Canonical transient receptor potential 3 channels regulate mitochondrial uptake

Shengjie Fenga,b,1, Hongyu Lia,b,1, Yilin Taia,1, Junbo Huanga,b, Yujuan Sua,b, Joel Abramowitzc, Michael X. Zhud, Lutz Birnbaumerc,2, and Yizheng Wanga,2

aLaboratory of Neural Signal Transduction, Institute of Neuroscience, Shanghai Institutes of Biological Sciences, State Key Laboratory of Neuroscience, Shanghai 200031, China; bUniversity of Chinese Academy of Sciences, Shanghai 200031, China; cLaboratory of Neurobiology, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709; and dDepartment of Integrative Biology and Pharmacology, University of Texas Health Science Center, Houston, TX 77030

Contributed by Lutz Birnbaumer, May 22, 2013 (sent for review April 2, 2013)

+ Mitochondrial Ca2 homeostasis is fundamental to regulation of organelles, including three well-established mitochondria-tar- mitochondrial membrane potential, ATP production, and cellular geted [heat shock 60 (HSP60); voltage-de- Ca2+ homeostasis. It has been known for decades that isolated pendent anion-selective channel protein (VDAC); and mitochondria can take up Ca2+ from the extramitochondrial so- cytochrome C], an ER marker (calnexin), an endosome marker + lution, but the molecular identity of the Ca2 channels involved [early endosome autoantigen 1 (EEA1)], a lysosome marker in this action is largely unknown. Here, we show that a fraction [lysosome associated membrane glycoprotein 1 (Lamp1)], and of canonical transient receptor potential 3 (TRPC3) channels is a plasma membrane-targeted protein [transferrin receptor localized to mitochondria, a significant fraction of mitochondrial (TrR); Fig. 1B]. The specificity of the anti-TRPC3 antibodies was + + Ca2 uptake that relies on extramitochondrial Ca2 concentra- verified and are shown in Fig. S1 B–D. The immunoreactivity of tion is TRPC3-dependent, and the up- and down-regulation of the TRPC3 protein was absent in the mitochondrial fraction and − − TRPC3 expression in the cell influences the mitochondrial mem- the whole homogenates isolated from the cerebellum of Trpc3 / brane potential. Our findings suggest that TRPC3 channels mice (Fig. 1B). Further, TRPC3 immunoreactivity was also lost 2+ − − contribute to mitochondrial Ca uptake. We anticipate our in cerebellum neurons from Trpc3 / mice (Fig. 1C). The ex- −/− observations may provide insights into the mechanisms of mi- pression of other members of the TRPC family in Trpc3 and CELL BIOLOGY 2+ tochondrial Ca uptake and advance understanding of the WT mice was similar (Fig. S1 E and F). physiological role of TRPC3. To confirm the mitochondrial localization of the TRPC3 protein in intact cells, we examined the subcellular localization + itochondrial Ca2 uptake is critical for regulation of nu- of TRPC3 by immunocytochemistry in HeLa cells using the anti- Mmerous cellular processes, including energy metabolism TRPC3 antibody. As shown in Fig. 1D, the endogenous TRPC3 + and cytosolic Ca2 homeostasis. Mitochondria undergo rapid protein was found in both the plasma membrane and the mito- 2+ 2+ changes in matrix Ca concentration ([Ca ]mito) on cell stim- chondria. Double staining using the Mitotraker and the anti- ulation to affect aerobic metabolism and cell survival (1–4). TRPC3 antibody further revealed that in HeLa cells, about + Mitochondrial Ca2 buffering also shapes the amplitude and 44.4% of the total TRPC3 is localized to mitochondria. Similarly, 2+ 2+ spatiotemporal patterns of cytosolic Ca concentration ([Ca ]cyt) 84.9% of HSP60 is localized to mitochondria. In contrast, only