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Neuroscience Letters 575 (2014) 7–12

Contents lists available at ScienceDirect

Neuroscience Letters

jo ur nal ho me page: www.elsev ier .com/ locate /neule t

olecular and functional identification of a mitochondrial ryanodineeceptor in neurons

egina Jakoba,1, Gisela Beutnera,1, Virendra K. Sharmaa, Yuntao Duana, Robert A. Grossa,b,tephen Hurstc, Bong Sook Jhunc, Jin O-Uchic,∗,2, Shey-Shing Sheua,c,∗,2

Department of Pharmacology and Physiology, University of Rochester, School of Medicine and Dentistry, Rochester, NY 14642, United StatesDepartment of Neurology, University of Rochester, School of Medicine and Dentistry, Rochester, NY 14642, United StatesCenter for Translational Medicine, Department of Medicine, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA 19107, United States

i g h l i g h t s

Dantrolene and ryanodine block mitochondrial Ca2+ uptake in striated neurons.Ryanodine receptor (RyR) is expressed in the inner mitochondrial membrane in neurons.Brain mitochondria bind [3H]ryanodine both in Ca2+- and caffeine-sensitive manner.Mitochondrial RyR takes part in the mitochondrial Ca2+ influx mechanism in neurons.

r t i c l e i n f o

rticle history:eceived 13 February 2014eceived in revised form 23 April 2014ccepted 13 May 2014vailable online 23 May 2014

eywords:triatal neuronsalciumyanodine receptor

3H]ryanodine bindingitochondria

a b s t r a c t

Mitochondrial Ca2+ controls numerous cell functions, such as energy metabolism, reactive oxygen speciesgeneration, spatiotemporal dynamics of Ca2+ signaling, cell growth and death in various cell types includ-ing neurons. Mitochondrial Ca2+ accumulation is mainly mediated by the mitochondrial Ca2+ uniporter(MCU), but recent reports also indicate that mitochondrial Ca2+-influx mechanisms are regulated notonly by MCU, but also by multiple channels/transporters. We previously reported that ryanodine receptor(RyR), which is a one of the main Ca2+-release channels at endoplasmic/sarcoplasmic reticulum (SR/ER) inexcitable cells, is expressed at the mitochondrial inner membrane (IMM) and serves as a part of the Ca2+

uptake mechanism in cardiomyocytes. Although RyR is also expressed in neuronal cells and works as aCa2+-release channel at ER, it has not been well investigated whether neuronal mitochondria possess RyRand, if so, whether this mitochondrial RyR has physiological functions in neuronal cells. Here we showthat neuronal mitochondria express RyR at IMM and accumulate Ca2+ through this channel in response to

2+

antrolene cytosolic Ca elevation, which is similar to what we observed in another excitable cell-type, cardiomy-ocytes. In addition, the RyR blockers dantrolene or ryanodine significantly inhibits mitochondrial Ca2+

uptake in permeabilized striatal neurons. Taken together, we identify RyR as an additional mitochondrialCa2+ uptake mechanism in response to the elevation of [Ca2+]c in neurons, suggesting that this channelmay play a critical role in mitochondrial Ca2+-mediated functions such as energy metabolism.

© 2014 Elsevier Ireland Ltd. All rights reserved.

Abbreviations: OMM, outer mitochondrial membrane; CS, contact sites; IMM,

arco/endoplasmic Ca2+-ATPase; VDAC, voltage-dependent anion channel; ANT, adeninerial Ca2+ concentration; RyR, ryanodine receptor; MTR, Mitotracker deep Red; Bodyipyodipy-conjugated thapsigargin.∗ Corresponding authors at: Center for Translational Medicine, Department of Mediuite543G/543D, Philadelphia, PA 19107, United States. Tel.: +1 215 503 1567/+1 215 503

E-mail addresses: jin.ouchi@jefferson.edu (J. O-Uchi), shey-shing.sheu@jefferson.edu (1 Both authors contributed equally.2 Both authors are co-corresponding authors.

ttp://dx.doi.org/10.1016/j.neulet.2014.05.026304-3940/© 2014 Elsevier Ireland Ltd. All rights reserved.

inner mitochondrial membrane; ER/SR, endo/sarcoplasmic reticulum; SERCA,-nucleotide translocase; [Ca2+]c , cytosolic Ca2+ concentration; [Ca2+]m , mitochon-, boron-dipyrromethene; Bodipy-Ry, Bodipy-conjugated ryanodine; Bodipy-Thap,

cine, Jefferson Medical College, Thomas Jefferson University, 1020 Locust Street, 5152; fax: +1 215 503 5731/+1 215 955 1690.S.-S. Sheu).

