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Subunit Composition of Mammalian Transient Receptor Potential Channels in Living Cells

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Subunit Composition of Mammalian Transient Receptor Potential Channels in Living Cells Subunit composition of mammalian transient receptor potential channels in living cells Thomas Hofmann*, Michael Schaefer†,Gu¨ nter Schultz†, and Thomas Gudermann*‡ *Institut fu¨r Pharmakologie und Toxikologie, Philipps-Universita¨t Marburg, Karl-von-Frisch-Strasse 1, D-35033 Marburg, Germany; and †Institut fu¨r Pharmakologie, Freie Universita¨t Berlin, Thielallee 67-73, D-14195 Berlin, Germany Edited by John H. Exton, Vanderbilt University School of Medicine, Nashville, TN, and approved March 21, 2002 (received for review November 7, 2001) Hormones, neurotransmitters, and growth factors give rise to the case of the vanilloid receptor (20), the crucial issue as to calcium entry via receptor-activated cation channels that are acti- which TRPC proteins can form multimeric ion channels remains vated downstream of phospholipase C activity. Members of the elusive. Considering that many TRPCs are ubiquitously ex- transient receptor potential channel (TRPC) family have been pressed and often coexpressed in a given cell (9), heteromul- characterized as molecular substrates mediating receptor- timerization in addition to homomultimerization among mem- activated cation influx. TRPC channels are assumed to be composed bers of this protein family represents an enticing possibility. A of multiple TRPC proteins. However, the cellular principles gov- deeper understanding of the selectivity of how TRPC subunits erning the assembly of TRPC proteins into homo- or heteromeric combine to form functional ion channel complexes is an essential ion channels still remain elusive. By pursuing four independent prerequisite to evaluate the contribution of a given TRPC experimental approaches—i.e., subcellular cotrafficking of TRPC channel to endogenous PLC-dependent cation currents. subunits, differential functional suppression by dominant-nega- Here we investigate the basic principles of TRPC channel tive subunits, fluorescence resonance energy transfer between homo- and heteromultimerization in living cells by means of four labeled TRPC subunits, and coimmunoprecipitation—we investi- independent cell biological and biophysical approaches. First, gate the combinatorial rules of TRPC assembly. Our data show that cellular trafficking-incompetent TRPC subunits are noted to be (i) TRPC2 does not interact with any known TRPC protein and (ii) translocated to the plasma membrane only when distinct addi- TRPC1 has the ability to form channel complexes together with tional TRPCs were coexpressed. Second, we find that a domi- TRPC4 and TRPC5. (iii) All other TRPCs exclusively assemble nant-negative mutant of TRPC6 selectively suppresses TRPC3 into homo- or heterotetramers within the confines of TRPC and TRPC6 but not TRPC4 and TRPC5 function. Third, we subfamilies—e.g., TRPC4͞5 or TRPC3͞6͞7. The principles of TRPC show that TRPC proteins C-terminally fused to fluorescent channel formation offer the conceptual framework to assess the proteins give rise to fluorescence resonance energy transfer physiological role of distinct TRPC proteins in living cells. (FRET) when coexpressed in certain combinations, thus allow- ing for the systematic determination of TRPC homo- and fter binding to their cognate receptors on the cell mem- heterotetramers. Finally, a set of coimmunoprecipitations con- Abrane, many hormones, neurotransmitters, and growth fac- firms the conclusions drawn from the FRET study. The system- tors induce increases in the intracellular free calcium concen- atical delineation of all permissive permutations of TRPC 2ϩ tration ([Ca ]i) subsequent to phospholipase C (PLC) channel assembly sets the framework to conclusively interpret stimulation. In addition to inositol-1,4,5-trisphosphate (InsP3)- TRPC-dependent ion fluxes in living cells. mediated calcium release from intracellular stores, plasma mem- brane ion channels are activated in a PLC-dependent manner in Materials and Methods most cells either by second messenger-mediated pathways or by Molecular Biology. To fuse TRPC1–6 with spectral mutants of the store depletion (1). Mammalian homologues of the Drosophila enhanced green fluorescent protein (GFP), the STOP codons melanogaster transient receptor potential (TRP) visual transduc- were replaced by in-frame XhoI (TRPC1, -2, -4, -5, -6, and -7) or tion channels (2), the TRP channel (TRPC) proteins, have been EcoR1 (TRPC3) restriction sites by PCR-mediated mutagenesis identified (3–8) and characterized as subunits of receptor- with the Expand High Fidelity (Roche Molecular Biochemicals) activated cation channels (9–12). Although it has been estab- PCR polymerase system and subsequent subcloning into the lished that TRPC proteins are