Use of ^(82)Br^- radiotracer to study transmembrane halide flux: The effect of a tranquilizing drug, chlordiazepoxide on channel opening of a GABA_A receptor

Abstract

We used the short-lived radionuclide, ^(82)Br^− 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 [^(81)Br^−(n,γ)^(82)Br^−] on irradiation of a natural isotope of bromine, ^(81)Br^− in a neutron flux. ^(82)Br^− decays by β-emission with secondary γ-emission. Possible advantages of ^(82)Br^− over ^(36)Cl^− in anion tracer measurements include, (a) a short lifetime (t_(1/2) = 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 GABA_A receptor. The radiotracers, ^(82)Br^− and ^(36)Cl^− 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, ^(82)Br^− was translocated only marginally faster than ^(36)Cl^−. The effect of chlordiazepoxide (CDPX) (2–250 μm) on the progress of GABA (10 μm)-mediated ^(82)Br^− 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 ^(82)Br^− 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 = P_mA/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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