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R. Jakob et al. / Neurosci

. Introduction

Mitochondria play an important role in shaping the intracel-ular Ca2+ concentration as they can take up Ca2+ in response tohysiological changes in the cytosolic Ca2+ concentration ([Ca2+]c)

n various cell-types/tissues including neurons [7,10,24]. Mito-hondrial Ca2+ accumulation was first recognized as an importantechanism for the acceleration of oxidative phosphorylation and

lectron transport chain activity, which results in the stimula-ion of ATP synthesis [12]. In addition, mitochondrial dysfunctionnd the loss of cellular Ca2+ homeostasis are frequently observedogether in pathophysiological conditions such as neuronal excito-oxicity, apoptosis and neurodegenerative disorders [8]. However,he detailed mechanisms of how altered mitochondrial Ca2+ hand-ing and/or mitochondrial dysfunction affect these neurologicalathogenesis are not yet fully understood.

Mitochondrial Ca2+ influx was originally considered as a singleransport mechanism through mitochondrial Ca2+ uniporter (MCU)hich can be inhibited by ruthenium red and lanthanides (see

eviews [7,24]). However, the molecular identities responsible foritochondrial Ca2+ accumulation have remained an unsolved ques-

ion until very recently. Recently, several groups have discoveredhe molecular identity of MCU and its regulatory proteins and con-rmed it as the main mitochondrial Ca2+ uptake mechanism (seeeviews [19,24]). Although in these studies MCU was confirmed ashe most dominant Ca2+ influx mechanism, previous studies havedentified additional Ca2+ uptake pathways, which display differ-nt physiological and pharmacological characteristics from MCUheory (see reviews [7,24]).

Among these studies, we reported that ryanodine receptor (RyR)s one of the mitochondrial Ca2+ influx mechanisms in anotherxcitable cell-type, cardiomyocytes, termed mRyR (mitochondrialyR) [2,3] (see also reviews [24,25]). Our group first identified that a

ow level of RyR is expressed in the mitochondrial inner membraneIMM) in cardiomyocytes through a combination of biochemical,ell biological and electrophysiological experiments. Since cardiacRyR exhibits a bell-shaped Ca2+-dependent activation (bimodal

ctivation) in the physiological range of [Ca2+]c, this unique prop-rty places mRyR as an ideal candidate for sequestering Ca2+ quicklynd transiently during physiological [Ca2+]c oscillation in excitableells. In addition, using not only native cardiomyocytes, but alsoyR overexpression/knock-out in cultured cardiac myoblasts andnock-out mouse hearts, we showed that the molecular identityf mRyR is possibly a skeletal-muscle type-isoform RyR type 1RyR1) and is required for Ca2+-dependent acceleration of ATP pro-uction in cardiomyocytes even though the expression level isuch lower than RyR2 which is the main RyR isoform expressed

n cardiac sarcoplasmic reticulum (SR)/endoplasmic reticulum (ER)3,23].

Although RyR is expressed [9,11,31] in the brain and serves as aa2+-release channel of the intracellular Ca2+ store (ER) in additiono inositol 1,4,5-trisphosphate (IP3) receptors [15,17,18], the inter-ction of between the RyR expression and mitochondrial functionsnder physiological and pathophysiological conditions in brain haseen completely unknown. Therefore, the objective of this studyas to investigate the possibility whether neuronal mitochondriaossess mRyR similar to cardiomyocytes and to assess if mRyRakes part in the mitochondrial Ca2+ influx mechanism. Our find-ng suggests that RyR is expressed at IMM and takes up Ca2+ into

itochondria in response to [Ca2+]c elevations.

. Materials and methods

An expanded Section 2 is available in the online Supplementaryle.

etters 575 (2014) 7–12

2.1. Reagents and antibodies

All reagents and antibodies were purchased from Sigma-AldrichCorporation (St. Louis, MO) unless otherwise indicated.

