A method for detection and characterization of GABA(A) receptor ligands using calcium-sensitive fluorescent probes

• Published in: Biophysics (Oxford) 2011-08, Vol.56 (4), p.660-667
• Main Authors: Berezhnov, A. V., Kononov, A. V., Fedotova, E. I., Zinchenko, V. P.
• Format: Article
• Language: English
• Subjects: Biological and Medical Physics; Biophysics; Cell Biophysics; Cell culture; Cellular biology; Fluorescence; Physics; Physics and Astronomy
• ISSN: 0006-3509; 1555-6654
• DOI: 10.1134/S0006350911040038

A method for detecting and characterizing possible ligands of neuronal GABA(A) receptors has been developed, which is based on measuring the calcium response to GABA by the fluorescence of a two-wavelength Ca-sensitive probe Fura-2. In a young (2–4 days) rat hippocampal cell culture, GABA induced depolarization and a transient increase in Ca 2+ concentration in the cytosol of neurons due to activation of voltage-dependent calcium channels. A brief application of GABA could attenuate the calcium response to a subsequent addition of depolarizing agents (GABA or KCl). However, at modest amplitudes of calcium response to GABA, the reduction of the subsequent effect of KCl was insignificant, and the amplitudes of responses to KCl and to GABA proved to be linearly correlated, with a slope of ∼3.4. Therefore, the GABA calcium signals could be normalized in order to compare experiments performed on different days and different cultures. With such normalization, we estimated the EC 50 for GABA in neurons at ∼2.23 μM and the Hill coefficient at ∼1.9. A blocker of voltage-dependent calcium channels nifedipine suppressed the calcium responses both to KCl and to GABA, so that the linear relationship between their amplitudes was retained. To further validate the method, the IC50 and the type of inhibition were verified for known noncompetitive and competitive antagonists of GABA(A) receptors.