyte receptor-activated Ca2+ inflow system.

Progress in elucidation of the properties of the hepatocyte receptor-activated Ca2+ inflow system (RACIS) has been hampered by difficulties in measuring rates of Ca2+ inflow to hepatocytes. These difficulties have led, for example, to different conclusions about the relationship between the extracellular Ca2+ concentration and the movement of Ca2+ through the RACIS. The hepatocyte RACIS admits Mn2+ and a number of other divalent cations as well as Ca2+. Many of these cations also inhibit the movement of Ca2+ through this system. While the RACIS is inhibited by high concentrations of verapamil and by some other Ca2+ antagonists, it is relatively insensitive to inhibition by organic compounds which inhibit other Ca2+ channels and Ca2+ transporters. There is circumstantial evidence which suggests that the hepatocyte RACIS is an exchange system, possibly one which catalyses Ca(2+)-H+ exchange or the co-transport of Ca2+ and OH-. Other circumstantial evidence suggests that the RACIS is a channel, with some similarities to voltage-operated Ca2+ channels in excitable cells. However, experiments using the patch-clamp technique have not yet detected agonist-stimulated Ca2+ movement across the hepatocyte plasma membrane. The molecular components of the RACIS probably differ from those which facilitate the large inflow of Ca2+ to hepatocytes which occurs in the absence of an agonist. The mechanism by which agonists activate the RACIS has not been elucidated.(ABSTRACT TRUNCATED AT 250 WORDS)     

TRP channels in hypertension

Pulmonary and systemic arterial hypertension are associated with profound alterations in Ca(2+) homeostasis and smooth muscle cell proliferation. A novel class of non-selective cation channels, the transient receptor potential (TRP) channels, have emerged at the forefront of research into hypertensive disease states. TRP channels are identified as molecular correlates for receptor-operated and store-operated cation channels in the vasculature. Over 10 TRP isoforms are identified at the mRNA and protein expression levels in the vasculature. Current research implicates upregulation of specific TRP isoforms to be associated with increased Ca(2+) influx, characteristic of vasoconstriction and vascular smooth muscle cell proliferation. TRP channels are implicated as Ca(2+) entry pathways in pulmonary hypertension and essential hypertension. Caveolae have recently emerged as membrane microdomains in which TRP channels may be co-localized with the endoplasmic reticulum in both smooth muscle and endothelial cells. Such enhanced expression and function of TRP channels and their localization in caveolae in pathophysiological hypertensive disease states highlights their importance as potential targets for pharmacological intervention.     

Intracellular trafficking of TRP channels

Thirteen years ago, it was suggested that exocytotic insertion of store-operated channels into the plasma membrane lead to increased Ca(2+) entry in non-excitable cells upon G protein-coupled or tyrosine kinase receptor stimulation. Since the discovery of the TRP channel superfamily and their involvement in receptor-induced Ca(2+) entry, many studies have shown that different members of the TRP superfamily translocate into the plasma membrane upon stimulation. While the exact molecular mechanism by which TRP channels insert into the plasma membrane is unknown, TRP-binding proteins have been shown to directly regulate this trafficking. This review summarizes recent advances related to the mechanism of TRP channel trafficking, focusing on the role of TRP-binding proteins.     

Voltage-dependent calcium-permeable channels in the plasma membrane of a higher plant cell.

Numerous biological assays and pharmacological studies on various higher plant tissues have led to the suggestion that voltage-dependent plasma membrane Ca2+ channels play prominent roles in initiating signal transduction processes during plant growth and development. However, to date no direct evidence has been obtained for the existence of such depolarization-activated Ca2+ channels in the plasma membrane of higher plant cells. Carrot suspension cells (Daucus carota L.) provide a well-suited system to determine whether voltage-dependent Ca2+ channels are present in the plasma membrane of higher plants and to characterize the properties of putative Ca2+ channels. It is known that both depolarization, caused by raising extracellular K+, and exposure to fungal toxins or oligogalacturonides induce Ca2+ influx into carrot cells. By direct application of patch-clamp techniques to isolated carrot protoplasts, we show here that depolarization of the plasma membrane positive to -135 mV activates Ca(2+)-permeable channels. These voltage-dependent ion channels were more permeable to Ca2+ than K+, while displaying large permeabilities to Ba2+ and Mg2+ ions. Ca(2+)-permeable channels showed slow and reversible inactivation. The single-channel conductance was 13 pS in 40 mM CaCl2. These data provide direct evidence for the existence of voltage-dependent Ca2+ channels in the plasma membrane of a higher plant cell and point to physiological mechanisms for plant Ca2+ channel regulation. The depolarization-activated Ca(2+)-permeable channels identified here could constitute a regulated pathway for Ca2+ influx in response to physiologically occurring stimulus-induced depolarizations in higher plant cells.     

