| Transient receptor potential Vanilloid 1(TRPV1 channel) is a nonselective cation channel with six transmembrane segments, is gated by its responsiveness to capsaicin? inflammatory mediators?low pH?the endocanabinoid anandamide, and noxious heat(>42?) as well. TRPV1 channel serves as a polymodal receptor due to its activation by a variety of stimulations. TRPV1 channels are widely distributed in the brain, spinal cord, kidneys, pancreas and other tissues. TRPV1 channel is involved in pain transmission and modulation of pain as well as the integration of different information. Despite the clear physiological importance of TRPV1, the most known stimuli of TRPV1 channels are exogenous physical and chemical stimulations, thus far little is known about TRPV1-associated endogenous proteins.We constructed the mouse DRG cDNA Library by SMARTTM. After library titer test, the good cDNA library was used for our screening experiements. We next performed a yeast 2-hybrid screen using the N-terminal region of TRPV1 channel as the bait and mice DRG cDNA bibrary as the fish. After three rounds of screening by the reporter genes, His, Ade, and LacZ. We got 120 positive colonies in total, then we retransformed the bait plasmid and fish libray into the yeast and thereafter the yeasts were grown on the SD/-Trp/-Leu/-His/-Ade plates. Each positive colony was picked out and amplified, then the cDNA was extracted and sequenced. By the end, we have got eight cDNA clones encoding different proteins. One of them, p85?, the subunit of PI3K(PI3K-p85?) was selected for further examination. To confirm the interaction between the TRPV1 cahnnel protein and PI3K-p85?, we constructed two different expression plasmids, TRPV1 tagged with FLAG and PI3K-p85? fused with GFP. After expressing the two proteins in HEK293 T cells, we carried out the the coimmunoprecipitation experiments, and the results showed that PI3K-p85? physically interacts with TRPV1 channel protein in vitro. In addition, we also constructed two fusion proteins with RFP and GFP as fluorescent tags, TRPV1-RFP and PI3K-p85?-GFP. After coexpression of TRPV1-RFP and PI3K-p85?-GFP in HEK293 T cells, we found that green and red color imaged by confocal microscopy were overlay on the cell membrane, which suggests that TRPV1 colocalized with PI3K-p85? in vivo.Dr. Gordon group in University of Washington once performed a yeast 2-hybrid screen in human fetal brain libray and found the p85? subunit of PI3K(PI3K-p85?) was physically and functionally coupled to TRPV1 channel. In their report, they proposed a new modle for NGF-mediated sensitiation of TRPV1 in which physical coupling of TRPV1 and PI3 K in a signal transduction complex facilitates trafficking of TRPV1 to the plasma membrane. However, our experiments were performed in DRG neurons which is a different tissue from theirs. We then asked whether PI3 K found in DRG can regulate the function of TRPV1 in the same way, although our above experiments indeed showed PI3 K physically colocalized with TRPV1. We thus carried out the patch-clamp recordings. Our whole-cell recordings in HEK293 T cells transfected with TRPV1, PI3K-p85? and trkA showed that 0.1 ?M capsaicin-evoked currents were increased by 5-fold after treatment by a 6-min treatment with 100 ng/mL NGF. Moreover the TRPV1 currents elicited by 1 ?M capsaicin were also enhanced as comparing to the control data. We thus conclude that the regulatory effects of PI3K-p85? on the function of TRPV1 channel is general in different tissues.The above results demonstrate that the mouse DRG cDNA library we constructed was successful and suitable for the yeast two-hybrid screening experiments. Moreover, our functional data showed that PI3K-p85? indeed has a general regulatory effects on the function of TRPV1 channel. The rest candidate proteins potentially for interacting with TRPV1 channel we got in this research lay a good foundation for further study. |
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