Abstract
We used the short-lived radionuclide, 82Br- to follow gamma-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,gamma)82Br-] on irradiation of a natural isotope of bromine, 81Br- in a neutron flux. 82Br- decays by beta-emission with secondary gamma-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 gamma-counter as well as a beta-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 microM) on the progress of GABA (10 microM)-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 microM 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 microM CDPX.
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