TRP, TRPL and Cacophony Channels Mediate Ca(2+) Influx and Exocytosis in Photoreceptors Axons in Drosophila

In Drosophila photoreceptors Ca(2+)-permeable channels TRP and TRPL are the targets of phototransduction, occurring in photosensitive microvilli and mediated by a phospholipase C (PLC) pathway. Using a novel Drosophila brain slice preparation, we studied the distribution and physiological properties of TRP and TRPL in the lamina of the visual system. Immunohistochemical images revealed considerable expression in photoreceptors axons at the lamina. Other phototransduction proteins are also present, mainly PLC and protein kinase C, while rhodopsin is absent. The voltage-dependent Ca(2+) channel cacophony is also present there. Measurements in the lamina with the Ca(2+) fluorescent protein G-CaMP ectopically expressed in photoreceptors, revealed depolarization-induced Ca(2+) increments mediated by cacophony. Additional Ca(2+) influx depends on TRP and TRPL, apparently functioning as store-operated channels. Single synaptic boutons resolved in the lamina by FM4-64 fluorescence revealed that vesicle exocytosis depends on cacophony, TRP and TRPL. In the PLC mutant norpA bouton labeling was also impaired, implicating an additional modulation by this enzyme. Internal Ca(2+) also contributes to exocytosis, since this process was reduced after Ca(2+)-store depletion. Therefore, several Ca(2+) pathways participate in photoreceptor neurotransmitter release: one is activated by depolarization and involves cacophony; this is complemented by internal Ca(2+) release and the activation of TRP and TRPL coupled to Ca(2+) depletion of internal reservoirs. PLC may regulate the last two processes. TRP and TRPL would participate in two different functions in distant cellular regions, where they are opened by different mechanisms. This work sheds new light on the mechanism of neurotransmitter release in tonic synapses of non-spiking neurons.     

Some assembly required: constructing the elementary units of store-operated Ca2+ entry

The means by which Ca(2+) store depletion evokes the opening of store-operated Ca(2+) channels (SOCs) in the plasma membrane of excitable and non-excitable cells has been a longstanding mystery. Indirect evidence has supported local interactions between the ER and SOCs as well as long-range interactions mediated through a diffusible activator. The recent molecular identification of the ER Ca(2+) sensor (STIM1) and a subunit of the CRAC channel (Orai1), a prototypic SOC, has now made it possible to visualize directly the sequence of events that links store depletion to CRAC channel opening. Following store depletion, STIM1 moves from locations throughout the ER to accumulate in ER subregions positioned within 10-25nm of the plasma membrane. Simultaneously, Orai1 gathers at discrete sites in the plasma membrane directly opposite STIM1, resulting in local CRAC channel activation. These new studies define the elementary units of store-operated Ca(2+) entry, and reveal an unprecedented mechanism for channel activation in which the stimulus brings a channel and its activator/sensor together for interaction across apposed membrane compartments. We discuss the implications of this choreographic mechanism with regard to Ca(2+) dynamics, specificity of Ca(2+) signaling, and the existence of a specialized ER subset dedicated to the control of the CRAC channel.     

Emerging perspectives in store-operated Ca2+ entry: roles of Orai, Stim and TRP

Depletion of intracellular Ca2+ stores induces Ca2+ influx across the plasma membrane through store-operated channels (SOCs). This store-operated Ca2+ influx is important for the replenishment of the Ca2+ stores, and is also involved in many signaling processes by virtue of the ability of intracellular Ca2+ to act as a second messenger. For many years, the molecular identities of particular SOCs, as well as the signaling mechanisms by which these channels are activated, have been elusive. Recently, however, the mammalian proteins STIM1 and Orai1 were shown to be necessary for the activation of store-operated Ca2+ entry in a variety of mammalian cells. Here we present molecular, pharmacological, and electrophysiological properties of SOCs, with particular focus on the roles that STIM1 and Orai1 may play in the signaling processes that regulate various pathways of store-operated entry.     

Abnormal calcium homeostasis in fibroblasts from patients with Leigh disease

Recently, we reported that in various cell lines under conditions of deenergization of the mitochondrial membrane, the release of Ca(2+) from the endoplasmic reticulum (ER) does not produce the expected activation of store-operated calcium channels (SOCs) in the plasma membrane. In the present work, we examined the activation of SOCs in fibroblasts derived from three patients with Leigh disease (LS). We identified mutations in the SURF-1 gene in all these cells. Consequently, cytochrome oxidase (COX) deficiency was found in all these (LS(COX)) cell lines and, thus, the main mitochondrial mechanism of generation of the electrochemical proton gradient on the mitochondrial membrane was naturally depressed. We demonstrated that, in untreated LS(COX) fibroblasts, the rate of Ca(2+)-inflow through SOCs was low compared to the fibroblasts from healthy individuals even after thapsigargin-induced maximal release of Ca(2+) from the ER. Moreover, the pretreatment of LS(COX) fibroblasts with a protonophore did not modify this rate. Thus, in LS(COX) fibroblasts, the activation of SOCs was naturally impaired. Our findings suggest that altered calcium metabolism, apart from severe energy production failure, may also contribute to developing pathological conditions in patients with COX-deficient Leigh disease related to SURF-1 gene mutation.