Abstract
BackgroundTRPM4 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.ResultsWe 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.ConclusionThese 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.
Highlights
TRPM4 channels are Ca2+-activated nonselective cation channels which are deeply involved in physiological and pathological conditions
Identification of 14-3-3γ as a binding partner of TRPM4b We previously reported that TRPM4 isoforms have differential expression on the plasma membrane: the full-length TRPM4b channels are highly localized on the plasma membrane, in contrast, TRPM4a lacking the N-terminal 174 amino acids is rarely present on the plasma membrane [22]
The results showed that (Green Fluorescent Protein) (GFP)-TRPM4b was strongly associated with FLAG-14-3-3γ (Figure 1C)
Summary
TRPM4 channels are Ca2+-activated nonselective cation channels which are deeply involved in physiological and pathological conditions. TRPM4 and TRPM5 have been identified as Ca2+-activated, Ca2+-impermeable monovalent cation channels whose activities have been first reported more than three decades ago [12,13,14,15,16] They conduct Na+ and K+ according to the concentration gradient of individual ion when activated by membrane depolarization and an increase in intracellular calcium ([Ca2+]i) [14,15,16,17]. They belong to a subgroup (melastatin-related TRPM) of TRP channels with six transmembrane domains which forms a functional channel as a tetramer [18,19]. Understanding trafficking mechanism of TRPM4 channels to the plasma membrane may open the therapeutic window to intervene the underlying TRPM4-related diseases
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