Interaction Of Rat TRPV4with Annexin A2and Tubulin β5

BackgroundTransient receptor potential vanilloid receptor4(TPRV4) is a kind of non-selective cation channels. It is mechanosensitive and widely expressed in the brain, DRG, kidney, and lungs. TRPV4could response to many kinds of stimuli, and cause an obvious increase of intracellular Ca2+. TRPV4plays a role in nociception and mechanical allodynia. The full length of rat TRPV4protein is871aa. Like the other TRP channels, TRPV4possesses six transmembrane domains and two flanking tails in the N-and C-termini. The intracellular terminal domains are considered to be essential in the regulation of channel function and in channel assembly.Our previous studies suggested that TRPV4contributes to mechanical allodynia after CCD. In CCD model, we found an increased expression and sensibilized function of TRPV4channel, with an increased expression of annexin A2and decreased expression of tubulin β5. We supposed that annexin A2and tubulin β5played a role in the regulation of TRPV4channel expression in the transduction of nociceptive stimuli.Annexin A2is a Ca2+-dependent membrane-binding protein. Annexin A2is associated with endosomal membrane dynamics and vesicular trafficking, and participates in fibrinolysis, blood clotting and angiogenesis. The S100A10-annexin A2complex could bind to TRPV5and TRPV6and regulate the channel activities. Tubulin β5is a subtype of β tubulin. Microtubules consisting of α/β tubulin heterodimers are important part of cytoskeleton and play important roles in key cellular events. Microtubules are related to the neuronal structure and function, and changes in microtubule dynamics can induce pathological neurological conditions. Tubulin dimers, as well as polymerized microtubules, can bind to TRPV1in a Ca2+sensitive manner. There were evidences that annexin A2and tubulin β5acted in the mechanotransduction, but the molecular mechnisms were still unknown.The aim of this part is to construct recombinant eukaryotic expression vectors through gene engineering techniques, and to provide necessary vector models for the further studies on the functions and interactions of TRPV4, annexin A2and tubulin β5.ObjectiveTo obtain TRPV4, TRPV4-Nt and TRPV4-Ct genes and construct epitope-tagged eukaryotic expression vectors with gene engineering techniques.To observe the expressions of TRPV4, annexin A2and tubulin β5in the transfected HEK293cells.MethodsThe full-length TRPV4cDNA were provided as a present. Specific primer pairs were designed to construct TRPV4, and its Nt, Ct epitope-tagged expression vectors, following PCR, restriction endonuclease digestion, ligation, transformation and plasmid extraction. The recombinant plasmids of TRPV4, annexin A2and tubulin β5were transfected into HEK293cells.48hours after transfection, western blot and immunofluorescence were used to detect the expression and localization of the proteins in the transfected cells in vitro.Results1. Construction of eukaryotic expression vectors for rat TRPV4and its N&C termini.The size of PCR amplified product of TRPV4and its N&C termini were both consistent with the expected fragments. The recombinant plasmids were verified with PCR, restriction endonuclease digestion and automated nucleotide sequencing.2. Expression of eukaryotic expression vectors of TRPV4, annexin A2and tubulin β5in transfected HEK293cells.TRPV4, annexin A2and tubulin (35plasmids transfected cell protein samples were detected single protein bands at98kD,39kD,55kD respectively. There were none specific bands in the there control groups. Immunofluorescence was used to obserbe the localization of the exogenous proteins. TRPV4protein was detected on the cell membranes, as well as in the cytoplasm. Annexin A2distributed extensively in the cell membranes and cytoplasm. Tubulin beta5was present on both the cell membrane and cytoplasm. FLAG or Myc protein was not detected in pcDNA3.1(+) transfected and un-transfected HEK293cells.ConclusionsWe successfully constructed the eukaryotic expression vectors pcDNA3.1-TRPV4-Myc, His, pcDNA3.1-TRPV4-Nt-Myc, His, pcDNA3.1-TRPV4-Ct-Myc, His, pcDNA3.1(+)-Anxa2-FLAG, and pcDNA3.1(+)-Tubb5-FLAG, which could effectively expressed in HEK293cells. This would facilitate the following invitro study of the molecular mechanisms of TRPV4, annexin A2and tubulin β5in the nociceptive pain conduction and their interactions.