2.2. Cells

All procedures involving animal use were in accordance withthe NIH Guide for the Care and Use of Laboratory Animals, and wereapproved by the University Committee on Animal Resources. Pri-mary striatal neurons were prepared from Sprague-Dawley ratembryos (embryonic day 18) [1].

2.3. Preparation of rat brain mitochondria and mitochondrialsubfractions

Mitochondria-enriched protein fractions and mitochondrialsubfractions were prepared using differential centrifugation [3,23].

2.4. Fluorescence microscopy

Localization of RyR was observed in live or fixed cultured striatalneurons using confocal microscopy (Leica, Heidelberg Germany).For live cells, RyR, SR/ER and mitochondria were labeled withboron-dipyrromethene (Bodipy)-conjugated ryanodine (Bodipy-Ry), Bodipy-conjugated sarco/endoplasmic Ca2+-ATPase (SERCA)inhibitor thapsigargin (Bodipy-Thap) and Mitotracker Deep Red(MTR) (Molecular Probes, Eugene, OR), respectively. The pre-treatment of unlabeled ryanodine completely abolished theBodipy-Ry staining, confirmed that Bodipy-Ry specifically bindsto RyRs in live striatal neurons (Supplementary Fig. 1). For time-lapse studies, live cell images were collected by TILL system(TILL Photonics, München, Germany). For detection of RyR infixed striatal neurons, cells were probed with primary antibod-ies against RyR (Santa Cruz biotechnology, Santa Cruz, CA) andcytochrome c oxidase (Cox) (for labeling mitochondria) (Molec-ular Probes) followed by fluorescence-conjugated secondaryantibodies. Scatter 2D plots of pixel intensities and Pearson’scorrelation coefficient were obtained by ImageJ software (NIH)[23].

2.5. Measurements of cytosolic and mitochondrial Ca2+

concentration

[Ca2+]c in permeabilized striatal neurons was measured byCa2+ indicator Fura-2 with a fluorescent microscope (TILL Pho-tonics) [26]. For mitochondrial Ca2+ concentration ([Ca2+]m)measurements, cells were skinned by saponin and then stainedby an acetoxymethyl (AM) ester form of Fura-2 or Rhod-2[26]. Fura-2 calibration was performed as previously reported[1].

2.6. [3H]ryanodine binding

[3H]ryanodine binding assays were performed as we previouslydescribed [3].

2.7. Data and statistical analysis

All results are shown as mean standard error or otherwiseindicated. Unpaired Student’s t-test was performed. Statistical sig-nificance was set as a p value of <0.05.

R. Jakob et al. / Neuroscience L

Fig. 1. Dantrolene inhibits mitochondrial Ca2+ uptake induced by IP3-mediated Ca2+

release from the ER. (A) Representative time course of the changes in [Ca2+]c inresponse to 10 �M IP3 treatment in saponin-permeabilized striatal neurons. First,permeabilized cells were exposed to IP3 to evoke first [Ca2+]c elevation. Then IP3

was washed out and the cells were treated with 10 �M dantrolene (Dan) for 10 min,fin

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ollowed by a second exposure to IP3. (B) Representative time course of the changesn [Ca2+]m in response to 10 �M IP3 treatment in saponin-permeabilized striataleurons under the same protocols in (A).

. Results

.1. Dantrolene and ryanodine block mitochondrial Ca2+ uptaken striated neurons

To test whether RyR is involved in the mitochondrial Ca2+ uptakeechanism in neurons, the changes in [Ca2+]m in response to

Ca2+]c elevation were measured in permeabilized neurons in theresence and absence of a RyR blocker, dantrolene using Fura-

[3]. First, we stimulated the cells with IP3 and mobilized IP3eceptor-based SR Ca2+ release. Because RyRs were expressed atR [3,11,21], this protocol is enable to match the magnitude ofytosolic Ca2+ transient in the presence and absence of dantro-ene [23]. We confirmed that the application of 10 �M IP3 induceda2+ release from intracellular stores, resulted in an increase ofhe [Ca2+]c (from 108 ± 11.4 to 550 ± 47.3 nM) (Fig. 1A). We alsoonfirmed that magnitude of Ca2+ release from ER by IP3 treat-ent did not changed significantly changed in the presence or in

he absence of dantrolene (490 ± 51.2 vs 550 ± 47.3 nM, p = 1.00).nder this experimental condition, we next observed the changes

