Low Cytosolic Ca2 Research Articles

Defective autophagy and Alzheimer's disease: is calcium the key?

Defective autophagy and Alzheimer's disease: is calcium the key?

Dimerization of MICU Proteins Controls Ca2+ Influx through the Mitochondrial Ca2+ Uniporter

The mitochondrial Ca2+ uniporter complex (MCUC) is responsible for Ca2+ influx into the mitochondrial matrix, playing critical roles in various mitochondrial functions. Eukaryotic MCUC consists of multiple subunits, and its Ca2+ influx activity is controlled by regulatory subunits, including mitochondrial Ca2+ uptake 1 (MICU1) and its paralogs (MICU2 and MICU3). However, the underlying mechanism remains unclear. Here, we determined multiple crystal structures of MICU2 and MICU3 from Homo sapiens. Our data demonstrate that distinct MICU protein N-domains determine the specific type of MICU dimers that perform the opposing roles in mitochondrial Ca2+ uptake at low cytosolic Ca2+ levels. In contrast, at high cytosolic Ca2+ levels, all MICU proteins undergo dimer rearrangement induced by Ca2+ binding, which releases the suppression of the MCUC pore-forming subunit and promotes the influx of large amounts of Ca2+. Altogether, our results elucidate the delicate mechanism of mitochondrial Ca2+ uptake regulation by MICU proteins.

Effects of calcium and its interaction with phosphorus on the nutrient status and growth of three Lupinus species.

Phosphorus (P)-deficiency symptoms are known for Lupinus species grown in calcareous soil, but we do not know if this is due to a high calcium (Ca) availability or a low P availability in the soil. To address this problem, we explored both the effects of Ca and its interactions with P on nutrient status and growth of three Lupinus species. Two Ca-sensitive genotypes (L. angustifolius L. P26723 and L. cosentinii Guss. P27225) and two Ca-tolerant genotypes (L. angustifolius L. cv Mandelup and L. pilosus Murr. P27440) were grown hydroponically at two P (0.1 and 10 μM) and three Ca (0.1, 0.6 and 6 mM) levels. Leaf symptoms and biomass were recorded, whole leaf and root nutrient concentrations were analysed, and leaf cellular P and Ca concentrations were determined. Phosphorus-deficiency symptoms were only observed in the Ca-sensitive genotypes. Among all the genotypes in this study, the Ca-tolerant L. pilosus showed an ability to maintain stable leaf Ca and P concentrations whereas the Ca-tolerant L. angustifolius cv Mandelup did not maintain a stable whole leaf Ca concentration, but maintained a low cytosolic Ca2+ concentration through effective Ca compartmentation. However, the two Ca-sensitive genotypes, L. angustifolius P26723 and L. cosentinii, did not exhibit an ability to maintain a stable whole leaf Ca concentration or effectively compartmentalise Ca. Therefore, having the capacity to maintain a stable whole leaf Ca concentration or effectively compartmentalising Ca in leaves are likely critical for Lupinus species to be Ca tolerant.

G4941K substitution in the pore-lining S6 helix of the skeletal muscle ryanodine receptor increases RyR1 sensitivity to cytosolic and luminal Ca2+

The ryanodine receptor ion channel RyR1 is present in skeletal muscle and has a large cytoplasmic N-terminal domain and smaller C-terminal pore-forming domain comprising six transmembrane helices, a pore helix, and a selectivity filter. The RyR1 S6 pore-lining helix has two conserved glycines, Gly-4934 and Gly-4941, that facilitate RyR1 channel gating by providing S6 flexibility and minimizing amino acid clashes. Here, we report that substitution of Gly-4941 with Asp or Lys results in functional channels as indicated by caffeine-induced Ca2+ release response in HEK293 cells, whereas a low response of the corresponding Gly-4934 variants suggested loss of function. Following purification, the RyR1 mutants G4934D, G4934K, and G4941D did not noticeably conduct Ca2+ in single-channel measurements. Gly-4941 replacement with Lys resulted in channels having reduced K+ conductance and reduced selectivity for Ca2+ compared with wildtype. RyR1-G4941K did not fully close at nanomolar cytosolic Ca2+ concentrations and nearly fully opened at 2 μm cytosolic or sarcoplasmic reticulum luminal Ca2+, and Ca2+- and voltage-dependent regulation of RyR1-G4941K mutant channels was demonstrated. Computational methods and single-channel recordings indicated that the open G4941K variant results in the formation of a salt bridge to Asp-4938. In contrast, wildtype RyR1 was closed and not activated by luminal Ca2+ at low cytosolic Ca2+ levels. A model suggested that luminal Ca2+ activates RyR1 by accessing a recently identified cytosolic Ca2+-binding site in the open channel as the Ca2+ ions pass through the pore.

