Canonical transient receptor potential 3 channels regulate mitochondrial calcium 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 proteins [heat shock protein 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.