Binding of GABA receptor channel drugs to a putative voltage-dependent chloride channel in torpedo electric organ

Abstract

In muscle and neuronal cells, there are two major types of Cl − channels: the voltage-dependent Cl − channels, which are activated by changes in voltage across the membrane and contribute a major share to the resting membrane conductance (1), and the chemically operated Cl − channels which are parts of the inhibitory receptor systems of γ-aminobutyric acid (GABA) or glycine, and are activated by binding of the transmitter (2). Although much is known about the biochemical and pharmacological nature of these receptors, very little such information is available on the voltage-dependent Cl − channels. The opportunity to study one such channel became feasible when it was discovered that the electric organ of electric rays, which is a rich source for nicotinic acetylcholine (ACh) receptors, may also be rich in Cl − channels. An anion-selective channel was discovered in the electric organs of Torpedo californica as shown by increased anion conductance in reconstituted membrane vesicles in planar phospholipid bilayer (3) and in liposomal membrane vesicles (4), and of Narke japonica as shown by changes in osmotic volume and efflux of 35SO 4 − (5). These effects were inhibited by the specific Cl − channel inhibitor 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS). This Cl − channel was also expressed in frog Xenopus oocytes that had been injected with mRNA fraction from Torpedo electric organ, while another fraction of mRNA induced the incorporation of ACh receptors (6). This electric organ Cl − channel is highly anion selective (at least 20-fold more permeable to Cl − than to K +) compared to a 2-fold selectivity for a mitochondrial Cl − channel (3). The present study was initiated to attempt identification of the Cl − channel in Torpedo electric organ, using [ 35 S]t- butylbicyclophosphorothionate (TBPS) as a probe since it binds with a high affinity to the GABA receptor Cl − channel (7), and to determine and compare the drug specificities of these different types of Cl − channels.