n [Ca2+]m in response to IP3 treatment (Fig. 1B). We confirmedhat the application of IP3 quickly increased [Ca2+]m (from 110 ± 0.6o 700 ± 59.6 nM), but 10-min pretreatment of dantrolene signifi-antly inhibited IP3-induced increase in [Ca2+]m. In addition, theP3-induced increase in [Ca2+]m (from 90 ± 7.8 to 250 ± 19.6 nM)artially recovered after washing out dantrolene, suggesting thathis inhibitory effect by dantrolene is reversible. We also observedhat the treatment of another RyR blocker, ryanodine, also sig-ificantly inhibited mitochondrial Ca2+ uptake in response to thepplication of Ca2+ into the extracellular solution (Supplementaryig. 2). These results indicate that in neurons, RyR is involved in theitochondrial Ca2+ uptake mechanism.

.2. RyR is expressed in the inner mitochondrial membrane ineurons

We next tested whether RyR is expressed in mitochondriai.e. mRyR) using biochemical approaches. Using specific anti-ody against RyR, we confirmed that RyR was detectable initochondria-enriched protein fractionation obtained from whole

rain (Fig. 2A and B). Because RyR is mainly expressed in SR/ER,

he purity of our mitochondria-enriched protein fractionation wasvaluated by detection of voltage-dependent anion channel (VDAC)nd calnexin by Western blotting as mitochondrial and ER/SRarkers, respectively [3]. The mitochondria-enriched protein

etters 575 (2014) 7–12 9

fractionation obtained from whole brain showed that RyR is foundin both in cytosolic (including SR)- and mitochondria-enrichedprotein fraction (Fig. 1B). To determine the submitochondrial local-ization of RyR in brain mitochondria, the IMM-enriched proteinswere separated by osmotic shock from the outer mitochondrialmembrane (OMM) and the contact site (CS) fractions. Separationof OMM-, CS-, and IMM-enriched fractions was confirmed by thedetection of the levels of marker proteins for IMM and OMM,adenine-nucleotide translocator (ANT) and VDAC, respectively(Fig. 2B). RyR was detectable mainly from IMM, which is similar tothe results we reported in cardiomyocytes [3].

Expression of RyR in mitochondria was further confirmedin cultured live striatal neurons. First, we labeled RyR withfluorescence-dye (Bodipy) conjugated ryanodine (Bodipy-Ry) inlive cultured striatal neurons (Fig. 2C). Bodipy-Ry showed a mesh-like staining pattern with punctuate staining region. We also foundthat punctuate staining regions of Bodipy-Ry were colocalizedwith MTR staining, suggesting that RyR is partially expressed inmitochondria. Time-lapse imaging showed that these spots moved,which further verified that this punctuate structures were indeedmitochondria (Supplementary Fig. 3). For comparison, we alsostained the neuron cells with Bodipy-Thap for labeling ER. TheBodipy-Thap signals showed almost no colocalization with MTR(Fig. 2C). Scatter 2D plots of pixel intensities in red (MTR) and green(Bodipys) channels indicated that the cells stained by Bodipy-Rypossessed higher amount of co-localized pixels (yellow in segment3 in Fig. 2C) compared to those observed in the cells stained byBodipy-Thap. We next quantitatively assessed an efficiency of themitochondrial colocalization by calculating the values of Pearson’scorrelation [23] (Fig. 2C). The Pearson’s value for Bodipy-Ry-MTRstaining was higher than that in Bodipy-Thap-MTR staining (0.716vs 0.322 in Fig. 2C), suggesting that RyR was not only localized atER, but also partially localized at mitochondria. In contrast to neu-rons, no colocalization of the Bodipy-Ry and MTR was observed inglia cells (Supplementary Fig. 4) (Pearson’s value = 0.276). In addi-tion, red-range Bodipy-Ry (Bodipy-TR-Ry) was almost colocalizedwith Bodipy-Thap, suggesting that RyR is expressed in ER, but notin mitochondria in glia cells (Pearson’s value = 0.828).

Expression of RyR in mitochondria was further confirmedby immunocytochemistry using the same RyR-specific anti-body used in Western blotting (Fig. 2D, see also Fig. 2Aand B). Confocal analysis revealed a mesh-like staining pat-tern with punctuate staining region for RyR. Double labelingagainst Cox (an IMM protein) confirmed that RyR partially local-ized at mitochondrial localization (see fluorescence profiles inFig. 2D).

