Multiple mechanisms of picrotoxin block of GABA‐induced currents in rat hippocampal neurons.

Abstract

SUMMARY1. WAe have examined the effect of picrotoxin on GABA-induced currents in dissociated rat hippocampal neurons. In addition, we used the putative picrotoxin receptor antagonist, ox-isopropyl-Lx-methyl-y-butyrolactone (cIMGBL), and the picrotoxin agonist, /?-ethyl-/I-methyl-y-butyrolactone (,/EMGBL) to explore the mechanisms of picrotoxin's interaction with the GABA-Cl-receptor-ionophore complex.2. The picrotoxin block of GABA current was use dependent, suggesting that the site of picrotoxin block is exposed by the conformational change initiated by GABA binding to the receptor.3. The alkyl-substituted butyrolactone antagonist, aIMGBL, selectively blocked the use-dependent mechanism of picrotoxin effect. After the apparent complete inhibition of the use-dependent effect, there was a residual picrotoxin effect that was independent of the time or concentration of GABA application. This indicates that the picrotoxin block of the GABA current is mediated by two different mechanisms. xIMGBL influences just one of these mechanisms.4. The picrotoxin receptor agonist, 8JEMGBL, exclusively blocked the GABA current in a use-dependent manner. Consistent with a use-dependent mechanism, the rate of onset of block increased with GABA concentration. Surprisingly, the fraction of GABA current block decreased with increasing GABA concentration.5. These results suggest that the relationship of picrotoxin and y-butyrolactones with the GABA-Cl-receptor-ionophore is quite complex. They are consistent with at least two possible models of agonist-antagonist interactions. Both cases require different antagonist affinities for the various kinetic states of the GABA-Clreceptor-ionophore. However, there is no need to require that either picrotoxin or

Identification of a Novel Residue within the Second Transmembrane Domain That Confers Use-facilitated Block by Picrotoxin in Glycine α1 Receptors

“…This may account for some of the reduction of glycine current amplitude following picrotoxin incubation. It is worth noting that the picrotoxin-site ligand ␤-ethyl-␤-methyl-␥-butyrolactone shows a prominent use-dependent block in GABA A receptors and can also readily access its site in the absence of channel opening (7). Indeed, in many respects, the block of GABA A receptors by ␤-ethyl-␤-methyl-␥-butyrolactone closely resembles the block of ␣1(S15ЈQ) receptors by picrotoxin described here.…”

“…Use-facilitated block was concluded based on the following criteria: 1) peak glycine current was unaffected by picrotoxin, but it greatly enhanced current decay kinetics, 2) glycine current decay rate was picrotoxin concentration-dependent, 3) current decay rate in the presence of picrotoxin was dependent on glycine concentration, and 4) the magnitude of picrotoxin block was dependent on glycine concentration. None of these traits is present in wild type glycine ␣1 receptors, and all are observed in GABA A and glutamate-gated Cl Ϫ channels (3,7,11). Thus, the introduction of Asn or Gln at the 15Ј position of TMII appears to be sufficient to confer use-facilitated block by picrotoxin.…”

“…Thus, picrotoxin might bind at an allosteric site to stabilize a closed or desensitized state of these channels (11,12,27). To resolve the complexity of the picrotoxin interaction with these channels, the existence of multiple binding sites for picrotoxin has been suggested (7,10). Upon analyzing the actions of picrotoxin and related compounds, Yoon et al (7) suggested that picrotoxin might bind to both use-dependent and use-independent sites in GABA A receptors (7).…”

“…To resolve the complexity of the picrotoxin interaction with these channels, the existence of multiple binding sites for picrotoxin has been suggested (7,10). Upon analyzing the actions of picrotoxin and related compounds, Yoon et al (7) suggested that picrotoxin might bind to both use-dependent and use-independent sites in GABA A receptors (7). Regardless of whether picrotoxin inhibition results from an interaction at one or two sites (discussed below), a molecular basis for the usefacilitated block has not been described.…”

Characterization of the Cytoprotective Action of Peroxynitrite Decomposition Catalysts

“…Dissociated cultures of rat hippocampal neurons were grown on a glial feeder layer as described previously (30,31) with the following modifications. Briefly, 1-day old Sprague-Dawley (Charles River, MA) rat pups were sacrificed, and their hippocampi were surgically removed and then dissociated both mechanically and enzymatically by papain digestion.…”