changes (5, 6). Mitochondrial calcium uniporter (MCU) has 17.6% of the immunostaining for β-actin, a cytoskeletal protein, + been shown to affect mitochondrial Ca2 uptake (7, 8). is associated with mitochondria (Fig. 1D; Fig. S2A). The fraction + However, mitochondrial Ca2 uptake remains evident when of mitochondrial TRPC3 staining ranged from 28.4% to 56.4% MCU is down-regulated (7, 8), suggesting that other routes re- of the total TRPC3 labels in different cells, including human + sponsible for its Ca2 uptake might exist. Transient receptor embryonic kidney 293 (HEK 293) cells, Chang-liver cells, mouse fi potential (TRP) channels have emerged as important cellular embryonic broblast (MEF) cells, and hippocampal neurons B C sensors, and although many TRP channels are expressed on the (Fig. S2 and ). Mitochondrial fractionation and proteinase K plasma membrane, some members of the TRP channel proteins protection assay further showed that in rat liver cells, TRPC3 are also found in the intracellular organelles such as endoplasmic mainly localized to the inner membrane of mitochondria (IMM) E reticulum (ER), secretory vesicles, granules, endosomes, and (Fig. 1 ). We then performed immunoelectron microscopy lysosomes (9–14). Recently, a proteomics study showed that using the antibody against TRPC3 on mouse cerebella. Gold canonical TRP 3 (TRPC3) interacts with a large number of particles were found on the inner mitochondrial membrane of −/− F mitochondrial proteins (15); we thus studied whether the TRPC3 WT, but not Trpc3 , cerebella (Fig. 1 ). The number of gold fi −/− ± protein localizes to mitochondria and plays a role in maintaining particles per eld on IMM in WT or Trpc3 mice was 2.15 2+ 0.79 or 0.13 ± 0.46, respectively. Similarly, the number of gold mitochondrial Ca homeostasis. − − particles per field on all membrane structures in WT or Trpc3 / Results and Discussion mice was 4.77 ± 0.68 or 1.71 ± 0.44, respectively. With heter- ologous expression, although most of the C-terminal myc-tagged TRPC3 Is Also Localized to Mitochondria. First, we examined the myc presence of TRPC3 in purified mitochondria prepared from rat TRPC3 (TRPC3 ) was localized to the plasma membrane liver and brain using the Percoll density gradient centrifugation, a method well established to obtain high purity mitochondria (16). In both tissues, Western blotting showed that TRPC3, but Author contributions: S.F., H.L., Y.T., M.X.Z., L.B., and Y.W. designed research; S.F., H.L., Y.T., J.H., and Y.S. performed research; J.A. and L.B. contributed new reagents/analytic not other members of the TRPC subfamily, including TRPC4, tools; S.F., H.L., Y.T., and L.B. analyzed data; and S.F., H.L., Y.T., and Y.W. wrote the paper. TRPC5, and TRPC6, were enriched in the mitochondrial frac- The authors declare no conflict of interest. tion (Fig. 1A). We used four anti-TRPC3 antibodies from dif- 1 A S.F., H.L., and Y.T. contributed equally to this work. ferent sources (Fig. S1 ), including a monoclonal anti-TRPC3 2 To whom correspondence may be addressed. E-mail: [email protected] or antibody generated in our laboratory (Fig. 1B); all of them [email protected]. detected TRPC3 in the mitochondrial fraction. The purity of This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. mitochondria was confirmed by a series of markers for cell 1073/pnas.1309531110/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1309531110 PNAS Early Edition | 1of6 Downloaded by guest on October 3, 2021 results suggest that, in addition to the plasma membrane, TRPC3 is also localized to mitochondria.