Gain-of-Function of TMEM16E/ANO5 Scrambling Activity Caused by a Mutation Associated with the Bone Genetic Disease Gnathodiaphyseal Dysplasia

Members of the TMEM16 or anoctamin membrane protein family are involved in a variety of functions that include ion transport, phospholipid scrambling and regulation of other membrane proteins. In particular, TMEM16A, 16B and 16F have a clear plasma membrane localization but while 16A and 16B work as ‘pure’ ion channels, TMEM16F shows both ion channel and scramblase activities. Recent data suggest that also TMEM16E, the closest relative of TMEM16F, may work as a phospholipid scramblase, although a direct confirmation is missing. TMEM16E is highly expressed in bone tissue and skeletal muscle and different mutations in the human TMEM16E (ANO5) gene are associated with the bone disease gnathodiaphyseal dysplasia (GDD) or to different forms of muscular dystrophy (LGMD2L, limb-girdle muscular dystrophy-2l and distal MMD3, miyoshi muscular dystrophy-3). Our data show that human TMEM16E, when overexpressed in mammalian cell lines, displays partial plasma membrane localization and give rise to phospholipid scrambling as well as non-selective ionic currents with slow time-dependent activation at highly depolarized membrane potentials and in presence of high intracellular Ca2+. We address the effect of the GDD-causing mutation T513I, located in the second extracellular loop, on TMEM16E and we show phospholipid scrambling activity and large time-dependent ion currents even at low cytosolic Ca2+ concentrations. Our data provide the first direct demonstration of Ca2+-dependent current and phospholipid scrambling activity for TMEM16E and suggest a gain-of-function phenotype related to a GDD mutation.

Dimerization of MICU Proteins Controls Ca 2 Influx Through the Mitochondrial Ca 2 Uniporter

The mitochondrial Ca2 uniporter complex (MCUC) is responsible for Ca2 influx into the mitochondrial matrix, playing critical roles in various mitochondrial functions. Eukaryotic MCUC consists of multiple subunits and its Ca2 influx activity is controlled by regulatory subunits, including mitochondrial Ca2 uptake 1 (MICU1) and its paralogs (MICU2 and MICU3). However, the underlying mechanism remains unclear. Here, we determined multiple crystal structures of MICU2 and MICU3 from Homo sapiens. Our structural analysis together with cell biology data demonstrate that distinct MICU protein N-domains determine the specific type of MICU dimers that perform the opposing roles in mitochondrial Ca2 uptake at low cytosolic Ca2 levels. In contrast, at high cytosolic Ca2 levels, all MICU proteins undergo dimer rearrangement induced by Ca2 binding, which releases the suppression of the MCUC pore-forming subunit and promotes the influx of large amounts of Ca2 . Altogether, our results elucidate the delicate mechanism of mitochondrial Ca2 uptake regulation by MICU proteins.

