Mol Brain. 2014 Jul 22;7(1):52. doi: 10.1186/s13041-014-0052-3.
Depletion of 14-3-3gamma reduces the surface expression of Transient Receptor Potential Melastatin 4b (TRPM4b) Channels and attenuates TRPM4b-mediated glutamate-induced neuronal cell death.
Cho, C. H., Kim, E., Lee, Y. S., Yarishkin, O., Yoo, J. C., Park, J. Y., Hong, S. G., Hwang, E. M.,
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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 14-3-3gamma 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 14-3-3gamma binding by showing GST pull-down and co-immunoprecipitation. Heterologous overexpression of 14-3-3gamma 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 14-3-3gamma. 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 14-3-3gamma or TRPM4b in these cells. Finally, glutamate-induced cell death was greatly prevented by treatment of 9-phenanthrol or 14-3-3gamma shRNA. CONCLUSION: These results showed that the cell surface expression of TRPM4 channels is mediated by 14-3-3gamma binding, and the specific inhibition of this trafficking process can be a potential therapeutic target for glutamate-induced neuronal cell death.
PMID: 25047048
 Screening
Validation: In vitro validation
Validation: In vivo validation
Characterization
Functional consequence
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Screening
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| Experimental screening | Non-experimental screening | Reference | ||||||||
| TRP channel construct | Interactor source | |||||||||
| TRP channel | Interactor | Method | Species | Region | Species | Organ/tissue | Sample type | |||
| TRPM4 |
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14-3-3ƣ | Yeast two-hybrid | Human | 1-174 | Human | cDNA libray | 25047048 | ||
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click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
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Screening
Validation: In vitro validation
Validation: In vivo validation
Characterization
Functional consequence
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| Validation: In vivo validation
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| Assay with endogenous proteins | Assay with overexpressed proteins | Reference | ||||||||
| Cell or tissue | Cell or tissue | TRP channel construct | Interactor construct | |||||||
| TRP channel | Interactor | Method | Species | Region | Species | Region | ||||
| TRPM4 |
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14-3-3ƣ | Co-immunoprecipitation | HEK293T | Human | 1-174 | Human | Full-length | 25047048 | |
| TRPM4 |
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14-3-3ƣ | Co-immunoprecipitation | HEK293T | Human | Full-length | Human | Full-length | 25047048 | |
| TRPM4 |
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14-3-3ƣ | Co-immunofluorescence staining | HEK293T | Human | Full-length | Human | Full-length | 25047048 | |
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click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
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click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
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Screening
Validation: In vitro validation
Validation: In vivo validation
Characterization
Functional consequence
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| Characterization
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| Binding region mapping | Stoichiometry | Affinity (Kd) | Reference | |||||||
| TRP channel | Interactor | |||||||||
| TRP channel | Interactor | Method | Species | Region | Species | Region | ||||
| TRPM4 |
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14-3-3ƣ | Co-immunoprecipitation | Human | 1-174 | Human | Full-length | 25047048 | ||
(:
click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
:
click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
|
Screening
Validation: In vitro validation
Validation: In vivo validation
Characterization
Functional consequence
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top
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| Functional consequence
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| TRP channel | Interactor | Method | Post-translational modification | Subcellular trafficking | Activity | Reference | ||||||
| TRPM4 |
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14-3-3ƣ | Patch clamp | Activation | 25047048 | |||||||
(:
click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
:
click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)