TRPC3 Regulates Mitochondrial Ca2+ Homeostasis. We next tested + whether TRPC3 regulates mitochondrial Ca2 homeostasis. For the + loss-of-function experiments, we examined the mitochondrial Ca2 − − signals in MEF cells isolated from WT and Trpc3 / mice. For the gain-of-function experiments, we used HeLa cells that stably over- expressed human TRPC3 (HeLa-TRPC3). The MEF cells derived − − from Trpc3 / mice appeared normal, and their mitochondrial morphology was not different from that of the WT cells (Fig. S2F). The expression of mitochondrial fusion and fission proteins in − − Trpc3 / mice was not different from that in WT mice (Fig. S2G). + Mitochondrial Ca2 elevation was assessed with Pericam, a mito- + chondria-targeted Ca2 -sensitive fluorescent protein (17) (Fig. S2H), and Rhod 5N. Treatment of MEF cells from WT mice with + ATP elevated mitochondrial Ca2 concentration, and this elevation − − was greatly reduced in the MEF cells from Trpc3 / mice (Fig. 2A; + Fig. S3A). By contrast, histamine-induced mitochondrial Ca2 ele- vation in HeLa-TRPC3 was markedly enhanced (Fig. 2B; Fig. S3B). + The Ca2 influx through plasma membrane TRPC3 may + contribute to cytosolic Ca2 elevation and subsequent mito- + chondrial Ca2 uptake. To distinguish the effect of plasma membrane TRPC3 and mitochondrial TRPC3 on mitochondrial + Ca2 uptake, we stimulated MEF and HeLa cells in the absence + Fig. 1. Localization of TRPC3 in mitochondria. (A) TRPC3 was found in the of extracellular Ca2 . As shown in Fig. 2C and Fig. S3C, under homogenate (h), mitochondrial (m), or cytosolic (c) fractions isolated from 2+ rat liver (Left) and brain (Right). HSP60, VDAC protein, and cytochrome this condition, the mitochondrial Ca uptake remained evident (cyto) C served as mitochondrial markers, calnexin as an ER marker and β-actin as a loading control. Polyclonal anti-TRPC antibodies were used. (B) TRPC3 immunoreactivity detected by the monoclonal anti-TRPC3 antibody in homogenates (h), cytosolic (c), and mitochondrial (m) fractions of the cere- − − bellum from the WT but not Trpc3 / (KO) mice. HSP60, VDAC, and cyto C served as mitochondrial markers, calnexin as an ER marker, EEA1 as an endosome marker, Lamp1 as a lysosome marker, TrR as plasma membrane marker, and β-actin as a loading control. For Western blotting, all isolated fractions from WT and Trpc3−/− tissues were run on the same gel and blotted on the same membrane. (C) Immunocytochemical staining of cerebella from − − WT and Trpc3 / mice using the polyclonal anti-TRPC3 antibody (green; Alomone. Cat. No. ACC016, Lot No. AN0702) and monoclonal anti-MAP2 antibody (red). Hochest33258 was used to label nuclei (blue). (Scale bars, 20 μm.) (D) Immunofluorescent labeling of TRPC3 in mitochondria. Represen- tative images of HeLa cells stained with the polyclonal anti-TRPC3 antibody (green; from C. Montell) and loaded with 10 nM MitoTraker red (red). (Scale bars, 5 μm.) 40×,40× objective; 40× 3×,40× objective with 3× zoom. (Right) Summary of percent fluorescence intensity of actin, TRPC3, or HSP60 colo- calized with MitoTraker red over the total fluorescence intensity of the corresponding protein, n = 10 for actin and TRPC3, respectively, n = 3for HSP60. Representative images for actin and HSP60 are shown in Fig. S2A. (E)(Left) Immunobloting of TRPC3 in the intact mitochondria and mitoplast of rat liver cells. (Right) Immunobloting of TRPC3 in the