Gain of function of TMEM16E/ANO5 scrambling activity caused by a mutation associated with gnathodiaphyseal dysplasia

Mutations in the human TMEM16E (ANO5) gene are associated both with the bone disease gnathodiaphyseal dysplasia (GDD; OMIM: 166260) and muscle dystrophies (OMIM: 611307, 613319). However, the physiological function of TMEM16E has remained unclear. We show here that human TMEM16E, when overexpressed in mammalian cell lines, displayed partial plasma membrane localization and gave rise to phospholipid scrambling (PLS) as well as non-selective ionic currents with slow time-dependent activation at highly depolarized membrane potentials. While the activity of wild-type TMEM16E depended on elevated cytosolic Ca2+ levels, a mutant form carrying the GDD-causing T513I substitution showed PLS and large time-dependent ion currents even at low cytosolic Ca2+ concentrations. Contrarily, mutation of the homologous position in the Ca2+-activated Cl− channel TMEM16B paralog hardly affected its function. In summary, these data provide the first direct demonstration of Ca2+-dependent PLS activity for TMEM16E and suggest a gain-of-function phenotype related to a GDD mutation.

Regulation of RhoA/ROCK and sustained arterial contraction by low cytosolic Ca2 + levels during prolonged depolarization of arterial smooth muscle

The role of L-type Ca2+ channels (LTCCs) and RhoA/Rho kinase (ROCK) on depolarization-induced sustained arterial contraction lasting several minutes is already known. However, in vivo, vascular smooth muscle cells can be depolarized for longer periods, inducing substantial inactivation of LTCCs and markedly reducing Ca2+ influx into the myocytes. We have examined, in femoral arterial rings, the role of LTCCs and RhoA/ROCK during long-lasting depolarization. Our results reveal a new vasoreactive response after 20–30min of depolarization in 2.5mM external Ca2+ that has not been identified previously with shorter stimuli. Prolonged depolarization-induced arterial contraction was permanently abolished when arterial rings were treated with 100nM external Ca2+ or 20nM nifedipine. However, when Ca2+ influx was restricted, applying ~7μM external Ca2+ solution or 3nM nifedipine, vasorelaxation was transient, and isometric force slowly increased after 30min and maintained its level until the end of the stimulus. Under these conditions, arterial contraction showed the same temporal course of RhoA activity and was sensitive to fasudil, nifedipine and cyclopiazonic acid. Ca2+-response curve in β-escin permeabilized arteries was also sensitive to ROCK inhibitors. Thus, although long-lasting depolarization inactivates LTCCs, the reduced Ca2+ entry can induce a detectable arterial contraction via RhoA/ROCK activation.

The human two-pore channel 1 is modulated by cytosolic and luminal calcium

Two-pore channels (TPC) are intracellular endo-lysosomal proteins with only recently emerging roles in organellar signalling and involvement in severe human diseases. Here, we investigated the functional properties of human TPC1 expressed in TPC-free vacuoles from Arabidopsis thaliana cells. Large (20 pA/pF) TPC1 currents were elicited by cytosolic addition of the phosphoinositide phosphatidylinositol-(3,5)-bisphosphate (PI(3,5)P2) with an apparent binding constant of ~15 nM. The channel is voltage-dependent, activating at positive potentials with single exponential kinetics and currents are Na+ selective, with measurable but low permeability to Ca2+. Cytosolic Ca2+ modulated hTPC1 in dual way: low μM cytosolic Ca2+ increased activity by shifting the open probability towards negative voltages and by accelerating the time course of activation. This mechanism was well-described by an allosteric model. Higher levels of cytosolic Ca2+ induced a voltage-dependent decrease of the currents compatible with Ca2+ binding in the permeation pore. Conversely, an increase in luminal Ca2+ decreased hTPC1 activity. Our data point to a process in which Ca2+ permeation in hTPC1 has a positive feedback on channel activity while Na+ acts as a negative regulator. We speculate that the peculiar Ca2+ and Na+ dependence are key for the physiological roles of the channel in organellar homeostasis and signalling.

