Monensin inhibits the binding of ³H‐flunitrazepam to and reveals the intracellular passage of GABA_A/benzodiazepine receptor

Abstract

Effects of monensin were examined on the intracellular processing of the GABAA/benzodiazepine receptor (GABAA/BZDR) in neuron cultures derived from embryonic chicken brain, using 3H-flunitrazepam as the probe for the benzodiazepine modulator site on the receptor. Incubation of cultures with 0.1 or 1 microM monensin for 3 h blocked the binding of 3H-flunitrazepam by about 18%. Loss of ligand binding was due to a reduction in the number of binding sites, with no significant changes in receptor affinity. The general cellular protein synthesis and glycosylation in the cells were inhibited by 26% and 56%, respectively, in the presence of 1 microM monensin, as detected by assaying the incorporation of 3H-leucine and 3H-galactose. In contrast, an increase was observed for mannose incorporation by the cultures in the presence of the drug. Moreover, the results from in situ trypsinization of the cultures following monensin treatment showed that monensin did not alter the distribution of intracellular and surface receptors. The data suggest that monensin induces the down-regulation of GABAA/BZDR by generating abnormal glycosylation of the receptor and interrupting its transport within the Golgi apparatus, as well as from the Golgi apparatus to the intracellular pool and cell membrane. The galactosylation of receptor proteins may be important for the maturation of the receptor.

Downregulation and subcellular redistribution of the γ-aminobutyric acidA receptor induced by tunicamycin in cultured brain neurons

“…Our previous study has declared that the galactosylation of GABA A R is implicated in its functional expression but is independent of the regulation to subcellular localization [Yin, 1992]. Thus N-glycosylation is likely to participate in regulating the transport of the receptor, in addition to maintaining its normal function.…”

“…3 H-leucine (153 Ci/mmol) at 0.4 µCi/ml, 3 H-mannose (13.9 Ci/ml) at 10 µCi/ml, or 3 H-galactose (25.5 Ci/ml) at 5 µCi/ml was applied to the cultures 2 h before terminating the drug treatment. Subsequent to washing the cultures with phosphatebuffered salt solution (PBSS) 4 times, each dish received 1 ml sodium dodecyl sulfate (SDS)/ Nonidet P-40/urea (0.2%/2%/8 M) and was then placed on a shaker for 1 h. Proteins in the lysate were precipitated on ice by using 10% trichloroacetic acid (TCA) for 1 h, before the addition of 0.1 N NaOH [Yin, 1992]. TCA-precipitable proteins were collected by filtration of the samples through Whatman filters and washed with PBSS containing 5% TCA.…”

“…We have previously shown, using a Na ϩ /H ϩ ionophore monensin and 3 H-flunitrazepam (FNZ) as the probe for GABA A R, that the galactosylation, i.e., the maturation of receptor glycoprotein, plays a role in maintaining regular binding capacity of the receptor of embryonic chicken brain neurons in culture [Yin, 1992]. In an earlier study, the long-term administration of tunicamycin (TM), a specific inhibitor to N-glycosylation, interrupted almost completely the Cl Ϫ current elicited by activating GABA A R in Xenopus oocytes transfected by brain mRNAs [Sumikawa et al, 1988].…”

Regulation of the subcellular distribution and gene expression of GABAA receptor by microtubules and microfilaments in cultured brain neurons

“…It appears that the receptor protein passes through the Golgi apparatus for maturation of its oligosaccharides [Yin, 1992;Yin and Yang, 1992;Lin et al, 1998]. Moreover, cytoskeletal ß…”

“…The subcellular distribution of the receptor was investigated by a macromolecule, trypsin, in combination with the radioligandbinding assay, because the sensitivity of surface GABA A R to trypsin digestion allows the determination of the proportion of the receptors located intracellularly or on the cell surface [Czajkowski and Farb, 1986;Yin, 1992]. The cellular expression of GABA A R a subunits was also studied by immuncytochemistry as well as in situ hybridization assays.…”