Drug Screening And Functional Characterization Of TRP Channels

Calcium ions are very important for the regulation of physiological functions of cells,including proliferation,growth,programmed cell death,and etc.Transient Receptor Poential（TRP）ion channels are mostly Ca²⁺ permeable and consist of seven subfamilies,Transient Receptor Potential Ankyrin（TRPA）,Transient Receptor Potential canonical（TRPC）,Transient Receptor Potential melastatin（TRPM）,Transient Receptor Potential mucolipin（TRPML）,Transient Receptor Potential polycystin（TRPP）,Transient Receptor Potential vanilloid（TRPV）,no mechanoreceptor potential（TRPN）.Using patch clamp electrophysiology and related molecular biology techniques,we investigated TRP channels in three main aspects: screening for small molecular modulators of TRPC channels;functional characterization of charged residuals in the pore helix region of TRPV1 channel;studying the possible involvement of TRPV4 channel in calcium response of Gastric Cancer（GC）cells,and obtained the corresponding experimental results.TRPC channels are expressed in many tissues,such as kidney,smooth muscle,nervous system and etc.TRPC channels help maintain the physiological function of cells by regulating Ca²⁺ influx into cells.Dysfunction of TRPC proteins has been linked to a number of diseases.Therefore,specific small molecular modulators of TRPC channels should be important for not only research on the functions of TRPC channels but also therapeutic development against TRPC-related diseases.However,specific small molecular modulators for TRPC channels are very scarce,highlighting the importance of screening for these molecules for TRPC channels.Based on a lead compound for TRPC6 identified through high throughput screening of a large chemical library,we performed electrophysiological characterizations to confirm the compound as a novel selective activator of TRPC3/6/7.In addition,the compound revealed differential sensitivity to extracellular Ca²⁺ by the three TRPC channels.TRPV1 channel was first cloned in rat dorsal root ganglion neurons and later shown to mediate pain sensation triggered by noxious heat and capsaicin.The cloning of TRPV1 channel had promoted research on the molecular mechanisms of inflammatory and neuropathic pain in sensory neurons.There have been many studies on mechanisms of TRPV1 activation by different ligands,such as capsaicin,protons,temperature,voltage,and so on.Our study aims to find the role of the pore helix in TRPV1 channel gating.We found that the charged residues in the pore helix contribute significantly to the low pH-induced and voltage-dependent TRPV1 channel gating.Our study enriched the understanding of mechanisms of TRPV1 channel gating.Ca²⁺ homeostatsis deregulation is common in GC cells,which represents one of the main factors that affect the occurrence and development of GC.Ca²⁺ supplements are frequently used by both health individuals and cancer patients.A direct effect of taking Ca²⁺ supplements is to increase the extracellular Ca²⁺ concentration of epithelial cells lining the gastrointestinal track.However,it is unclear whether the elevated extracellular Ca²⁺ concentration is beneficial or detrimental to the growth of GC cells.Ca²⁺ sensing receptor（CaSR）is a G protein-coupled receptor expressed on the plasma membrane that responds to the elevation of extracellular Ca²⁺ concentration.CaSR is expressed in GC cells,but its function in cancer growth was unknown.The goal for this part of our study was to examine the function of CaSR in GC cells and how it is coupled to the ion channel,TRPV4,to regulate Ca²⁺ influx and consequently the growth and proliferation of GC.Our results reveal important functions of CaSR in GC and suggest that targeting the CaSR-TRPV4 coupling may be a viable new strategy to treat GC.