L-Met Activates Arabidopsis GLR Ca2+ Channels Upstream of ROS Production and Regulates Stomatal Movement

Plant glutamate receptor homologs (GLRs) have long been proposed to function as ligand-gated Ca2+ channels, but no in planta evidence has been provided. Here, we present genetic evidence that Arabidopsis GLR3.1 and GLR3.5 form Ca2+ channels activated by L-methionine (L-Met) at physiological concentrations and regulate stomatal apertures and plant growth. The glr3.1/3.5 mutations resulted in a lower cytosolic Ca2+ level, defective Ca2+-induced stomatal closure, and Ca2+-deficient growth disorder, all of which involved L-Met. Patch-clamp analyses of guard cells showed that GLR3.1/3.5 Ca2+ channels are activated specifically by L-Met, with the activation abolished in glr3.1/3.5. Moreover, GLR3.1/3.5 Ca2+ channels are distinct from previously characterized ROS-activated Ca2+ channels and act upstream of ROS, providing Ca2+ transients necessary for the activation of NADPH oxidases. Our data indicate that GLR3.1/3.5 constitute L-Met-activated Ca2+ channels responsible for maintaining basal [Ca2+]cyt, play a pivotal role in plant growth, and act upstream of ROS, thereby regulating stomatal aperture.

Calcium and ATP control multiple vital functions.

Life on Planet Earth, as we know it, revolves around adenosine triphosphate (ATP) as a universal energy storing molecule. The metabolism of ATP requires a low cytosolic Ca(2+) concentration, and hence tethers these two molecules together. The exceedingly low cytosolic Ca(2+) concentration (which in all life forms is kept around 50-100 nM) forms the basis for a universal intracellular signalling system in which Ca(2+) acts as a second messenger. Maintenance of transmembrane Ca(2+) gradients, in turn, requires ATP-dependent Ca(2+) transport, thus further emphasizing the inseparable links between these two substances. Ca(2+) signalling controls the most fundamental processes in the living organism, from heartbeat and neurotransmission to cell energetics and secretion. The versatility and plasticity of Ca(2+) signalling relies on cell specific Ca(2+) signalling toolkits, remodelling of which underlies adaptive cellular responses. Alterations of these Ca(2+) signalling toolkits lead to aberrant Ca(2+) signalling which is fundamental for the pathophysiology of numerous diseases from acute pancreatitis to neurodegeneration. This paper introduces a theme issue on this topic, which arose from a Royal Society Theo Murphy scientific meeting held in March 2016.This article is part of the themed issue 'Evolution brings Ca(2+) and ATP together to control life and death'.

Calcium signaling mediates the response to cadmium toxicity in Saccharomyces cerevisiae cells

The involvement of Ca2+ in the response to high Mn2+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+, and Hg2+ was investigated in Saccharomyces cerevisiae. The yeast cells responded through a sharp increase in cytosolic Ca2+ when exposed to Cd2+, and to a lesser extent to Cu2+, but not to Mn2+, Co2+, Ni2+, Zn2+, or Hg2+. The response to high Cd2+ depended mainly on external Ca2+ (transported through the Cch1p/Mid1p channel) but also on vacuolar Ca2+ (released into the cytosol through the Yvc1p channel). The adaptation to high Cd2+ was influenced by perturbations in Ca2+ homeostasis. Thus, the tolerance to Cd2+ often correlated with sharp Cd2+-induced cytosolic Ca2+ pulses, while the Cd2+ sensitivity was accompanied by the incapacity to rapidly restore the low cytosolic Ca2+.

Altered Ion Channel Properties of Ryanodine Receptor from Heart Mice Lacking Calstabin2

