title: Depletion of 14-3-3γ reduces the surface expression of Transient Receptor Potential Melastatin 4b (TRPM4b) channels and attenuates TRPM4b-mediated glutamate-induced neuronal cell death.
abstract: BACKGROUND
  TRPM4 channels are Ca2+-activated nonselective cation channels which are deeply involved in physiological and pathological conditions. However, their trafficking mechanism and binding partners are still elusive.
  
  
  RESULTS
  We have found the 14-3-3γ as a binding partner for TRPM4b using its N-terminal fragment from the yeast-two hybrid screening. Ser88 at the N-terminus of TRPM4b is critical for 14-3-3γ binding by showing GST pull-down and co-immunoprecipitation. Heterologous overexpression of 14-3-3γ in HEK293T cells increased TRPM4b expression on the plasma membrane which was measured by whole-cell recordings and cell surface biotinylation experiment. Surface expression of TRPM4b was greatly reduced by short hairpin RNA (shRNA) against 14-3-3γ. Next, endogenous TRPM4b-mediated currents were electrophysiologically characterized by application of glutamate and 9-phenanthrol, a TRPM4b specific antagonist in HT-22 cells which originated from mouse hippocampal neurons. Glutamate-induced TRPM4b currents were significantly attenuated by shRNAs against 14-3-3γ or TRPM4b in these cells. Finally, glutamate-induced cell death was greatly prevented by treatment of 9-phenanthrol or 14-3-3γ shRNA.
  
  
  CONCLUSIONS
  These results showed that the cell surface expression of TRPM4 channels is mediated by 14-3-3γ binding, and the specific inhibition of this trafficking process can be a potential therapeutic target for glutamate-induced neuronal cell death.
authors.forename: Chang-Hoon
authors.initials: CH
authors.lastname: Cho
authors.forename: Eunju
authors.initials: E
authors.lastname: Kim
authors.forename: Young-Sun
authors.initials: YS
authors.lastname: Lee
authors.forename: Oleg
authors.initials: O
authors.lastname: Yarishkin
authors.forename: Jae Cheal
authors.initials: JC
authors.lastname: Yoo
authors.forename: Jae-Yong
authors.initials: JY
authors.lastname: Park
authors.forename: Seong-Geun
authors.initials: SG
authors.lastname: Hong
authors.forename: Eun Mi
authors.initials: EM
authors.lastname: Hwang
mesh_terms: D048948:14-3-3 Proteins / Q000172:deficiency* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D016923:Cell Death / Q000187:drug effects; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D002470:Cell Survival / Q000187:drug effects; D055785:Gene Knockdown Techniques; D005971:Glutamates / Q000633:toxicity*; D057809:HEK293 Cells; D006624:Hippocampus / Q000166:cytology; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects; D051379:Mice; D008969:Molecular Sequence Data; D009474:Neurons / Q000166:cytology* / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects; D034741:RNA, Small Interfering / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D013329:Structure-Activity Relationship; D050053:TRPM Cation Channels / Q000737:chemistry / Q000378:metabolism*
chemical_list: D048948:14-3-3 Proteins; D005971:Glutamates; D034741:RNA, Small Interfering; D050053:TRPM Cation Channels; C437888:TRPM4 protein, human; C476766:TRPM4 protein, mouse; D012694:Serine