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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

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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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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Key words

  • Membrane vesicles
  • Quench flow
  • Kinetics
  • Bromide permeability
  • Ion flux
  • GABA receptor