swelled mitochon- dria incubated with or without proteinase K in 0.5% TritonX-100 or not. Bcl-2 served as an outer membrane marker, cyto C a mitochondrial in- termembrane space marker, PRODH a mitochondrial inner membrane marker, and Hsp60 a matrix marker. Cytosolic (c), mitochondrial (m), mito- plasts (mp), and proteinase K (Prot. K) (F) Electron microscopic images (Left) of immunogold labeling by the polyclonal anti-TRPC3 antibody (Alomone Cat. No. ACC016, Lot No. AN0702) of mitochondrial inner membranes (red − − / + + arrows) of the cerebellum from WT, but not Trpc3 (KO), mice. Labeling to Fig. 2. TRPC3 regulates mitochondrial Ca2 uptake. (A) Mitochondrial Ca2 other membrane structures, presumably plasma membrane, is indicated (red 2+ −/− dynamics ([Ca ]mito)inWTorTrpc3 MEF cells treated with 100 μM ATP in 0.3 + + arrowhead). (Scale bar, 200 nm.) Statistics of gold particles associated with mM extracellular Ca2 .[Ca2 ] was monitored by changes in Pericam fluo- fi mito mitochondrial inner membranes per eld (Center) and that associated with rescence. (B) Similar measurements in HeLa cells or HeLa cells stably transfected fi ± all membrane structures per eld (Right) are shown as means SEM of 50 with human TRPC3 (HeLa-TRPC3) treated with 1 μM histamine in 0.3 mM ex- fi + EM elds from three mice for each genotype. tracellular Ca2 .(C and D) Same experiments as A and B, but cells treated + with 200 μM ATP or 10 μM histamine in a Ca2 -free extracellular solution. (E) 2+ [Ca ]mito changes in digitonin-permeabilized WT and KO MEF cells challenged μ 2+ 2+ (Fig. S2D), a substantial level of the TRPC3myc proteins was with cytosolic loading of 300 MCa .(F)[Ca ]mito changes in digitonin- permeabilized HeLa and HeLa-TRPC3 cells challenged with cytosolic loading of also evident in the mitochondria. At similar total expression 50 μMCa2+.(B–F)(Left) Representative traces from single experiments. (Right) levels, the immunoreactivity of TRPC3myc associated with the 2+ Means ± SEM of peak [Ca ]mito changes normalized to that of control (WT, mitochondrial fraction in HeLa cells was 13.0 ± 3.9-fold higher MEF, or HeLa) of three independent experiments with the total number of MEF (n = 4) than that of TRPC4myc (Fig. S2E). Together, these cells, n = 30, control HeLa and HeLa-TRPC3 cells, n = 186. *P < 0.05, **P < 0.01.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1309531110 Feng et al. Downloaded by guest on October 3, 2021 − − + − − in WT MEF cells, whereas it was greatly suppressed in Trpc3 / mitochondrial Ca2 signals in the Trpc3 / MEF cells were − − MEF cells. Mirroring the results of Trpc3 / MEF cells, the substantially reduced compared with the WTs. The mitochon- + + mitochondrial Ca2 uptake in HeLa-TRPC3 showed a marked drial Ca2 signals in the HeLa-TRPC3 cells were significantly increase compared with control cells (Fig. 2D; Fig. S3D). To higher than those in the control cells (Fig. 2F; Fig. S3F). Further, further demonstrate the contribution of the mitochondrial in the HeLa-TRPC3 cells or the permeabilized HeLa-TRPC3 + TRPC3 to its Ca2 uptake, we permeabilized the plasma mem- cells, in which MCU was down-regulated by siRNA and in- + brane with digitonin (10 μM) and then applied Ca2 to the cubated with cyclosporine A to inhibit mPTP opening, the en- + + permeabilized cells. As shown in Fig. 2E and Fig. S3E, the hancement in mitochondrial Ca2 signals after histamine or Ca2

