Abstract
BackgroundCa2+-activated Cl- channels (CaCCs) participate in many important physiological processes. However, the lack of effective and selective blockers has hindered the study of these channels, mostly due to the lack of good assay system. Here, we have developed a reliable drug screening method for better blockers of CaCCs, using the endogeneous CaCCs in Xenopus laevis oocytes and two-electrode voltage-clamp (TEVC) technique.ResultsOocytes were prepared with a treatment of Ca2+ ionophore, which was followed by a treatment of thapsigargin which depletes Ca2+ stores to eliminate any contribution of Ca2+ release. TEVC was performed with micropipette containing chelerythrine to prevent PKC dependent run-up or run-down. Under these conditions, Ca2+-activated Cl- currents induced by bath application of Ca2+ to oocytes showed stable peak amplitude when repetitively activated, allowing us to test several concentrations of a test compound from one oocyte. Inhibitory activities of commercially available blockers and synthesized anthranilic acid derivatives were tested using this method. As a result, newly synthesized N-(4-trifluoromethylphenyl)anthranilic acid with trifluoromethyl group (-CF3) at para position on the benzene ring showed the lowest IC50.ConclusionOur results provide an optimal drug screening strategy suitable for high throughput screening, and propose N-(4-trifluoromethylphenyl)anthranilic acid as an improved CaCC blocker.
Highlights
Ca2+-activated Cl- channels (CaCCs) participate in many important physiological processes
Cl- current elicited by Ca2+ influx in oocytes permeabilized with ionomycin To find optimal conditions for drug screening using CaCCs in Xenopus oocytes, we first characterized the CaCC currents in Xenopus oocytes and compared various treatment conditions
Ifast and Islow were not induced by substituting Ca2+ with Ba2+ in each condition (Figure 1E, F, I, J, N, O), indicating that both of these currents were activated by Ca2+ entry
Summary
Ca2+-activated Cl- channels (CaCCs) participate in many important physiological processes. CaCCs serve a number of important physiological roles in a variety of cell types These functions include vascular tone regulation, cardiac excitability, smooth muscle contraction, fast block of polyspermy in certain eggs [1]. CaCC currents in non-sensory neurons of the spinal cord and the autonomic nervous system were reported, and further investigation may prove an even more extensive expression in the nervous system [2]. Despite this physiological importance of CaCC, the channel remains poorly understood at the molecular, biophysical and pharmacological level, owing to the lack of specific pharmacologic tools with high potent and selectivity.
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