3.3. Characterization of mRyR in brain mitochondria

Finally, we further characterized the neuronal mRyR using[3H]ryanodine binding assays (Fig. 3). [3H]ryanodine bound withhigh affinity to isolated brain mitochondria in [3H]ryanodine-concentration dependent manner (Fig. 3A). The apparent affinity(Kd) of [3H]ryanodine binding was 8.5 nM in the presence of20 �M Ca2+ and the maximal number of binding sites (Bmax) was103 fmol/mg protein obtained by Scatchard plot (Fig. 3B). We nextinvestigated modulation of [3H]ryanodine binding by Ca2+ and caf-feine, which are well known modulators of ryanodine binding toRyR of ER/SR [3]. Indeed, brain mitochondria showed both Ca2+-and caffeine-sensitive manner in [3H]ryanodine binding (Fig. 3C).We found a bell-shaped curve for Ca2+ dependence with maximal[3H]ryanodine binding occurring at pCa 5.2. In addition, application

of caffeine increased [3H]ryanodine binding (Fig. 3C). This increasein [3H]ryanodine binding by caffeine was also Ca2+-dependent.Maximal increase (∼=50%) in [3H]ryanodine binding was obtainedat pCa 6.

10 R. Jakob et al. / Neuroscience Letters 575 (2014) 7–12

Fig. 2. RyR is expressed in neuronal mitochondria. (A) Representative Western blots of mitochondria-enriched fractions from rat liver and brain (from three different prepa-rations). (B). Representative Western blots of mitochondria-enriched fraction and mitochondrial subfractions from rat brain. Cytosolic/ER-protein enriched fraction from ratbrain and mitochondria-enriched fraction from rat heart were shown as a comparison. (C) Labeling of RyR or SERCA in live striatal neurons using Bodipy-Ry or Bodipy-Thap,respectively. Images were taken from the areas of neurites. Top: neurons were also labeled with MTR (red emission) to visualize the localizations of mitochondria. Color scatterplottings obtained from the representative pictures at left were shown in right. Region 1 and 2 pixels represent signal in channel 1 (green) or 2 (red) only, respectively, andregion 3 represents co-localized pixels. Scale bars, 5 �m. (D) Immunocytochemical detection of RyR. Images were taken from the areas of neurites. The fluorescence profilef cationt n of th

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rom a dot square region at each image is shown at the bottom. Arrows show the lohe references to color in this figure legend, the reader is referred to the web versio

. Discussion

In the present study, we report the molecular and func-ional identification of mRyR in neuron. First, we showed inultured primary cells that neuronal mitochondrial Ca2+ accumu-ation is sensitive to both ryanodine and dantrolene (Fig. 1 and

of peak fluorescence. Scale bars, 20 �m. A.U., arbitrary unit. (For interpretation ofis article.)

Supplementary Fig. 2). Second, using brain mitochondria andcultured primary cells, we found that RyR is not only expressed

in ER, but also in mitochondria possibly at IMM (Fig. 2). Third,we characterized neuronal mRyR and found that the propertiesof ryanodine binding are similar to those we observed in cardiacmRyR [3] (Fig. 3). These results indicate that, in addition to MCU,

R. Jakob et al. / Neuroscience Letters 575 (2014) 7–12 11

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ig. 3. [3H]ryanodine binding assay in isolated brain mitochondria. (A) [3H]ryanodin3H]ryanodine binding. (C) [3H]ryanodine binding in various free Ca2+ concentrationompared to the control in each pCa.

yR is involved in the mitochondrial Ca2+ influx mechanism ineurons, similar to our previous observation in another excitableell-type, cardiomyocytes [3].

