title: Close spatio-association of the transient receptor potential canonical 4 (TRPC4) channel with Gαi in TRPC4 activation process.
abstract: TPRC channels are Ca(2+)-permeable, nonselective cation channels that are activated by a wide variety of stimuli, including G protein-coupled receptors (GPCRs). TRPC4 is commonly assumed to be activated by Gq/phospholipase C-coupled receptors. However, the other molecular mechanisms by which Gα proteins regulate TRPC4 remain unclear. Here, we found that Gαi2 regulates TRPC4 activation by direct binding. To investigate this mechanism, we used whole patch clamp and fluorescence resonance energy transfer (FRET). We tagged an isoform of mTRPC4 and G protein with CFP and YFP, respectively, and transiently transfected cells with the FRET pair. The FRET efficiency between TRPC4β-CFP and the constitutively active mutant form of Gαi2 was nearly 15% and was greater than that observed with wild-type Gαi2 (nearly 5%). Gβγ and the TRPC4 channel showed a FRET efficiency lower than 6%. In HEK293 cells transfected with the M2 muscarinic receptor, the application of carbachol increased the FRET efficiency between TRPC4β-CFP and Gαi2(WT)-YFP from 4.7 ± 0.4% (n = 7) to 12.6 ± 1.4% (n = 7). We also found that the TRPC4 channel directly interacts with Gαi2, but not with Gαq, when the channel is open. We analyzed the calcium levels in HEK293 cells expressing the channels and Gαi2 or Gαq using the calcium indicator YC6.1 (Yellow Cameleon 6.1). In response to the muscarinic agonist carbachol, M2-, Gαi2-, and TRPC4-expressing cells showed a prolonged Ca(2+) influx compared with cells expressing only M2. Together, these data suggest that Gαi2 activates the TRPC4 channel by direct binding, which then induces Ca(2+) entry.
authors: Department of Physiology, Seoul National University College of Medicine, Seoul, Republic of Korea; and.
authors: JongYun
authors: J
authors: Myeong
authors: Department of Physiology, Seoul National University College of Medicine, Seoul, Republic of Korea; and.
authors: Misun
authors: M
authors: Kwak
authors: Department of Integrative Biology and Pharmacology, The University of Texas Health Science Center at Houston, Houston, Texas.
authors: Jae-Pyo
authors: JP
authors: Jeon
authors: Department of Physiology, Seoul National University College of Medicine, Seoul, Republic of Korea; and.
authors: Chansik
authors: C
authors: Hong
authors: Department of Physiology, Seoul National University College of Medicine, Seoul, Republic of Korea; and.
authors: Ju-Hong
authors: JH
authors: Jeon
authors: Department of Physiology, Seoul National University College of Medicine, Seoul, Republic of Korea; and insuk@plaza.snu.ac.kr.
authors: Insuk
authors: I
authors: So
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects / Q000235:genetics*; D002135:Calcium-Binding Proteins / Q000737:chemistry; D002217:Carbachol / Q000494:pharmacology; D018679:Cholinergic Agonists / Q000494:pharmacology; D031541:Fluorescence Resonance Energy Transfer; D051960:GTP-Binding Protein alpha Subunit, Gi2 / Q000235:genetics* / Q000378:metabolism; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D050052:TRPC Cation Channels / Q000235:genetics* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D002135:Calcium-Binding Proteins; D018679:Cholinergic Agonists; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D050052:TRPC Cation Channels; C489571:TRPC4 ion channel; C503159:cameleon YC6.1; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; D002217:Carbachol; D051960:GTP-Binding Protein alpha Subunit, Gi2; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D002118:Calcium
keywords: FRET; G protein; TRPC4; calcium