1.5μM Histamine +0.3mM Ca2+ 1.5μM Histamine +0.3mM Ca2+ 1.5μM Histamine +0.3mM Ca2+ HeLa A ## 1200 HeLa-TRPC3 1200 1200 2.5 ## ) 0 800 2.0 ##

/F 800 800 X 1.5 *

(1-F 400 X 400 400 1.0 F * * 0 HeLa-Scramble 0 HeLa-MCU RNAi-1 0 HeLa-MCU RNAi-2 0.5 HeLa-TRPC3-Scramble HeLa-TRPC3-MCU RNAi-1 HeLa-TRPC3-MCU RNAi-2

Relative Calcium Uptake 0 0 1.0 2.0 3.0 4.0 0 1.0 2.0 3.0 0 1.0 2.0 3.0 Sc RNAi-1 RNAi-2 Sc+CsA Time (min) Time (min) Time (min) B HeLa ## ## HeLa-TRPC3 15μM Histamine 15μM Histamine 15μM Histamine 800 800 800 * # 600 1.2 ) 600 600 0 /F X 400 400 400 1.0 CELL BIOLOGY (1-F X

F 200 200 * * 200 0.8 0 HeLa-Scramble HeLa-MCU RNAi-1 HeLa-MCU RNAi-2 HeLa-TRPC3-Scramble 0 HeLa-TRPC3-MCU RNAi-1 0 HeLa-TRPC3-MCU RNAi-2 Relative Calcium Uptake 0.6 0 1.0 2.0 3.0 0 1.0 2.0 3.0 0 1.0 2.0 3.0 Sc RNAi-1 RNAi-2 Sc+CsA Time (min) Time (min) Time (min)

HeLa ## C HeLa-TRPC3 75µM Ca2+ 75µM Ca2+ 75µM Ca2+ 1.6 ## ** # 1600 1200 1200

) 1.2 12000 /F

X 800 800 800 0.8 **

(1-F *** X 400 400 400 F 0.4 0 HeLa-Scramble HeLa-MCU RNAi-1 HeLa-MCU RNAi-2 HeLa-TRPC3-Scramble 0 HeLa-TRPC3-MCU RNAi-1 0 HeLa-TRPC3-MCU RNAi-2

Relative Calcium Uptake 0 0 1.0 2.0 3.0 4.0 5.0 0 1.0 2.0 3.0 4.0 5.0 0 1.0 2.0 3.0 4.0 5.0 Sc RNAi-1 RNAi-2 Sc+CsA Time (min) Time (min) Time (min)

D E TRPC3 KO MEF 2+ # TRPC3 KO MEF-TRPC3 1000 SC 0.3mM Ca 2.5 pcDNA3.1 MCU-myc i-1 800 SC+C3 ## i-1+C3 2.0

Ctrl SC i-1 i-2 ) 0 600 /F MCU-1 X 1.5 400 (1-F X 1.0 ¢-Tubulin F 200 0 0.5 *** Relative Calcium Uptake -200 0 0 1.0 2.0 3.0 SC i-1 Time (min)

2+ 2+ Fig. 3. In the absence of MCU, mitochondrial Ca uptake remains regulated by TRPC3. [Ca ]mito changes in HeLa cells or HeLa-TRPC3 cells transfected with + scrambled (sc) or indicated siRNAs in the absence or presence of 4 μM cyclosporine A (CsA) and evoked by 1.5 μM histamine in the presence of 300 μMCa2 (A), by 15 μM histamine in a Ca2+-free extracellular solution (B), or by 75 μMCa2+ in digitonin-permeabilized HeLa cells (C). (A–C)(Right) Means ± SEM of peak 2+ [Ca ]mito changes normalized to that of sc HeLa of four independent experiments with the total number of HeLa and HeLa-TRPC3 cells, n = 103. **P < 0.01, ***P < 0.001 vs. sc HeLa. #P < 0.05, ##P < 0.01. (D) Down-regulation of MCU by its RNAi-1 or -2 (i-1 or 2) in HEK 293 cells, in which MCU-myc was overexpressed. (E) 2+ −/− −/− [Ca ]mito changes in Trpc3 MEF cells or Trpc3 MEF-TRPC3 cells transfected with scramble (sc) or i-1, treated with 10 μM digitonin, and evoked by 300 μM 2+ 2+ −/− Ca .(Right) Means ± SEM of peak [Ca ]mito changes normalized to that of sc of four independent experiments with the total number of Trpc3 MEF and − − − − Trpc3 / MEF-TRPC3 cells, n = 69. ***P < 0.001, vs. sc Trpc3 / MEF. #P < 0.05, ##P < 0.01.