Our finding that neurons express mRyR adds a new compo-ent to the complexity of mitochondrial Ca2+ uptake in neuron.he prevailing view is that the ruthenium red-inhibitive MCU isxclusively responsible for mitochondrial Ca2+ influx (see review19]). However, the growing evidence also advances the idea that

ultiple Ca2+ influx mechanisms coexist, including our findings ofRyR [24,25]. Indeed, we showed that ryanodine or dantrolene

reatment significantly inhibited the mitochondrial Ca2+ accumu-ation in neuron, but did not completely abolish, suggesting thatther ion channel/transporters are engaged in mitochondrial Ca2+

echanism and mRyR serves as a part of this mechanism (Fig. 1nd Supplementary Fig. 2). Elucidation of the unique biophysi-al characteristics and physiological functions for each Ca2+ influxechanism is significant for our understanding of mitochondrial

a2+ signaling in regulating physiology and pathophysiology inxcitable cells including neurons. Recent studies revealed thatantrolene treatment is significantly protective from cytotoxicity

n neurons and/or the progression of neurodegenerative disorders5,14,22], suggesting the potential importance of RyR in neu-onal cell death signaling under the pathophysiological conditions.uture studies will address our next hypothesis: mRyR in neurons isnvolved in the regulation of ATP production through Ca2+ accumu-ation, as we previously showed in the heart, [3,23] and how mRyRxpression levels and functions might be altered under pathophy-iological conditions.

In brain, all three RyR subtypes (RyR1, 2 and 3) are expressed9,11,27,31]. However, in our biochemical and cell biological stud-es (Figs. 2 and 3), we used RyR-isoform non-specific antibody andodipy-Ry to identify mRyR in neuronal cells, which could not

s to isolated mitochondria from rat brain with high affinity. (B) Scatchard plot of presence (gray circles) or in the absence (black squares) of 20 mM caffeine. p < 0.05,

distinguish which isoform(s) is (are) expressed in mitochondria.We previously reported that RyR2 is expressed at SR and RyR1can be expressed only in mitochondria, but not in SR in cardiaccells, suggesting that the molecular identity of mRyR in cardiomy-ocytes is RyR1 [3,23]. RyR2 is not only expressed in SR/ER, butalso at the plasma membrane [20] or in the Golgi complex [6],which supports our idea of RyR isoform-dependent subcellular dis-tribution/retention. Thus, it is a reasonable idea that multiple RyRsubtypes are expressed in a single neuronal cell and each isoformhas a different subcellular localization as shown in cardiomyocytes.Further studies will be required to precisely identify the isoform(s)of mRyR in neuron (whether neuronal mRyR is RyR1 or not) usinggenetic knockdown or overexpression as we performed in cardiaccells [3,23].

In addition, RyR subtypes show different spatial and temporalexpression patterns among various brain regions [9,11,27,31]. Themain RyR isoform is RyR2 [15,17,18], which is evenly expressedthroughout the brain at relatively high levels. RyR3 is also ubiq-uitously expressed in the brain at lower levels compared to RyR2[9,11,31] and serves as a part of the Ca2+-release mechanism inER [21,29]. Interestingly, skeletal-muscle type isoform RyR1 isexpressed in the brain [11,31], particularly at the cerebellum, cor-tex, and hippocampus [16,30], but the physiological relevance ofthe uneven distribution pattern of RyR1 is completely unknown.The brain is an organ with a high metabolic demand and energyavailability determines the power and speed of the neuronal trans-mission system. Since the cerebellum, cortex, and hippocampushave a high number of synapses [28] and use ATP to acceler-

ate synaptic transmission, act as a neurotransmitter [4], generateaction potential, and maintain resting potentials [13]: it is a feasibleidea that the neuronal cells in these regions possess an additionalmolecular mechanism to enhance the efficiency of Ca2+-dependent

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TP production in mitochondria (mRyR), similar to cardiomy-cytes. Further studies will address the different expression levelsf mRyR between various brain regions and also will clarify whetherhe mRyR expression pattern in the brain has a correlation to the

itochondrial bioenergetics and cellular energy demands in theentral nervous system.

. Conclusion

Our studies show the molecular and functional identificationf mRyR in neuronal mitochondria. Our results suggest that mRyRay function to sequester Ca2+ to mitochondria in response to the

levation of [Ca2+]c in neurons.

cknowledgments

The authors thank Mr. Mark Gallagher for culturing thetriatal neurons. This work was supported by NIH grants (RO1HL-33333, RO1HL-093671, NS37710, and R21HL-110371 to S.-S.S.nd 5T32AA007463-26 to S.H.) and AHA grants (0335425T to Y.D.nd 14BGIA18830032 to J.O.-U.).

ppendix A. Supplementary data

Supplementary data associated with this article can beound, in the online version, at http://dx.doi.org/10.1016/j.neulet.014.05.026.

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