Screening method of calcium-activated chloride channel modifier
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A calcium-activated chloride channel and screening method technology, applied in the field of cell channel regulator screening, can solve problems such as poor accuracy, low efficiency, and complicated experiments, and achieve the effects of simple and easy operation, high sensitivity, and accurate and reliable test results.
Inactive Publication Date: 2013-09-11
HEBEI MEDICAL UNIVERSITY
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[0004] The purpose of the present invention is to provide a simple and efficient screening method for calcium-activated chlor
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[0037] Example 1: Screening and verification of CaCCs channel blockers
[0038] a) Prepare incubation solution, washing solution and channel opening solution:
[0039] Incubation solution: Weigh or measure 50mmol of sodium chloride, 100mmol of potassium chloride, 1mmol of magnesium chloride, 0.8mmol of sodium dihydrogen phosphate dihydrate, 4mmol of calcium chloride, 10mmol of Hepes, and 15μmol of calcium ionophore to dissolve in deionized water and dilute to 1000ml, then adjust the pH to 7.4 with sodium hydroxide solution;
[0040] Washing solution: weigh 150mmol of sodium gluconate, 5.4mmol of potassium gluconate, 1mmol of magnesium gluconate, 0.8mmol of sodium dihydrogen phosphate dihydrate and 20mmol of Hepes, dissolved in deionized water, dilute to 1000ml, and then adjust the pH with sodium hydroxide solution Value to 7.4;
[0041] Channel opening solution: Weigh 54mmol of sodium gluconate, 100mmol of potassium gluconate, 1mmol of magnesium gluconate, 4mmol of calcium gluconate,...
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[0068] Example 2: Screening and verification of CaCCs channel openers
[0069] a) Prepare incubation solution, washing solution and channel opening solution:
[0070] Incubation solution: Weigh or measure 50mmol of sodium chloride, 100mmol of potassium chloride, 1mmol of magnesium chloride, 0.8mmol of sodium dihydrogen phosphate dihydrate, 4mmol of calcium chloride, 10mmol of Hepes, and 15μmol of calcium ionophore to dissolve in deionized water and dilute to 1000ml, then adjust the pH to 7.4 with sodium hydroxide solution;
[0071] Washing solution: weigh 150mmol of sodium gluconate, 5.4mmol of potassium gluconate, 1mmol of magnesium gluconate, 0.8mmol of sodium dihydrogen phosphate dihydrate and 20mmol of Hepes, dissolved in deionized water, dilute to 1000ml, and then adjust the pH with sodium hydroxide solution Value to 7.4;
[0072] Channel opening solution: Weigh 54mmol of sodium gluconate, 100mmol of potassium gluconate, 1mmol of magnesium gluconate, 4mmol of calcium gluconate, ...
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Abstract
The invention discloses a screening method of a calcium-activated chloride channel modifier. The screening method comprises the following steps of: (a), preparing an incubation liquid, a washing liquid and a channel opening liquid; (b), dissolving a to-be-screened compound in the incubation liquid to prepare a positive incubation liquid and incubating cells; (c), adding the cleaning liquid, cleaning for three times to four times after the incubation, and adding the channel opening liquid for carrying out stewing reaction to obtain a reaction liquid; (d), adding a silver nitrate solution in the reaction liquid, uniformly mixing and keeping in the dark for stewing for 24 hours, taking liquid supernatant to ICR8000 to measure the concentration of silver ions. According to the screening method of the calcium-activated chloride channel modifier disclosed by the invention, a to-be-detected compound is added in the incubation stage to judge the opening-closing condition of the ion channel by changing the number of chloride ions entering the cells, so that not only can the chloride ion concentration in the cells be improved, but also the flexibility can be increased and a to-be-detected drug can be prevented from directly contacting with the silver irons to generate reaction. The screening method of the calcium-activated chloride channel modifier is high in flexibility, accurate and reliable in detected result and simple and easy to operate, and therefore, the screening method is a simple and efficient CaCCs channel modifier screening method with better advantages.
Description
technical field [0001] The invention relates to a screening method for a cell channel regulator, in particular to a screening method for a calcium-activated chloride channel regulator. Background technique [0002] Calcium-activated chloride channels (CaCCs) were first discovered in oocytes of Xenopus laevis. They are excitable in non-excitable cells such as endothelial cells, epithelial cells, and blood cells, as well as in cardiomyocytes, nerve cells, and vascular smooth muscle cells. widely distributed on cells. The physiological functions of CaCCs include: ① as a channel for chloride ions to pass through epithelial cells, changing the electrochemical gradient inside and outside the cells; ② participating in the regulation of nerve excitation and cardiac excitability, and playing an important role in the depolarization of myocardial action potential; ③ TMEM16A in Highly expressed in tumor cells, it is a potential therapeutic target and a marker of tumor biology. In 2008...
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