Feng et al. PNAS Early Edition | 3of6 Downloaded by guest on October 3, 2021 treatment remained evident (Fig. 3 A–C). Moreover, in the − − permeabilized Trpc3 / MEF cells, in which MCU was down- regulated (Fig. 3D), expressing TRPC3 greatly increased the + mitochondrial Ca2 uptake (Fig. 3E). Together, these results suggest that in addition to MCU, TRPC3 also contributes to + mitochondrial Ca2 uptake. As mitochondria play a critical role + in shaping cytosolic Ca2 dynamics (1), we examined agonist- + evoked changes in cytosolic Ca2 transient in the absence of + extracellular Ca2 . As shown in Fig. 4A, overexpression of TRPC3 in HeLa cells led to a substantial reduction in the cy- + + tosolic Ca2 transient evoked by histamine. By contrast, the Ca2 transient was enhanced in the cells that expressed the dominant-

Fig. 5. TRPC3 contributes to the mitochondrial membrane potential (ΔΨm). − − Representative images of WT or Trpc3 / MEF cells (A) and of control HeLa or HeLa-TRPC3 cells (B) loaded with JC-1 (Left). Red fluorescence indicates mi- tochondria with formation of J-aggregates at high transmembrane poten- tials; green fluorescence shows mitochondria with formation of JC-1 monomers at low transmembrane potentials. FCCP treatment (Rightmost) was used as a positive control. Statistics of the JC-1 ratio of red/green fluo- rescence intensity (Right). Data are means ± SEM of four independent − − experiments. Changes in TMRE fluorescence in WT and Trpc3 / MEF cells treated with 500 μMCa2+ (C) or in HeLa and HeLa-TRPC3 cells treated with 300 μMCa2+ (D). The fluorescence values were normalized to those of the baseline. The dotted lines represent the slope (K) of the curve before and + after Ca2 treatment. The K values of the baseline were calculated by the + linear fitting. After Ca2 treatment, the K values were calculated by fitting the normalized TMRE fluorescence during the first 20 s immediately fol- + lowing Ca2 addition with a linear function. FCCP was used as a positive + control. Shown are representative traces from single experiments (Left), Fig. 4. Reduction in extramitochondrial Ca2 by TRPC3 in HeLa cells or in − − + statistics of the changes in K values normalized to those of WT or HeLa cells isolated liver mitochondria from Trpc3 / mice. (A) Cytosolic Ca2 dynamics + (Center), and statistics of maximal TMRE fluorescence decreases induced by ([Ca2 ] ) in HeLa cells transfected with control vector (CTRL), WT-TRPC3, cyto 2+ ± μ Ca (Right). Data in bar graphs are means SEM of three independent and TRPC3 mutants (E630Q or E630K) treated with 10 M histamine in the −/− + experiments with 128 WT cells, 122 Trpc3 cells, 352 control HeLa cells, and absence of extracellular Ca2 were monitored by changes in Fura 2 fluo- 337 HeLa-TRPC3 cells. *P < 0.05, **P < 0.01. rescence. Representative traces from a single experiment (Left) and means ± 2+ SEM of peak [Ca ]cyto changes normalized to CTRL of four independent = = experiments (Right) with the total number: CTRL, n 58; WT-TRPC3, n 50; 2+ E630K, n = 58; E630Q, n = 27. *P < 0.05, **P < 0.01 vs. CTRL, #P < 0.001 vs. negative nonpermeant mutant (E630K) or the Ca -impermeable + WT-TRPC3. (B–D) Reduction of medium Ca2 by isolated liver mitochondria mutant of TRPC3 (E630Q) (18). Together, these results suggest − − + from WT and Trpc3 / mice. Purified liver mitochondria were incubated in that TRPC3 is indeed involved in mitochondrial Ca2 uptake. + + aCa2 -free buffer in the presence of Fluo-5N (1 μM). Ca2 was added at To further support the notion that mitochondrial TRPC3 a final concentration of 10 (B), 50 (C), or 100 μM(D) and changes in extra- 2+ 2+ channels contribute to mitochondrial Ca uptake, we performed mitochondrial [Ca ] monitored by Fluo-5N fluorescence for 65 s. Shown are 2+ fl Ca uptake experiments in isolated liver mitochondria. The representative traces of time courses of relative uorescence changes (Left) + + 2 and means ± SEM (n = 4) of overall Ca2 uptake during the entire experi- maximal quantity of Ca that can be sequestered by mitochon- 2+ mental period, normalized to that of WT. *P < 0.05 vs. WT. dria was determined by monitoring the changes of free Ca in

