| 研究概要 |
S-palmitoylation is a common post-translational modification of many proteins. It has been shown to control protein surface expression, spatial organization, protein-protein interaction, and functionalactivity. TRP channels are non-selective cation channels. They are expressed in most tissues and cell types, are activated by a wide range of stimuli and play important roles. Using 2-bromopalmitate, a S-palmitoylation inhibitor, we found that the expressions of several TRP channels were changed when exogenously expressed in HEK293T cells, which indicates that these TRP channels could be regulated by S-palmitoylation. In addition, when co-expression of These TRP channels with palmitoylation enzyme such as zDHHC7, the expressions were significantly increased. Moreover, similar results were confirmed using cultured mouse dorsal root ganglia neurons, which endogenously express these TRP channels. Specifically, after incubation with either palmitoylation inhibitor (2-bromopalmitate) or thioester chemical (N-(tert-Butyl) hydroxylamine), the Calcium influx was signifcantly reduced in response to specific TRP agonists. These result could be explained by changing in the channel expression or translocation similar in HEK393T cells. In deed, using DRG immunocytochemistry method, we found that the plasma membrane expression of these TRP channels were strongly reduced after incubation with these chemicals. In summary, current results indicates that these TRP channels are regulated by S-palmitoylation both in HEK393T cells and DRG neurons.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
3: やや遅れている
理由
Most of the experiments go smoothly, except the experiment to localization of the cysteine residue which is involved in S-palmitoyation. Since there are many cysteine residues in these TRP channels, we have to make many cysteine point mutant to find out which is the most important site for S-palmitoylation. Hopefully, we can finish the screening in the near future.
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| 今後の研究の推進方策 |
In the future, we will confirm the plasma membrane expression of these TRP channels incubation with 2-BP and NtBHA using biotinylation method. At the same time, whether the channel property is changed or not will be examined by single-channel analysis. Most important, animal behavior study will carried out to examine this involvement of palmitoylation in acute and chronic pain conditions.
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