Investigation Of The Mechanisms Underlying Upregulation Of Thermal TRPV1-3 Channels By Acid And 1,4-Dioxane

Transient receptor potential (TRP) channels are vanguards of the human sensory systems. They can response to a variety of intracellular and extracellular stimuli. TRP channels are widely expressed and the regulatory mechanisms are diverse. In recent years, it has been clear that TRP channels play important roles in temperature sensing. The thermo-TRP channels are expressed in the terminals of sensory nerve. They are activated by a variety of physical or chemical stimuli including temperature. Thermo-TRP channels not only mediate temperature sensing, but also modulate pain, itch and skin inflammation process. Therefore, thermo-TRP channels have become the new molecular targets for the treatment of pain, skin inflammatory disease and itch.Ion channels are involved in the regulations of intracellular and extracellular ion concentrations and the establishing of membrane potential and they play important roles in many physiological processes. Ion channel diseases are usually caused by electrophysiological abnormality of ion channels. For example, the deficiency of heat sensitive TRPV3 channel lead to abnormal hair growth and the increased incidence of skin diseases, while the gain of function mutations of TRPV3 channel lead to human genetic diseases (Olmsted syndrome). The thermo-TRPV1 channel is involved in the generation and transduction of pain. TRPV1-knockout mice lost the sensing of allergic pain caused by inflammatory substances. Therefore, the regulations of ion channel gating (open and close) are not only important for the research on ion channels themselves, but also for the understanding of pathogenesis of ion channel disease and provide theoretical guidance for clinical treatment. Based on the important roles of heat sensitive TRPV1 and TRPV3 channels in maintaining body health, we used the techniques of patch clamp electrophysiology, molecular biology and biochemistry to investigate the gating modulation of TRPV3 and TRPV1 channels.The first part consists of below:regulatory effect of H+ on thermal TRPV3 channel. Thermo-TRPV3 channels are abundantly expressed in keratinocytes, trigeminal ganglion and brain tissue. Temperature and chemical stimulations can induce the opening of the channel. Tissue acidification is a common phenomenon in physiological and pathological conditions. Anaerobic glycolysis in skeletal muscle caused by strenuous exercise result in the accumulation of lactic acid and lead to the decrease of extracellular pH; tissue injury, hypoxia, epilepsy and the infection are often accompanied by extracellular acidification. Previous studies have indicated that acid can not only directly activate TRPV1 channels, but also sensitize the response of TRPV1 channels to other agonists. Previous researches reported that acid could not directly activate TRPV3 channels. However whether acid can regulate TRPV3 channel remained unclear. For these reasons, we carried out research on the regulatory effect of H+ on thermal TRPV3 channel. The results included the following: 1) Acid potentiates the responses of TRPV3 to 2-APB. Under acidic conditions, the dose response of TRPV3 to 2-APB shifts left, so acid can enhance the activity of TRPV3 channel in response to 2-APB; 2) we then individually neutralized all extracellular negatively charged amino acid. Patch clamp recordings were performed. Results showed that all the point mutations fail to alter the acid potentiation of 2-APB-evoked TRPV3 currents; 3) Acid can directly activate TRPV3 channel from intracellular side, but not the extracellular side, and cannot activate the TRPV1 and TRPV2 channel from intracellular side. Based on the different actions of acid on TRPV3 and TRPV1, we established a series of chimeras between TRPV3 and TRPV1, and proved that four amino acid residues (L508, D512, S518 and A520) in linker 2-3 played key roles in the activation of acid on TRPV3; 4) In excised patch, acid also potentiated the responses of TRPV3 to 2-APB from the intracellular side no matter the intracellular side was in neutral or acidic environment. All the results indicated that the modulation of acid on TRPV3 channel functioned on the agonist but not the channel protein; 5) The potentiation of acid on TRPV3 channel is agonist specific. Protons only enhanced the activity of 2-APB and its analogues on TRPV3 channel; we performed 1H-NMR and 2H-NMR experiments to examine their structural features at different pH (pH=8.5,7.4 or 5.5) and found that the hydrogen atoms in the aromatic areas of 2-APB, DPBA, and DPB were shifted downfield while not for DPM. The NMR and MS results suggest that the acid-induced configuration switch involves either an addition of a proton or a loss of hydroxyl (OH-) group from the compound. Being a Lewis acid, the boron compound can accept a hydroxyl (OH-) group from water in neutral to alkaline conditions, but will lose it in the acidic solution due to competition