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1309531110 Feng et al. Downloaded by guest on October 3, 2021 extramitochondrial media using Fluo-5N (19). Liver mitochondria manner, TRPC3 likely plays an important role in mitochondrial + isolated from the WT littermates exhibited a higher ability to take Ca2 uptake in some physiological conditions with high extra- + − − + up Ca2 from the medium than that from Trpc3 / mice. More- mitochondrial Ca2 concentrations. Indeed, TRPC3 exhibits + + + + over, the TRPC3-dependent mitochondrial Ca2 uptake was de- a low permeability to Ca2 over Na . When cytoplasmic Ca2 is + + pendent on extramitochondrial Ca2 concentrations. With 10 μM low (<5 μM) and cytoplasmic Na is ∼10 mM, TRPC3 does + + + Ca2 added to isolated liver mitochondria, Ca2 sequestration not support much Ca2 flux into the matrix (28). When high, local −/− 2+ was similar between Trpc3 and WT preparations (Fig. 4B). Ca emanates from the InsP3 receptors (29), and TRPC3 + + + However, with 50 and 100 μMCa2 in the medium, Ca2 se- in mitochondria located within 20–200 nm of ER can uptake Ca2 . questration by mitochondria was substantially reduced for sam- A recent study demonstrated that, during IP3-mediated store re- − − + ples from Trpc3 / mice compared with the WT (Fig. 4 C and D). lease, [Ca2 ] at the ER-mitochondrial interface often exceeds 10 + These results suggest that TRPC3 is particularly relevant in mi- μM (30). Cytosolic Ca2 levels within the nanodomain of the hot + + tochondrial Ca2 uptake when extramitochondrial Ca2 concen- spot region are transiently expected to be high (>50 μM) (31). tration is relatively high (≥50 μM). Whether the mitochondrial TRPC3 channels are constitutively + open or modulated by extramitochondrial Ca2 concentration is + TRPC3 Channels Affect Mitochondrial Membrane Potential. A large unclear at present. The Ca2 uptake studies in isolated mito- negative membrane potential (ΔΨm) across the inner mito- + chondria suggest that TRPC3 channels in mitochondria can be chondrial membrane generated by H gradient and cations, such 2+ + + either directly activated by Ca , which could be similar to the as Ca2 and K (20), is important for mitochondrial functions. 2+ 2+ + + activation of Ca uniporter (32) or modulated by other Ca Mitochondrial depolarization induces Ca2 release from Ca2 sensing proteins. Although there is no evidence until now to show + + -loaded mitochondria (21, 22) and prevents mitochondrial Ca2 that Ca2 itself can gate the plasma membrane TRPC3, several 2+ ΔΨ uptake (5). In contrast, Ca uptake may collapse the m (23, TRP channels (TRPA1, TRPM4, and TRPM5) can be directly + 24). To test whether TRPC3 channels affect mitochondrial gated by Ca2 (33–35). Our results are consistent with previous ΔΨ ′ ’ membrane potential, we measured m using 5,5 ,6,6 -tetra- findings showing that plasma membrane TRPC3 channels are + chloro-1,1’,3,3′-tetrethyl benzimidalyl carbocyanine iodide (JC-1) stimulated in a Ca2 -dependent manner (36). Phosphitidylinositol- and tetramethylrhodamine ethyl ester (TMRE), known as sen- 4,5-bisphosphate (PIP2) and phospholipase C (PLC) are located in sitive probes of changes in the ΔΨm. As shown in Fig. 5 A and B mitochondria (37, 38). It is also possible that the activation of mi- ΔΨ −/− by JC-1 staining, the resting m in Trpc3 MEF cells was tochondrial TRPC3 involves the PIP2-PLC pathway. Furthermore,