The cardiac ryanodine receptor (RyR2) is the main channel for the release of intracellular calcium during excitation-contraction coupling in cardiac muscle. Calstabin2 (i.e. FKBP12.6), a member of the FK506-binding protein family, has high affinity to RyR2, stabilizing RyR2 in the closed state and reducing its activity. The goal of the present work was first to examine the ion channel properties of RyR2 from calstabin2-/- mice reconstituted into lipid bilayers in resting condition. The second aim was to evaluate the effect of adding recombinant calstabin2 on RyR2 ion channel properties. RyR2 channels from 9 months old calstabin2 deficient mice exhibited an increase of opening probability compared to RyR2 from wild-type mice in conditions of low free cytosolic Ca2+ of 150 nM to approximate the conditions in the heart during diastole. The activity of RyR2 calstabin2-/- channels was also higher with 350 nmol/L and 700 nmol/L cytosolic Ca2+. When channels were activated by higher cytosolic Ca2+ concentration (> 700 nmol/L free Ca2+), no differences in open probability were found between RyR2 calstabin2-/- and WT channels. Addition of 1 mmol/L Na2-ATP increased the number of long openings to the maximum conductance in both native and calstabin2-/- channels with a prevalent sub-conductance activity for the depleted calstabin2 RyR2 channels. Addition of calstabin2 did neither increase nor decrease RyR2 calstabin2-/- open probability and had no effect in preventing the sub-conductance activity. This suggests that in vitro addition of calstabin2 is not sufficient to restore the RyR2/calstabin2 complex. This conclusion should further supported by the biochemical analyzes of RyR2 macromolecular complex remodeling, performed along with age-dependency.

The Mechanism of Uncoupling ATP Hydrolysis and Calcium Transport in Serca by Sarcolipin

The interactions of sarcolipin (SLN) with the sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) causes uncoupling of SERCA's ATP hydrolysis and Ca2+ -transport activities. SERCA is an integral component of proper muscle function. It is one of the key proteins responsible for re-establishing the low cytosolic Ca2+ concentrations needed for normal muscle contraction. When SLN is bound to SERCA, fewer moles of Ca2+ are transported per mole of ATP hydrolyzed, which leads to an increase in the heat released. By using solid-state nuclear magnetic resonance spectroscopy and functional assays, we demonstrate that it is the topological coupling between these proteins that is important for the regulation of SERCA. It is through the interactions of their transmembrane regions as well as the interactions of SLN's lumenal tail that lead to uncoupling. These results also provide insight into the mechanism for non-shivering, muscle-based thermogenesis.

Loss-of-function mutations in MICU1 cause a brain and muscle disorder linked to primary alterations in mitochondrial calcium signaling

Mitochondrial Ca(2+) uptake has key roles in cell life and death. Physiological Ca(2+) signaling regulates aerobic metabolism, whereas pathological Ca(2+) overload triggers cell death. Mitochondrial Ca(2+) uptake is mediated by the Ca(2+) uniporter complex in the inner mitochondrial membrane, which comprises MCU, a Ca(2+)-selective ion channel, and its regulator, MICU1. Here we report mutations of MICU1 in individuals with a disease phenotype characterized by proximal myopathy, learning difficulties and a progressive extrapyramidal movement disorder. In fibroblasts from subjects with MICU1 mutations, agonist-induced mitochondrial Ca(2+) uptake at low cytosolic Ca(2+) concentrations was increased, and cytosolic Ca(2+) signals were reduced. Although resting mitochondrial membrane potential was unchanged in MICU1-deficient cells, the mitochondrial network was severely fragmented. Whereas the pathophysiology of muscular dystrophy and the core myopathies involves abnormal mitochondrial Ca(2+) handling, the phenotype associated with MICU1 deficiency is caused by a primary defect in mitochondrial Ca(2+) signaling, demonstrating the crucial role of mitochondrial Ca(2+) uptake in humans.

Calcium, mitochondria, and the pathogenesis of ALS: the good, the bad, and the ugly

Calcium, mitochondria, and the pathogenesis of ALS: the good, the bad, and the ugly

Endoplasmic reticulum stress induces sarco/endoplasmic reticulum calcium ATPase and alters calcium homeostasis in the vasculature

