The Study Of The Effect Of TRP Channel On The Gallbladder Smooth Muscle Contraction And The Screening Of Its Small Molecule Modulators

The screening of small molecule modulators for TRPC4 ion channel Objective Transient receptor potential canonical(TRPC) channels are Ca2+-permeable nonselective cation channels widely expressed in various tissues in the body. These channels are involved in diverse physiological functions, including neurotransmission, and regulation of cardiovascular, respiratory, gastrointestinal, and urinary systems. At present, the small molecule regulators of TRPC channels are less and their specificity are poor. In this study, we aim to find specific small molecule modulators of TRPC channels. These findings will facilitate the studies on the physiological functions and regulatory mechanisms of TRPC channels as well as their implications in clinical applications. Methods 1. Chemical synthesis: Based on the core structure of ML401, a series of 1-H-benzimidazole-2-amine derivatives were designed and synthesized. 2. PCR and protein mutagenesis: A mutant of TRPC4 was made. 3. Calcium imaging assay: A series of compounds were identified by a cell-based high throughput screening assay. 4. The patch clamp recording: several good inhibitory compounds against TRPC4 areidentified using whole-cell patch clamp recording. Results 1. We designed and synthesized 30 1-H-benzimidazole-2-amine derivatives. 2. The mutant of TRPC4(TRPC4 G503L) was made. 3. The most improved potency was achieved by compound 21(IC50=0.65μM) compared with ML401(IC50=1.05 μM). 4. In addition, G1224 displayed a potentiateeffect onthe agonist-induced TRPC4 activity(EC50=0.78μM). Conclusion We find a number of derivatives displayed good inhibitory potency against TRPC4. G1224 displayed have a potentiate effect on the agonist-induced TRPC4 activity.The role of TRPP2 regulating guinea pig gallbladder smooth muscle contractionObjective TRPP2 channels belong to the superfamily of transient receptor potential channels and are widely expressed in various tissues, including smooth muscle of digestive gut. Accumulative evidences indicate that TRPP2 is able to mediate Ca2+ release from Ca2+ store. However, the functional role of TRPP2 in gallbladder smooth muscle contraction still remained unclear. In the present study, we investigate the physiological function of TRPP2 in gallbladder smooth muscle contraction and its relevant mechanism.Methods 1. Preparation of gallbladder muscle strips: The guinea pigs were killed by smothering method and the gallbladder was removed. After clearing the fat tissue and serosa, the gallbladder was cut along the longitudinal axis into 1.0 × 0.3 cm muscle strips. Every longitudinal muscle strip was suspended in an organ bath containing Krebs solution(p H 7.4) gassed with 95% O2 and 5% CO2.2. Tension measurement of gallbladder muscle strips: The isometric tension was recorded and analyzed by a data acquisition and analysis system(BL-420S). We used different agonists to test smooth muscle contraction of guinea pig gallbladder.3. Calcium imaging in gallbladder smooth muscle: TRPP2 si RNA was transfected into gallbladder smooth muscle, and the Leica SP5 confocal microscopy system was used todetect agonist-induced intracellular Ca2+ concentration change.4. Western blot in gallbladder smooth muscle: To use the antibodies including anti-TRPP2 detected the expression of TRPP2 protein in gallbladder smooth muscle.Results 1. When TRPP2 protein was knocked down via TRPP2 specific si RNA transfection in gallbladder muscle strips from guinea pig, carbachol(CCh)-evoked Ca2+ release and extracellular Ca2+ influx were reduced significantly, and gallbladder contractions induced by agonists were suppressed markedly as well. 2. U73122, 2APB and TG significantly suppressed the CCh-induced gallbladder contraction with concentration-dependent manner. Pretreatment with U73122, 2APB or TG eliminated the difference of gallbladder contraction between TRPP2 knockdown and scramble si RNA control groups. Conclusion TRPP2 mediates Ca2+ release from gallbladder muscle cell Ca2+ store, which has an essential role in agonist-induced gallbladder muscle contraction.