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

Mol Brain. 2014 Jul 22;7:52. doi: 10.1186/s13041-014-0052-3.

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.

Conclusion: 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 14-3-3 Proteins / deficiency*
  • 14-3-3 Proteins / metabolism
  • Amino Acid Sequence
  • Animals
  • Cell Death / drug effects
  • Cell Membrane / drug effects
  • Cell Membrane / metabolism*
  • Cell Survival / drug effects
  • Gene Knockdown Techniques
  • Glutamates / toxicity*
  • HEK293 Cells
  • Hippocampus / cytology
  • Humans
  • Ion Channel Gating / drug effects
  • Mice
  • Molecular Sequence Data
  • Neurons / cytology*
  • Neurons / metabolism
  • Phosphorylation / drug effects
  • Protein Binding / drug effects
  • RNA, Small Interfering / metabolism
  • Serine / metabolism
  • Structure-Activity Relationship
  • TRPM Cation Channels / chemistry
  • TRPM Cation Channels / metabolism*

Substances

  • 14-3-3 Proteins
  • Glutamates
  • RNA, Small Interfering
  • TRPM Cation Channels
  • TRPM4 protein, human
  • TRPM4 protein, mouse
  • Serine