Calcium homeostasis is tightly regulated in the vasculature. The sarco/endoplasmic reticulum (ER) Ca2+‐ATPase (SERCA) is responsible for maintaining a relatively low cytosolic Ca2+ concentration. The ER is a multifunctional organelle and is easily perturbed resulting in ER stress. When ER stress occurs, the ER initiates a cellular signaling network, the unfolded protein response (UPR) to alleviate this stress. We tested the hypothesis that during ER stress increased SERCA function would lead to increased Ca2+‐induced contraction and elevated ER Ca2+ stores in the aorta of male mice. Male CD‐1 mice were treated with tunicamycin (TM, 100mg/kg, 8hrs, i.p.), an inducer of ER stress or vehicle (saline, 125ul, 8hrs, i.p.). TM treatment significantly upregulated aortic SERCA2b expression. Using a wire myograph, aortic rings of TM treated mice had an increased Ca2+‐induced contraction when compared to control (99.7%±2.6% vs. 69.4%±2.7%) in the presence of thapsigargin (TG, 1μM), an inhibitor of SERCA. Caffeine (10mM) induced depletion of intracellular Ca2+ stores measured as a transient contraction, was greater in aortic rings from TM treated mice (62.1%±5.1% vs. 44.1%±3.9%) compared to controls which was completely abolished in the presence of TG (6.8%±1.7% vs 7.4%±1.1%). Therefore augmented SERCA activity following ER stress may be a mechanisms altering Ca2+ homeostasis in the vasculature.

Calcium dysregulation, mitochondrial pathology and protein aggregation in a culture model of amyotrophic lateral sclerosis: Mechanistic relationship and differential sensitivity to intervention

The combination of Ca2+ influx during neurotransmission and low cytosolic Ca2+ buffering contributes to the preferential vulnerability of motor neurons in amyotrophic lateral sclerosis (ALS). This study investigated the relationship among Ca2+ accumulation in intracellular compartments, mitochondrial abnormalities, and protein aggregation in a model of familial ALS (fALS1). Human SOD1, wild type (SOD1WT) or with the ALS-causing mutation G93A (SOD1G93A), was expressed in motor neurons of dissociated murine spinal cord–dorsal root ganglia (DRG) cultures. Elevation of mitochondrial Ca2+ ([Ca2+]m), decreased mitochondrial membrane potential (Δψ) and rounding of mitochondria occurred early, followed by increased endoplasmic reticular Ca2+ ([Ca2+]ER), elevated cytosolic Ca2+ ([Ca2+]c), and subsequent appearance of SOD1G93A inclusions (a consequence of protein aggregation). [Ca2+]c was elevated to a greater extent in neurons with inclusions than in those with diffusely distributed SOD1G93A and promoted aggregation of mutant protein, not vice versa: both [Ca2+]c and the percentage of neurons with SOD1G93A inclusions were reduced by co-expressing the cytosolic Ca2+-buffering protein, calbindin D-28K; treatment with the heat shock protein inducer, geldanamycin, prevented inclusions but not the increase in [Ca2+]c, [Ca2+]m or loss of Δψ, and inhibiting proteasome activity with epoxomicin, known to promote aggregation of disease-causing mutant proteins including SOD1G93A, had no effect on Ca2+ levels. Both expression of SOD1G93A and epoxomicin-induced inhibition of proteasome activity caused mitochondrial rounding, independent of Ca2+ dysregulation and reduced Δψ. That geldanamycin prevented inclusions and mitochondrial rounding, but not Ca2+ dysregulation or loss of Δψ indicates that chaperone-based therapies to prevent protein aggregation may require co-therapy to address these other underlying mechanisms of toxicity.

Knockout of Na+/Ca2+ exchanger in smooth muscle attenuates vasoconstriction and L-type Ca2+ channel current and lowers blood pressure