by the increased free proton levels. This change in OH- binding should transform the boron from a tetrahedral structure with four sp3 hybrid orbitals to a triangular planar structure with three sp2 hybrid orbitals and an empty p orbital. Thus, the acidification alters not only the orientation of the phenol rings but also the bond length between the boron and each phenol ring.6) Finally, we also found that acid can enhance the inhibitory effect of 2-APB on TRPM8 channel.The second part includes:the regulations of 1,4-Dioxane on thermal TRPV1 channel. TRPV1 channels exist widely in many species from yeast to human and are mainly distributed in the brain tissue, primary sensory nerve terminal and some non neural tissues, such as skin keratinocytes, gastrointestinal tract, stem cells, fibroblasts, T cells, mast cells and vascular smooth muscle cells, where they take part in nociceptive transmission, modulation and integration of multiple kinds of pain information.1,4-Dioxane is widely used as solvent for practical applications or in the laboratory. It is found in cosmetics and personal care products as a byproduct of the ethoxylation process which is a route to some ingredients found in cleansing and moisturizing products. The toxicity of 1,4-Dioxane have been reported. Chronically exposed to 1,4-Dioxane in drinking water or acute inhalation exposure to high levels of 1,4-dioxane cause damages of central nervous system, liver and kidney in animals; acute respiratory exposure to high levels of 1, 4-dioxane has caused vertigo and irritation of the eyes, nose, throat, and lungs in humans and may also irritate the skin. While, the molecular target of 1,4-Dioxane is still not clear. Considering that TRPV1 channals are widely expressed in nervous system, digestive system, immune system and skin, we carried out the research on the regulation of 1, 4-Dioxane on TRPV1 channel. The results contain the following:1)1,4-Dioxane activate TRPV1 channel at high concentration while at low concentration, it can potentiate the response of TRPV1 channel to other agonists, especially reduce the temperature sensing threshold; 2) In trigeminal ganglion and dorsal root ganglion neurons,1,4-Dioxane can also activate TRPV1 channels and potentiate the TRPV1 current evoked by capsaicin; 3) Protonation and phosphorylation of TRPV1 channel (channel states in inflammation and hyperalgesia) sensitize the response of TRPV1 channel to 1,4-Dioxane, suggesting that, in the states of inflammation and hyperalgesia,1,4-Dioxane produces more harmful damage to the body; 4) Hot plate and Von Frey experiment of wild-type and TRPV1 knockout mice prove that 14-Dioxane can cause thermal hyperalgesia behavior, but has no effect on the mechanical pain of mice; 5) we further examined the action of 14-Dioxane on TRPV2, TRPV3 and TRPM8 channels which have similar sequential homology with TRPV1 channel. The results showed that 1,4-Dioxane can activate TRPV3, but inhibit the activity of TRPM8, and had no effect on TRPV2, so 1,4-Dioxane has specificity on different channels; 6) Based on the different actions of 1,4-Dioxane on TRPV1 and TRPV2, we establish a series of chimeras between TRPV1 and TRPV2, and proved that the Linker 4-5 of TRPV1 channel is responsible for the activation of 1,4-Dioxane on TRPV1 and E570 site plays an important role; 7) In the end, we detected the action of 1,3-Dioxane, tetrahydrofuran and 1,3,5-Trioxane, which have the similar structure with 1,4-Dioxane and are widely used in daily life. The results show that except for 1,3,5-Trioxane, the other two substances can activate TRPV1 channel, indicating that this kind of materials have similar effects on channels. The results in this part suggest that 1,4-Dioxane not only activates TRPV1 channel but also potentiates the sensitivity of TRPV1 to other agonists in endogenous or exogenous express system, indicating that 1,4-Dioxane produces hyperalgesia behavior by activating TRPV1 channel and causes other damages. Meanwhile, we found that 1,4-Dioxane can activate and sensitive TRPV3 channel. The dysfunction of TRPV3 cause various of skin diseases and affect the normal growth of hair, but in many toilet articles and cosmetics,1,4-Dioxane have been detected, suggesting that the concentration of 1,4-Dioxane should be strictly controlled in the use of cosmetics exposed to the skin.In summary, through the application of a variety of research methods, this paper clarifies the modular mechanisms of H+ on TRPV3 channel, which equally applies to TRPV1 and TRPV2 channels, that H+ potentiate the sensitivity of TRPV1-3 to 2-APB but regulating the conformation of 2-APB. We also found the activity sites of H+ on the inner side of TRPV3, L508, D512, S518 and A520; and we reveal that thermosensitive TRPV1 and TRPV3 channels are the molecular targets of 1,4-Dioxane in the body. Our results provide corresponding theoretical explanation for the damage of 1,4-Dioxane on body health and provide a theoretical basis for the specifications of 1,4-Dioxane.