subject to a gating mechanism that appropriate pcDNA3-CFP or -YFP (CFP, cyan fluorescent operates through PLC, the exact activation mechanism for protein; YFP, yellow fluorescent protein) fusion vectors as distinct TRPC family members is still a controversial issue. described (21). The dominant-negative TRPC6 mutant was Proposed models favoring store-dependent or InsP3 receptor- generated by PCR-mediated mutagenesis replacing three highly CELL BIOLOGY mediated channel gating (5, 6, 13, 14) were challenged (8, 15–18), conserved amino acids (L678-W680) within the putative pore and alternative hypotheses like the direct second messenger- region of wild-type TRPC6 channels for alanine residues and mediated activation of TRPC3, TRPC6, and TRPC7 by diacyl- confirmed by DNA sequencing. glycerol have been put forward (8, 16). TRPC channels and TRP-related protein families (11, 12) Cell Culture and Transfection. HEK293 cells were cultured in belong to the superfamily of hexahelical cation channels. There- Eagle’s minimal essential medium supplemented with 10% FCS. fore, several assumptions on the structure and function of TRPC For transfections, we used 4 ␮l of FuGene6 transfection reagent channels were made based on analogous reasoning. (i) TRPC (Roche Molecular Biochemicals) and 3 ␮g of DNA per 30-mm proteins are thought to span the plasma membrane 6 times with dish. When no other fluorescent construct was expressed, we a pore loop inserted between transmembrane segments 5 and 6. (ii) Functional channel complexes are believed to be composed of tetrameric TRPC proteins. (iii) Because the canonical TRPC This paper was submitted directly (Track II) to the PNAS office. family comprises seven distinct members, TRPC channels are Abbreviations: TRPC, transient receptor potential channel; PLC, phospholipase C; FRET, fluorescence resonance energy transfer; CFP, cyan fluorescent protein; YFP, yellow postulated to form homo- as well as heterotetramers giving rise fluorescent protein; GFP, green fluorescent protein; CHO, Chinese hamster ovary; HA, to biophysically and functionally discernible channel entities. hemagglutinin. Whereas the first assumption has been proven experimentally ‡To whom reprint requests should be addressed. E-mail: guderman@mailer. for TRPC channels (19) and the second has been confirmed in uni-marburg.de. www.pnas.org͞cgi͞doi͞10.1073͞pnas.102596199 PNAS ͉ May 28, 2002 ͉ vol. 99 ͉ no. 11 ͉ 7461–7466 Downloaded by guest on October 4, 2021 added 100 ng of peGFP-C1-encoding enhanced GFP (CLON- TECH) to allow detection of transfected cells. Cells were used 24–36 h after transfection. An inducible cell line stably expressing hTRPC6 was gener- ated with an HEK293 cell line that stably expresses the Dro- sophila ecdysone receptor under Zeocin selection (EcR293, Invitrogen). hTRPC6 was subcloned into the pIND(SP1)͞Hygro plasmid (Invitrogen), transfected into these cells, and selected by hygromycin B (400 ␮g͞ml). Clones were induced by 10 ␮M ponasterone A for 1 day and tested for receptor-activated Mn2ϩ Ϫ entry and AlF4 -induced whole-cell currents. Clone no. 6-3 was chosen for further studies. Chinese hamster ovary (CHO)-K1 cells were microinjected with mixtures of 5 ng͞␮l of enhanced GFP-C1, 5 ng͞␮l of guinea ͞␮ pig H1 histamine receptor in pCMV, 100 ng l of the respective wild-type channel cDNA in pcDNA3, and 400 ng͞␮l of a variable mixture of empty pcDNA3 vector with the dominant-negative TRPC6 mutant in pcDNA3. Confocal Fluorescence Microscopy. Living cells grown and trans- fected on glass coverslips were examined with an LSM 510 laser-scanning microscope (Zeiss). For detection of GFP or YFP fluorescence, the sample was excited with the 488-nm line of an argon laser applied through a 488-nm beamsplitter. A 505-nm long pass filter filtered emitted light. Pinhole settings were adjusted to allow sections thinner than 0.8 ␮M. Fig. 1. Co-trafficking of TRPC1 and TRPC6⌬131 by members of the TRPC family. C-terminally YFP-tagged hTRPC1A was expressed in HEK293 cells alone Assessment of Mn2؉ Entry. Measurements of Mn2ϩ quenching (a) or together with untagged hTRPC3 (b) or mTRPC4␤ (c), and the cellular were performed as described (16). Briefly, cells were loaded in localization was assessed by confocal laser-scanning microscopy. d shows a cell ͞ ͞ expressing TRPC6-GFP. An N-terminally truncated TRPC6 mutant C-terminally Hepes-buffered saline (HBS; 140 mM NaCl 6mMKCl1.25 ⌬131 ͞ ͞ ͞ ͞ tagged with GFP (TRPC6 -GFP) was expressed alone (e) or together with mM MgCl2 1.25 mM CaCl2 10 mM Hepes 5 mM glucose BSA untagged hTRPC3 (f), hTRPC6 (g), mTRPC4␤ (h), or mTRPC5 (i). Typical exam- 0.1%, pH 7.4) at 37°C for 30 min. After a 15-min incubation at ples from three independent transfections are shown. ne, nuclear envelope; room temperature, cells were rinsed with HBS and used for pm, plasma membrane. experiments within 2 h. Experiments were carried
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