Mice with smooth muscle (SM)-specific knockout of Na(+)/Ca(2+) exchanger type-1 (NCX1(SM-/-)) and the NCX inhibitor, SEA0400, were used to study the physiological role of NCX1 in mouse mesenteric arteries. NCX1 protein expression was greatly reduced in arteries from NCX1(SM-/-) mice generated with Cre recombinase. Mean blood pressure (BP) was 6-10 mmHg lower in NCX1(SM-/-) mice than in wild-type (WT) controls. Vasoconstriction was studied in isolated, pressurized mesenteric small arteries from WT and NCX1(SM-/-) mice and in heterozygotes with a global null mutation (NCX1(Fx/-)). Reduced NCX1 activity was manifested by a marked attenuation of responses to low extracellular Na(+) concentration, nanomolar ouabain, and SEA0400. Myogenic tone (MT, 70 mmHg) was reduced by approximately 15% in NCX1(SM-/-) arteries and, to a similar extent, by SEA0400 in WT arteries. MT was normal in arteries from NCX1(Fx/-) mice, which had normal BP. Vasoconstrictions to phenylephrine and elevated extracellular K(+) concentration were significantly reduced in NCX1(SM-/-) arteries. Because a high extracellular K(+) concentration-induced vasoconstriction involves the activation of L-type voltage-gated Ca(2+) channels (LVGCs), we measured LVGC-mediated currents and Ca(2+) sparklets in isolated mesenteric artery myocytes. Both the currents and the sparklets were significantly reduced in NCX1(SM-/-) (vs. WT or NCX1(Fx/-)) myocytes, but the voltage-dependent inactivation of LVGCs was not augmented. An acute application of SEA0400 in WT myocytes had no effect on LVGC current. The LVGC agonist, Bay K 8644, eliminated the differences in LVGC currents and Ca(2+) sparklets between NCX1(SM-/-) and control myocytes, suggesting that LVGC expression was normal in NCX1(SM-/-) myocytes. Bay K 8644 did not, however, eliminate the difference in myogenic constriction between WT and NCX1(SM-/-) arteries. We conclude that, under physiological conditions, NCX1-mediated Ca(2+) entry contributes significantly to the maintenance of MT. In NCX1(SM-/-) mouse artery myocytes, the reduced Ca(2+) entry via NCX1 may lower cytosolic Ca(2+) concentration and thereby reduce MT and BP. The reduced LVGC activity may be the consequence of a low cytosolic Ca(2+) concentration.

Presynaptic Mitochondria in Functionally Different Motor Neurons Exhibit Similar Affinities for Ca2+But Exert Little Influence as Ca2+Buffers at Nerve Firing RatesIn Situ

Mitochondria accumulate within nerve terminals and support synaptic function, most notably through ATP production. They can also sequester Ca(2+) during nerve stimulation, but it is unknown whether this limits presynaptic Ca(2+) levels at physiological nerve firing rates. Similarly, it is unclear whether mitochondrial Ca(2+) sequestration differs between functionally different nerve terminals. We addressed these questions using a combination of synthetic and genetically encoded Ca(2+) indicators to examine cytosolic and mitochondrial Ca(2+) levels in presynaptic terminals of tonic (MN13-Ib) and phasic (MNSNb/d-Is) motor neurons in Drosophila, which, as we determined, fire during fictive locomotion at approximately 42 Hz and approximately 8 Hz, respectively. Mitochondrial Ca(2+) sequestration starts in both terminals at approximately 250 nM, exhibits a similar Ca(2+)-uptake affinity (approximately 410 nM), and does not require Ca(2+) release from the endoplasmic reticulum. Nonetheless, mitochondrial Ca(2+) uptake in type Is terminals is more responsive to low-frequency nerve stimulation and this is due to higher cytosolic Ca(2+) levels. Since type Ib terminals have a higher mitochondrial density than Is terminals, it seemed possible that greater mitochondrial Ca(2+) sequestration may be responsible for the lower cytosolic Ca(2+) levels in Ib terminals. However, genetic and pharmacological manipulations of mitochondrial Ca(2+) uptake did not significantly alter nerve-stimulated elevations in cytosolic Ca(2+) levels in either terminal type within physiologically relevant rates of stimulation. Our findings indicate that presynaptic mitochondria have a similar affinity for Ca(2+) in functionally different nerve terminals, but do not limit cytosolic Ca(2+) levels within the range of motor neuron firing rates in situ.