Dataset Information

Depletion of 14-3-3? 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 14-3-3? 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 14-3-3? binding by showing GST pull-down and co-immunoprecipitation. Heterologous overexpression of 14-3-3? 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 14-3-3?. 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 14-3-3? or TRPM4b in these cells. Finally, glutamate-induced cell death was greatly prevented by treatment of 9-phenanthrol or 14-3-3? shRNA.

Conclusion

These results showed that the cell surface expression of TRPM4 channels is mediated by 14-3-3? binding, and the specific inhibition of this trafficking process can be a potential therapeutic target for glutamate-induced neuronal cell death.

SUBMITTER: Cho CH

PROVIDER: S-EPMC4115172 | biostudies-literature | 2014 Jul

REPOSITORIES: biostudies-literature

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Publications

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.

Cho Chang-Hoon CH Kim Eunju E Lee Young-Sun YS Yarishkin Oleg O Yoo Jae Cheal JC Park Jae-Yong JY Hong Seong-Geun SG Hwang Eun Mi EM

Molecular brain 20140722

<h4>Background</h4>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.<h4>Results</h4>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. Heterologo ...[more]

PMID: 25047048

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Project description:BackgroundMyalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is hallmarked by a significant reduction in natural killer (NK) cell cytotoxicity, a mechanism tightly regulated by calcium (Ca2+). Interestingly, interleukin-2 (IL-2) increases NK cell cytotoxicity. Transient receptor potential melastatin 2 (TRPM2) ion channels are fundamental for Ca2+ signalling in NK cells. This pilot investigation aimed to characterise TRPM2 and CD38 surface expression in vitro on NK cells in ME/CFS patients. This investigation furthermore examined the pharmaceutical effect of 8-bromoadenosine phosphoribose (8-Br-ADPR) and N6-Benzoyladenosine-3',5'-cyclic monophosphate (N6-Bnz-cAMP) on TRPM2 and CD38 surface expression and NK cell cytotoxicity between ME/CFS and healthy control (HC) participants.MethodsTen ME/CFS patients (43.45 ± 12.36) and 10 HCs (43 ± 12.27) were age and sex-matched. Isolated NK cells were labelled with fluorescent antibodies to determine baseline and drug-treated TRPM2 and CD38 surface expression on NK cell subsets. Following IL-2 stimulation, NK cell cytotoxicity was measured following 8-Br-ADPR and N6-Bnz-cAMP drug treatments by flow cytometry.ResultsBaseline TRPM2 and CD38 surface expression was significantly higher on NK cell subsets in ME/CFS patients compared with HCs. Post IL-2 stimulation, TRPM2 and CD38 surface expression solely decreased on the CD56DimCD16+ subset. 8-Br-ADPR treatment significantly reduced TRPM2 surface expression on the CD56BrightCD16Dim/- subset within the ME/CFS group. Baseline cell cytotoxicity was significantly reduced in ME/CFS patients, however no changes were observed post drug treatment in either group.ConclusionOverexpression of TRPM2 on NK cells may function as a compensatory mechanism to alert a dysregulation in Ca2+ homeostasis to enhance NK cell function in ME/CFS, such as NK cell cytotoxicity. As no improvement in NK cell cytotoxicity was observed within the ME/CFS group, an impairment in the TRPM2 ion channel may be present in ME/CFS patients, resulting in alterations in [Ca2+]i mobilisation and influx, which is fundamental in driving NK cell cytotoxicity. Differential expression of TRPM2 between NK cell subtypes may provide evidence for their role in the pathomechanism involving NK cell cytotoxicity activity in ME/CFS.