USE OF BR-82(-) RADIOTRACER TO STUDY TRANSMEMBRANE HALIDE FLUX - THE EFFECT OF A TRANQUILIZING DRUG, CHLORDIAZEPOXIDE ON CHANNEL OPENING OFA GABA(A) RECEPTOR

We used the short-lived radionuclide, Br-82(-) to follow gamma-aminobu tyrate (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 captu re [Br-81(-)(n,gamma)Br-82(-)] on irradiation of a natural isotope of bromine, Br-81(-) in a neutron flux. Br-82(-) decays by beta-emission with secondary gamma-emission. Possible advantages of Br-82(-) over Cl -36(-) 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 gamma-counter as well as a beta-counter, (d) a sim ple preparation not requiring subsequent purification steps giving a s pecific activity depending on the irradiation time. With 6 hr irradiat ion time the specific activity was sufficient to make measurements wit h <1 mM Br-, which is less than the bromide concentration known to aff ect the properties of GABA, receptor. The radiotracers, Br-82(-) and C l-36(-) could be compared with the same solution composition. In condi tions where a direct effect of binding of halide to receptor does not contribute to a difference in measured ion-flux, Br-82(-) was transloc ated only marginally faster than Cl-36(-). Th, effect of chlordiazepox ide (CDPX) (2-250 mu M) on the progress of GABA (10 mu M)-mediated Br- 82(-) uptake was measured in a time range of 200 msec to 20 sec using quench-flow technique. The two phases of anion exchange previously rep orted in this experimental model with GABA alone were observed. The ra te of Br-82(-) exchange was increased 2.3-fold at 30-60 mu M CDPX and was not further increased with increasing [CDPX]. The rate of halide e xchange is a measure of open channel concentration. The isotope exchan ge rate constant, J, in a membrane vesicle preparation, is a measure o f the membrane permeability per internal volume/surface area, J = P(M) A/V. Receptor desensitization rate was also increased by CDPX, but unl ike the isotope exchange rate, it continued to increase up to at least 250 mu M CDPX.