ABSTRACT
<p><b>AIM</b>To discover new regulators of potassium channel, an in vitro assay based on DiBAC4 (3) to determine the fluorescence was established for high throughput screening.</p><p><b>METHODS</b>A cell-based 96-well format fluorescence assay using DiBAC4 (3) in cultured PC12 cells was described. Cells were loaded with 5 mumol.L-1 DiBAC4 (3) and incubated at 37 degrees C for 30 min before adding KCl or several known potassium channel regulators. The cellular DiBAC4 (3) fluorescence responce was then detected. The fluorescence changes can be used to evaluate membrane potential changes, which are determined mainly by potassium channels.</p><p><b>RESULTS</b>Extracellular high K(+)-induced depolarization and several potassium channel blockers including 4-AP, TEA, E-4031, glibenclamide, quinidine and nifedipine all evoked increases in DiBAC4 (3) fluorescence response. The potassium channel opener, cromakalim, evoked decrease in DiBAC4 (3) fluorescence response. The fluorescence changes of 4-AP, TEA, glibenclamide, nifedipine and cromakalim were in a concentration-dependent manner. In 76 compounds screened by using the established DiBAC4 (3)-based assay, 9 compounds were found to change the fluorescence dose-dependently. Patch clamp technique is needed to further testify and screen their actions on potassium currents.</p><p><b>CONCLUSION</b>The DiBAC4 (3)-based assay is easily operated, economical and repeatable. So, it can be performed by high throughput screening for potassium channel regulators.</p>
Subject(s)
Animals , Rats , 4-Aminopyridine , Pharmacology , Barbiturates , Chemistry , Calcium Channel Blockers , Pharmacology , Cromakalim , Pharmacology , Isoxazoles , Chemistry , Membrane Potentials , Nifedipine , Pharmacology , PC12 Cells , Patch-Clamp Techniques , Piperidines , Pharmacology , Potassium Channel Blockers , Pharmacology , Potassium Channels , Pyridines , Pharmacology , Quinidine , PharmacologyABSTRACT
<p><b>AIM</b>To study mRNA expression alteration of two-pore potassium channels in the brain of beta-amyloid peptide25-35 (beta-AP25-35)-induced memory impaired rats.</p><p><b>METHODS</b>Memory impairments induced in rats by single icv injection of beta-AP25-35 (2 mmol.L-1) 5 microL were assessed in the Morris water maze test. The mRNA expression levels of three two-pore potassium channels TREK-1, TREK-2 and TRAAK were detected in rat cerebral cortex and hippocampus by reverse transcription-polymerase chain reaction (RT-PCR).</p><p><b>RESULTS</b>In the Morris water maze test, the escape latencies of the beta-AP25-35-treated rats were longer than those of the control group in 1st, 2nd and 4th training day, suggesting that the memory of beta-AP25-35-treated rats was obviously impaired. Compared with the control group, the mRNA levels of TREK-1, TREK-2 and TRAAK in the hippocampus of the beta-AP25-35-treated rats were increased by 40.0%, 27.9% and 18.9%, respectively; while no significant change of TREK-1, TREK-2 and TRAAK mRNA levels was observed in the cortex.</p><p><b>CONCLUSION</b>The mRNA expression levels of two-pore potassium channels were increased significantly in the brain of beta-AP25-35-induced memory impaired rats.</p>
Subject(s)
Animals , Male , Rats , Amyloid beta-Peptides , Cerebral Cortex , Metabolism , Hippocampus , Metabolism , Maze Learning , Memory Disorders , Metabolism , Peptide Fragments , Potassium Channels , Genetics , Potassium Channels, Tandem Pore Domain , RNA, Messenger , Genetics , Rats, Sprague-Dawley , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
<p><b>AIM</b>To study mRNA expression difference of voltage-dependent potassium channels in the brain of scopolamine-induced memory impaired rats.</p><p><b>METHODS</b>Memory impairments induced in rats by scopolamine (1 mg.kg-1) were assessed in the Morris water maze test. After rats were injected intraperitoneally with scopolamine for 6 days, the mRNA expression level of five voltage-dependent potassium channels, Kv1.4, Kv1.5, Kv2.1, Kv4.2 and Kv4.3 were detected in the rat cortex and hippocampus by RT-PCR.</p><p><b>RESULTS</b>Scopolamine (1 mg.kg-1) was shown to significantly induce memory impairment in rats. The mRNA levels of Kv4.2 were decreased by 28.8% and 33.9% in the cortex and hippocampus, respectively. The mRNA levels of Kv1.4 and Kv2.1 were increased in the hippocampus by 111.7% and 64.3%, respectively. There were no differences in the brain mRNA levels of other voltage-dependent potassium channels in scopolamine-induced memory impaired rat.</p><p><b>CONCLUSION</b>The mRNA expression levels of voltage-dependent potassium channels changed significantly in the brain of scopolamine-induced memory impaired rats.</p>