increased compared with that in WT MEF cells. Consistently, investigations on targeting of TRPC3 channels to mitochondria CELL BIOLOGY HeLa-TRPC3 cell mitochondria showed more depolarized ΔΨm and the activation mechanisms of mitochondrial TRPC3 channels than those of control cells. Then, we measured ΔΨm by TMRE in fi + will expand our understanding on the membrane traf cking response to Ca2 stimulation. As shown in Fig. 5C, the reduction mechanisms of plasma proteins and their ΔΨ −/− 2+ in m in Trpc3 MEF cells, in response to Ca stimulation, specific roles in different intracellular localizations in physio- was slower and to a lesser extent than that in WT MEF cells. The logical and pathological conditions. ΔΨm of HeLa-TRPC3 mitochondria decreased faster and to a greater extent than that of control cells (Fig. 5D). Together, Materials and Methods these results suggest that TRPC3 channels play an important More extensive descriptions can be found in SI Materials and Methods. role in regulating the mitochondrial membrane potential in re- + sponse to changes in extramitochondrial Ca2 levels. Isolation of Mitochondria. Mitochondria were isolated from adult rat or The results presented here reveal TRPC3 channels as mouse liver and brain, respectively, by Percoll density gradient centrifugation + a unique mitochondrial Ca2 uptake pathway. A fraction of the following the method described previously (16). For details, see SI Materials TRPC3 protein is localized to mitochondria and mediates mi- and Methods. 2+ 2+ tochondrial Ca uptake when extramitochondrial Ca con- + Measurement of Mitochondrial and Intracellular Ca2 Changes. Mitochondrial centrations are relatively high. TRPC3 channels appear to be Ca2+ uptake was measured with Pericam (gift from Atsudhi Miyawaki, Brain important in regulating the mitochondrial membrane potential. Science Institute, RIKEN, Wako City, Japan) or Rhod 5N-AM. Cytosolic Ca2+ + It has been reported that the MCU regulates mitochondrial changes were measured with fura-2. For details Ca2 imaging experiments, 2+ Ca uptake (7). However, silencing MCU did not abolish the see SI Materials and Methods. + mitochondrial Ca2 uptake (7), indicating that other routes are 2+ + + also responsible for its Ca uptake. We found that application Ca2 Uptake of Isolated Liver Mitochondria. Extramitochondrial Ca2 re- of , known as a putative MCU inhibitor, to the duction was measured with Fluo-5N according to the method described − − isolated liver mitochondria derived from the WT and Trpc3 / previously (19). See SI Materials and Methods for details. + mice blocked all of the mitochondrial Ca2 uptake in both − − WT and Trpc3 / mitochondria. Indeed, Ruthenium red has Statistical Analysis. All data were expressed as the means ± SEM. Data been shown to inhibit TRP channels (25). The lost portion of were analyzed using the SPSS 11.5 software. The means between two 2+ groups were analyzed by either paired or unpaired t tests. ANOVA followed the Ca uptake by TRPC3 was Ruthenium Red sensitive, by a post hoc least significant difference test was performed for statistical which suggested that TRPC3 might be another conduit besides fi < + comparison of several groups. Signi cance was taken at P 0.05. MCU. The MCU regulates mitochondrial Ca2 uptake with fi 2+ μ 2+ ahighafnity to Ca (26), and 2 M[Ca ] could initiate ACKNOWLEDGMENTS. We thank C. Montell and W. P. Schilling for TRPC3 2+ 2+ + MCU Ca uptake. Letm1, a mitochondrial Ca /H anti- antibodies, A. Miyawaki for Pericam, V. Flockerzi for comments, and Q. Hu + porter, contributes to mitochondrial Ca2 influx when extra- and Z. J. Fan for technical assistance. This work was supported in part by 2+ < μ a grant (81130081) from National Neural Science Foundation of China mitochondrial Ca concentrations are relatively low ( 1 M) (NNSF), the 973 program (2011CBA00400), the Intramural Research Program (27). Therefore, although TRPC3, MCU, and Letm1 affect of the National Institutes of Health (NIH; Z01-ES-101684; to L.B.), and NIH 2+ 2+ mitochondrial Ca uptake in a Ca concentration-dependent Grant R01 GM081658 (to M.X.Z.).

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