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Use of 82Br− radiotracer to study transmembrane halide flux: The effect of a tranquilizing drug, chlordiazepoxide on channel opening of a GABAA receptor

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Abstract

We used the short-lived radionuclide, 82Br− to follow γ-aminobutyrate (GABA) receptor-mediated halide exchange into membrane vesicles from rat cerebral cortex in millisecond and second time regions using quench-flow technique. The radioisotope was prepared by neutron capture [81Br−(n,γ)82Br−] on irradiation of a natural isotope of bromine, 81Br− in a neutron flux. 82Br− decays by β-emission with secondary γ-emission. Possible advantages of 82Br− over 36Cl− in anion tracer measurements include, (a) a short lifetime (t1/2 = 35.3 hr), which alleviates contamination and disposal problems, (b) high counting efficiency (1.54) due to the secondary radiation, (c) measurement with a γ-counter as well as a β-counter, (d) a simple preparation not requiring subsequent purification steps giving a specific activity depending on the irradiation time. With 6 hr irradiation time the specific activity was sufficient to make measurements with <1 mm Br−, which is less than the bromide concentration known to affect the properties of GABAA receptor. The radiotracers, 82Br− and 36Cl− could be compared with the same solution composition. In conditions where a direct effect of binding of halide to receptor does not contribute to a difference in measured ion-flux, 82Br− was translocated only marginally faster than 36Cl−. The effect of chlordiazepoxide (CDPX) (2–250 μm) on the progress of GABA (10 μm)-mediated 82Br− uptake was measured in a time range of 200 msec to 20 sec using quench-flow technique. The two phases of anion exchange previously reported in this experimental model with GABA alone were observed. The rate of 82Br− exchange was increased 2.3-fold at 30–60 μm CDPX and was not further increased with increasing [CDPX]. The rate of halide exchange is a measure of open channel concentration. The isotope exchange rate constant, J, in a membrane vesicle preparation, is a measure of the membrane permeability per internal volume/surface area, J = PmA/V. Receptor desensitization rate was also increased by CDPX, but unlike the isotope exchange rate, it continued to increase up to at least 250 μm CDPX.

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  1. Department of Biochemistry, School of Medicine, University of Missouri, 65211, Columbia, MO, USA

    D. J. Cash, P. Serfözö & K. Zinn

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The authors thank the staff of the University of Missouri Research Reactor Center, Columbia (MURR) for their encouragement and help and for provision of [82Br]NH4Br. This work was supported in part by a grant from the Research Council of the University of Missouri Medical School and in part by the Missouri Agricultural Experiment Station (No. BCHB0307). P.S. held a Missouri Institute of Psychiatry fellowship.

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Cash, D.J., Serfözö, P. & Zinn, K. Use of 82Br− radiotracer to study transmembrane halide flux: The effect of a tranquilizing drug, chlordiazepoxide on channel opening of a GABAA receptor. J. Membarin Biol. 145, 257–266 (1995). https://doi.org/10.1007/BF00232717

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